Wenche Jy

Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis.We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines.EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding.It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD106). Coronary MaVEC released significantly less EMP than MiVEC.EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium.

The molecular mechanisms by which extreme blood pressure elevation leads to vascular injury are not defined. To explore the hypothesis that activation of endothelium and platelets as manifested by increased concentrations of circulating endothelial microparticles and platelet microparticles could play a role in this target organ injury, we conducted a cross-sectional study of these markers in 3 groups: (1) untreated patients referred specifically for treatment of severe uncontrolled hypertension; (2) untreated patients with established mild hypertension; and (3) normotensive volunteer subjects. By ANOVA, endothelial (P=0.002) and platelet (P=0.01) microparticles were greatest in the severely hypertensive group. There was a significant correlation between both of these markers and blood pressure, even in the setting of multiple risk factors. Our results suggest that these markers may be useful and specific for pressure-induced endothelial and platelet activation in hypertension. Furthermore, because of the combined effects of endothelial and platelet microparticles on coagulation, leukocytes, and endothelium, it is possible that they may play a pathogenic role in mediating target organ injury in severe hypertension.

Endothelial injury plays a critical role in coronary artery disease (CAD), but the assessment of this injury has been problematical. Recently, it has been shown in vitro that endothelial cells (ECs) release endothelial microparticles (EMPs) on activation or apoptosis and that an assay of EMPs can provide useful information on EC status in patients with thrombotic disorders. This study is aimed at assessing possible correlations between EMPs, which are markers of endothelial injury, and clinical subgroups of patients with CAD. A prospective, case-controlled study was conducted on 84 patients with CAD and 42 control subjects to investigate EMP profiles. Included were 64 patients with acute coronary syndromes ([ACS], 38 with myocardial infarction [MI] and 26 with unstable angina [UA]) and 20 patients with stable angina (SA). EMPs in platelet-poor plasma were measured flow cytometrically with combinations of fluorescent antibodies (anti-CD31, -51, -42), allowing distinction of EMPs from platelet microparticles (PMPs). Clinical subgroups of patients were correlated with EMP and PMP levels in blood. Two species of EMPs (CD31+ and CD51+) were evaluated. Both were significantly higher in patients with CAD than in control subjects. CD31+ EMP was higher in ACS than SA. Among patients with first MI, CD31+ EMP was higher in patients with MI than in patients with UA and was significantly higher than in patients with recurring MI. CD51+ EMP did not discriminate ACS from SA. A simultaneous assay of PMP showed correlation between EMPs and PMPs. However, PMPs did not discriminate patients with SA from control subjects. EMP assay appears promising for assessing EC injury in CAD.

Cell-derived microparticles (MPs) are receiving increasing attention in recent years, both as a diagnostic aid and investigative tool [1-4]. Because they carry markers of the parent cell, including those induced by activation or apoptosis, endothelial MPs (EMPs) can provide valuable information on the status of the parent cell, obtainable in no other way. In addition, there is a growing belief that MPs can function as important diffusible vectors of specific adhesins and cytokines promoting cellular interactions and signal transmission [2]. Thus MP analysis constitutes a new avenue for investigation of pathologies in various diseases. Although still considered investigational [1-4], recent results from several laboratories suggest that MP analysis may be poised to enter the mainstream of clinical testing. However, a major impediment to that end is the wide variety of methodologies used by different laboratories in this field, few of which can be directly compared to the others, and results from which are sometimes inconsistent or conflicting. As a first step in addressing that problem, the Editor has organized this Forum article, consisting of a brief description of the preferred methods and rationality from each of six active laboratories in the field, including our own [5-10]. Table 1 lists some key features of the six methodological approaches. It is seen that major differences exist in the preparation of the MP samples (such as centrifugation), whether or not they are first sedimented and resuspended, means of generic MP detection (4 of 6 use annexin V), and cell lineage-specific antigenic markers. These differences probably account for some of the different findings among the groups.

The purpose of this research was to determine the levels of platelet, leukocyte, and endothelial activation and markers of cellular interactions in patients with venous thromboembolism (VTE).The details of interactions between endothelium, platelets, and leukocytes in VTE are not well understood.We studied 25 patients with VTE and compared 25 healthy controls. We used flow cytometry to measure: 1) endothelial microparticles (EMP) identified by CD31+/CD42b- (EMP(31)) or E-selectin (EMP(62E)); 2) platelet microparticles (CD31+/CD42b+); 3) surface expression of P-selectin in platelets and CD11b in leukocytes; 4) EMP-monocyte conjugates (percentage of monocytes positive for E-selectin); and 5) platelet-leukocyte conjugates (PLC) expressed as percentage of leukocytes positive for CD41.Patients with VTE had marked elevations of EMP(31) (2,193 vs. 383 counts/microl; p = 0.003), EMP(62E) (368 vs. 223 counts/microl; p = 0.001), and EMP-monocyte conjugates (3.3% vs. 2.5%; p = 0.002), as well as increased activation of platelets (35.2 vs. 5.0 fluorescence intensity units for P-selectin; p < 0.0001) and leukocytes (13.9 vs. 7.7 U for CD11b; p = 0.004). Also elevated in VTE were PLC (61.7% vs. 39.6%; p = 0.01). Expression of CD11b in leukocytes strongly correlated with PLC (r = 0.74; p < 0.0001).Marked activation of endothelium, platelets, and leukocytes occurs in VTE, and VTE, or the accompanying inflammatory process, involves the release of EMP and formation of EMP-monocyte conjugates and PLC. These findings support prior studies suggesting that release of EMP and their binding to monocytes are key events in thrombogenesis. Our findings also support the concept that the formation of PLC regulates leukocyte activation and participates in linking thrombosis with inflammation.

To assess endothelial dysfunction in patients with MS and to investigate whether plasma from patients with MS induces endothelial cell dysfunction in vitro.Endothelial cell dysfunction may contribute to the pathogenesis of MS. Elevations of soluble adhesion molecules intracellular adhesion molecule, vascular cell adhesion molecule, and platelet-endothelial cell adhesion molecule-1 (CD31) have been reported as markers of blood-brain barrier (BBB) damage in MS, but direct assay of endothelium has been difficult. Endothelial cells release microparticles < approximately 1.5 microm (EMP) during activation or apoptosis. The authors developed a flow cytometric assay of EMP and studied EMP as markers of endothelial damage in MS.Platelet-poor plasma (PPP) from 50 patients with MS (30 in exacerbation and 20 in remission) and 48 controls were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD31 and anti-CD51 (vitronectin receptor) antibodies, and two classes of EMP (CD31+ and CD51+) were assayed by flow cytometry. For in vitro studies, patients' plasma was added to the microvascular endothelial cell (MVEC) culture and release of CD31+ and CD51+ EMP were measured in the supernatant.Plasma from patients in exacerbation had 2.85-fold elevation of CD31+ EMP as compared with healthy controls, returning to near control value during remission. The CD31+ EMP concentration showed a positive association with gadolinium enhancement in patients with MS. In contrast, CD51+ EMP remained elevated in both exacerbation and remission. This suggests that CD31+ EMP is a marker of acute injury, whereas CD51+ EMP reflects chronic injury of endothelium. MS plasma induced release of both CD31+ and CD51+ EMP from MVEC culture in vitro.Endothelial dysfunction is evident during exacerbation of MS, evidenced by shedding of EMP expressing PECAM-1 (CD31). The in vitro data indicate contribution of one or more plasma factors in endothelial dysfunction of MS.

Platelets release microparticles (PMP) upon activation. Elevated levels of PMP were observed in patients with immune thrombocytopenic purpura (ITP), sometimes associated with a syndrome resembling transient ischemic attack (TIA), suggesting a thrombogenic potential for PMP. To determine if this association applies to TIA and other cerebrovascular accidents (CVA) without ITP, we studied PMP profiles in 71 patients with ischemic CVA: 28 with small vessel CVA (SCVA), either lacunar infarcts or TIA; 24 with large vessel CVA (LCVA); 19 with multiinfarct dementia (MID); 12 with Alzheimer's dementia (AD); and 31 healthy controls. The mean PMP values were: MID = 3.71 ±0.51; SCVA = 3.48 ±0.63; LCVA = 1.97 ±0.28; AD = 1.19 ±0.27; controls = 0.88 ±0.09, (all units × 107/mL). PMP values in all groups except AD were significantly above normal (p<0.01). However, the elevation in SCVA was more marked than in LCVA (p<0.01). Administration of the calcium channel blocker, nifedipine, to 11 TIA patients reduced PMP significantly.

The interaction of activated platelets with leukocytes are believed to play an important role in ischemic reperfusion injury and other thrombotic conditions. Upon activation, platelets shed platelet microparticles (PMP) and express activation markers. CD62P expressed on activated platelets mediates adhesion of platelets to leukocytes, chiefly neutrophils, but little is known of the interaction of PMP with neutrophils. We investigated this interaction as compared to platelet/ neutrophil interaction. PMP isolated from stored platelets or thrombin activated platelets was incubated with leukocytes and binding assessed by flow cytometry. FITC-labeled α-CD41 was used to assess platelet material associated with WBC. Like platelets, PMP bound preferentially to neutrophils rather than lymphocytes, and exhibited an absolute dependence on the presence of Ca2+. Binding was time- and concentration-dependent, reaching a plateau at 10 min at a ratio of PMP to neutrophils of 150:1. Fluorescence microscopy showed that most of the neutrophils were aggregated into clusters of 5-20 cells. Clustering of neutrophils was not observed to result from interaction with platelets. In these clusters the adherent PMP appeared to serve as bridges between the neutrophils. Addition of EGTA after brief incubation (5-10 min) released most of the bound PMP but if added after >10 min, only ≈60% of bound PMP were released. In contrast, nearly all bound platelets were released by EGTA at the same time of incubation. Incubation of neutrophils with PMP gave a significantly higher percentage of CD41a(+) neutrophils than did platelets incubated at the same numerical ratio. PMP association with neutrophils was less markedly inhibited by α-CD62P (AC1.2) than platelets, but binding of both PMP and activated platelets was inhibited ≈90% by anti-sialyl Lewis X. PMP binding to neutrophils induced a significant increase in both CD11b expression and phagocytic activity in a concentration-dependent manner. These findings suggest a possible role for PMP in addition to providing platelet factor 3, specifically, as an activator and mediator of neutrophils in ischemic injury, thrombosis, and inflammation.

Endothelial injury is believed to be a key initiating event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), leading to platelet activation and formation of platelet‐rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and clinical assays to rapidly and reliably monitor endothelial damage are not readily available. Using flow cytometry, we measured endothelial microparticles (EMPs) generated from cultured renal and brain microvascular endothelial cells (MVECs) during activation and apoptosis, and evaluated the effect of TTP plasma on them. EMPs were measured using positivity for monoclonal antibodies (mAbs) CD31 and CD51, and their procoagulant activity was assessed using a Russell viper venom assay. Both cell lines generated procoagulant EMPs when cultured with inducers of activation (tumour necrosis factor alpha; TNF‐α) or apoptosis (mitomycin C). TTP plasma induced a five‐ to sixfold increase of EMP generation and a two‐ to threefold increase of procoagulant activity in cultured brain and renal MVECs. TTP plasma induced a threefold and 13‐fold increase of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) expression, respectively, on renal MVECs. Procoagulant activity tended to parallel EMP numbers. The effect of TTP plasma on cell viability was similar to that of TNF‐α, implying that it induced activation rather than apoptosis. Control plasma and idiopathic thrombocytopenic purpura (ITP) plasma had little effect. In the clinical study, EMP assay of blood from acute TTP patients showed levels markedly elevated compared with normal controls, but values returned to normal in remission. In conclusion, TTP plasma activated and induced injury to MVECs in culture, judged by production of EMP and expression of activation markers. Released procoagulant EMP may play a role in the pathogenesis of TTP. Assay of EMP may be a useful marker of disease activity and endothelial injury in TTP and possibly other thrombotic disorders.

Objective: Mortality in sepsis is believed to be associated with exaggerated inflammatory responses, but recent evidence suggests that poor outcome is associated with reduced inflammation. To test this hypothesis, we measured several inflammatory markers to determine whether any of them or any combinations are associated with mortality or organ dysfunction. Design: Clinical study. Setting: School of medicine. Patients: Thirty-five patients with severe sepsis. Interventions: Markers of endothelial, platelet, and leukocyte activation were measured on days 1, 2, and 3 after enrollment. The markers were a) endothelial microparticles (EMPs) and their conjugates with monocytes (EMP/MONO); b) platelet microparticles (PMPs) and platelet activation marker CD62P; c) platelet-leukocyte conjugates (PLT/LEU) and leukocyte activation marker CD11b; and d) intracellular nitric oxide in leukocytes. Measurements and Main Results: The 28-day mortality rate was 51% (18 of 35). Significant differences between survivors and nonsurvivors on day 1 were found in PLT/LEU (p = .001), CD11b (p = 0.02), and EMP/MONO (p = .02) groups. Using logistic regression to assess if these markers predict mortality on day 1, we found that PLT/LEU had the best predictive value among the markers used (area under receiver operating characteristics curve = 0.82). All markers of cell activation and inflammation were significantly higher among survivors on days 2 and 3, except nitric oxide, which was lower. This marker showed significant negative correlation with the Sequential Organ Failure Assessment score throughout the study. Conclusions: Our data support the hypothesis that early increased, not decreased, inflammatory response as measured by our markers is associated with improved survival rate. A high negative correlation was found between some of these markers and Sequential Organ Failure Assessment score.

Accumulating evidence has shown a strong association between the metabolic syndrome (MS) and a chronic inflammatory state predisposing to atherosclerosis. We investigated leukocyte, platelet, and endothelial activation markers and cellular interactions in 33 patients with the MS and 25 healthy controls. Using flow cytometry, we measured: (1)P-selectin expression in platelets; (2) platelet microparticles identified by CD31 expression; (3) endothelial microparticles (EMPs) identified by expression of CD31 (EMP(31)), CD62E (EMP(62E)), and CD51 (EMP(51)); (4) conjugates of leukocytes with platelet microparticles/platelets and with EMPs identified by CD54 (EMP(54)); and (5) CD11b expression in leukocytes. Patients with the MS had markedly elevated EMP(31), although EMP(62E) levels were normal, suggesting that EMP(31) levels were increased because of endothelial cell apoptosis, rather than activation. EMP(51), EMP(54)-lymphocyte conjugates, platelet expression of P-selectin, CD11b expression in leukocytes, and platelet-lymphocyte conjugates were also increased in patients with the MS. Platelet-leukocyte conjugates correlated with leukocyte activation, suggesting that platelet binding to leukocytes regulates leukocyte activation in vivo. In conclusion, our data demonstrate endothelial cell microparticle release, platelet and leukocyte activation, and increased binding of EMPs and platelets to leukocytes in patients with the MS.

A fatality in one multiple sclerosis (MS) patient due to acute idiopathic thrombocytopenic purpura (ITP) and a near fatality in another stimulated our interest in platelet function abnormalities in MS. Previously, we presented evidence of platelet activation in a small cohort of treatment-naive MS patients.In this report, 92 normal controls and 33 stable, untreated MS patients were studied. Platelet counts, measures of platelet activation [plasma platelet microparticles (PMP), P-selectin expression (CD62p), circulating platelet microaggragtes (PAg)], as well as platelet-associated IgG/IgM, were carried out. In addition, plasma protein S activity was measured.Compared to controls, PMP were significantly elevated in MS (p < 0.001) and CD62p expression was also markedly elevated (p < 0.001). Both are markers of platelet activation. Platelet-associated IgM, but not IgG, was marginally elevated in MS (p = 0.01). Protein S in MS patients did not differ significantly from normal values.Platelets are significantly activated in MS patients. The mechanisms underlying this activation and its significance to MS are unknown. Additional study of platelet activation and function in MS patients is warranted.

This article reviews evidence for the involvement of cell‐derived microparticles (MPs) in transfusion‐related adverse events. The controversy concerning possible added risk of older versus fresher stored blood is also reviewed and is consistent with the hypothesis that MPs are involved with adverse events. Although all types of circulating MPs are discussed, the emphasis is on red blood cell–derived MPs (RMPs). The evidence is particularly strong for involvement of RMPs in transfusion‐related acute lung injury, but also for postoperative thrombosis. However, this evidence is largely circumstantial. Work in progress to directly test the hypothesis is also briefly reviewed.

<h3>Background</h3> In light of recent reports of diminished platelet serotonin concentration and increased plasma serotonin levels in patients with Alzheimer disease (AD), we hypothesized that a state of heightened platelet activation might be present in AD. <h3>Objective</h3> To compare baseline activation of unstimulated platelets in patients with AD with that in control subjects. <h3>Patients and Methods</h3> Flow cytometry was used to measure platelet activation in 91 patients with probable AD and 40 age-matched control subjects. Groups were compared for percentage of circulating platelet aggregates, expression of CD62p, formation of leukocyte-platelet complexes, and presence of circulating platelet microparticles, controlling for effects of demographic, clinical, physiological, and logistical factors. <h3>Results</h3> Multiple analysis of covariance on ranked data revealed a 39.5% increase in percentage of platelet aggregates (<i>P</i>=.0001), a 59.3% increase in expression of CD62p (<i>P</i>=.001), and a 53.3% increase in leukocyte-platelet complexes (<i>P</i>=.0001) in the group with AD but no differences in the number of platelet microparticles, overall platelet count, plasma fibrinogen level, or plasma platelet factor 3. Activation was weakly correlated with sex, but was independent of age, severity of disease, duration of disease, depression, agitation, and family history of dementia. <h3>Conclusions</h3> Platelets of patients with AD exhibit greater unstimulated activation than those of controls. Potential causes of such activation include possible stimulation of platelets by damaged cerebral endothelial cells or platelet activation induced by membrane abnormalities previously reported to be present in platelets of patients with AD. In light of recent evidence that platelets are the principal source of both amyloid precursor protein and β-amyloid peptide in human blood, it is possible that AD platelet activation may reflect or even contribute to the pathogenesis of the disease.

This study evaluated a possible relationship between levels of endothelial microparticles (EMPs), known to be a sensitive indicator of endothelial disturbance, and changes in postprandial lipid levels in healthy volunteers after a low- or high-fat meal.Eighteen healthy subjects without known cardiovascular risk factors were evaluated. Lipid and EMP levels were measured before and 1 and 3 hours after a single low- or high-fat isocaloric meal. The low-fat meal had no significant postprandial effect on EMPs or lipids compared with fasting levels. In contrast, a single high-fat meal significantly increased EMP levels after 1 and 3 hours, from 389+/-54 (thousands per milliliter) when fasting to 541+/-139 (P=0.0002) and 677+/-159 (P<0.0001), respectively, and correlated with a postprandial elevation in serum triglycerides.A single high-fat meal led to a significant elevation of plasma EMP levels in healthy, normolipidemic subjects and correlated with a postprandial elevation of serum triglycerides. EMPs may be an indirect marker of endothelial dysfunction or injury induced by postprandial triglyceride-rich lipoproteins.

Endothelial Microparticles (EMP) are small fragments of endothelial cell membrane shed during apoptosis or activation. Our group has previously reported elevations of EMP in patients with coronary artery disease (CAD), thrombotic thrombocytopenic purpura (TTP), pre-eclampsia, multiple sclerosis (MS), and severe hypertension (HTN). In the present study, we evaluate the possible relationship between EMP levels and the angiographic severity and characteristics of coronary obstructive lesions.We studied a total of 43 patients undergoing coronary angiography. Fifteen had presented with acute myocardial infarction (MI), 20 with unstable anginas (UA), 5 with stable angina (SA) and 3 with congestive heart failure. Coronary angiography was reviewed and coronary lesions were classified using the Ambrose classification. Coronary stenoses were classified as high and low risk. High-risk included lesions with eccentric appearance (type II), presence of thrombi, or multiple irregularities. Low-risk lesions were defined as concentric or type I. Lesions were also analyzed by degree of stenosis and history of acute coronary syndrome (ACS). EMP in plasma was assayed by flow cytometry.EMP in eccentric type II or multiple irregular lesions (high-risk) were 2.5-fold higher than in type I or concentric (low-risk) lesions, p<0.05. Lesions with thrombi had three-fold higher EMP than those without (p=0.05). Mild stenosis (>20%-<45%) had three-fold higher EMP than more severe (>45%), and five-fold higher than those without stenosis (p<0.01). Among patients with type II lesions, those with first ACS episode had four-fold higher EMP levels than those with recurrent ACS (p<0.01).High EMP was associated with high-risk angiographic lesions including eccentric type II, multiple irregular, and lesions with thrombi. Mild to moderate stenosis was associated with higher EMP levels than severe stenosis. EMP may be a useful marker in detecting endothelial injury and risk of ACS as defined by angiography.

Analysis of circulating cell-derived microparticles (MP) is becoming more refined and clinically useful. This review, stemming from lectures given at Tokyo late 2003, does not repeat prior reviews but focuses on new horizons. A major theme is the rising recognition of platelets and their MP (PMP) as key mediators of inflammation/immunity. Among the major concepts developed are that (i) many so-called soluble markers of inflammation are in reality MP-bound; (ii) PMP and other MP appear to serve important signaling and immune functions including antigen presentation. In conclusion, MP analysis is poised to enter the mainstream of clinical testing, measuring specific antigens rather than gross levels. However, more research is needed to decisively establish their functions, and international standards are needed to allow comparing results from different laboratories.

Platelet microparticles (PMPs) are vesicles derived from platelet membranes that are too small (less than 0.5 micron) to be detected in routine platelet counting. They arise in association with platelet activation and other unknown causes. Elevated PMPs have been observed in idiopathic thrombocytopenic purpura (ITP), a disorder in which autoantibody interacts with platelets and the opsonized platelets are destroyed by macrophages. However, the clinical significance of PMP has been unknown. Using flow cytometry, we examined PMP concentrations in 62 patients with ITP and in 33 normal control subjects to assess the clinical significance of PMP in ITP. When compared with PMP levels in control subjects, PMP levels were significantly higher (p less than 0.005) in patients with ITP, but considerable variation among individual patients was observed. Patients with platelet counts less than or equal to 60,000 were evaluated for correlation of PMP levels with manifestations of thrombocytopenias; patients without symptoms (free of petechiae or mucosal bleeding) are found to have significantly higher PMP levels (p less than 0.05) than patients with symptoms, suggesting hemostatic protection by PMP. Additionally, we identified a group of patients with ITP who experienced neurologic complications resembling transient cerebral ischemic attacks (TIAs): recurrent episodes of dizzy spells or weakness in mild cases, and coma, seizure, or progressive dementia in advanced cases. Small cerebral infarcts were demonstrated by computed axial tomography scan or magnetic resonance imaging in spite of severe thrombocytopenias. Patients with this syndrome are often found to have higher PMP levels (p less than 0.005) when compared with the group free of neurologic complications. It is concluded that PMPs play an important role in hemostasis in patients with thrombocytopenia, and that high concentrations of hemostatically active PMP can be thrombogenic in certain clinical settings. Quantitation and characterization of PMP is important in assessment and management of patients with thrombocytopenia.

Summary. It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3‐ to 10‐fold and 8‐ to 10‐fold respectively. However, CD31 and CD105 were reduced 40–60% in EC but increased twofold in EMP. VWF expression was found in 55 ± 15% of CD62E + EMP. Markers of apoptosis were negative. In TTP patients, CD62E + and CD31 + /CD42b − EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E + EMP (60 ± 20%) co‐expressed VWF and CD62E. The ratio of CD31 + /42b − to CD62E + EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co‐express VWF and CD62E could play a role in the pathogenesis of TTP.

Elevated plasma endothelial microparticles (EMP) have been documented in MS during exacerbation. However, the role of EMP in pathogenesis of MS remains unclear. We investigated the formation of EMP-monocyte conjugates (EMP-MoC) and their potential role in transendothelial migration of inflammatory cells in MS. EMP-MoC were assayed in 30 MS patients in exacerbation, 20 in remission and in 35 controls. EMP-leukocyte conjugation was investigated flowcytometrically by employing alpha-CD54 or alpha-CD62E for EMP, and alpha-CD45 for leukocytes. EMP-MoC were characterized by identifying adhesion molecules involved and their effect on monocyte function. In vivo (clinical): EMP-MoC were markedly elevated in exacerbation vs. remission and controls, correlating with presence of GD+ MRI lesions. Free CD54+ EMP were not elevated but free CD62E+ EMP were. In vitro: EMP bound preferentially to monocytes, less to neutrophils, but little to lymphocytes. Bound EMP activated monocytes: CD11b expression increased 50% and migration through cerebral endothelial cell layer increased 2.6-fold. Blockade of CD54 reduced binding by 80%. Most CD54+ EMP bound to monocytes, leaving little free EMP, while CD62+ EMP were found both free and bound. These results demonstrated that phenotypic subsets of EMP interacted differently with monocytes. Based on our observations, EMP may enhance inflammation and increase transendothelial migration of monocytes in MS by binding to and activating monocytes through CD54. EMP-MoC were markedly increased in MS patients in exacerbation compared to remission and may serve as a sensitive marker of MS disease activity.

Elevated plasma endothelial microparticle levels have been found to be elevated in women with preeclampsia. However, their role in distinguishing preeclampsia from gestational hypertension remains to be elucidated. The objectives of this study were to compare endothelial microparticle levels among patients with preeclampsia, gestational hypertension, and healthy pregnant control subjects and to evaluate the effect of plasma from women with preeclampsia and gestational hypertension on the release of endothelial microparticles by renal microvascular endothelial cells.A prospective study was conducted on 52 women with preeclampsia, 20 women with gestational hypertension, and 38 healthy pregnant control subjects. Endothelial microparticles were measured by flow cytometry with fluorescent monoclonal mouse anti-human antibodies against CD31, CD42b, and CD62E.CD31 + /42b - endothelial microparticle levels were 10497 +/- 5145 counts/microL in women with preeclampsia versus 6768 +/- 1810 counts/microL in women with gestational hypertension ( P < .01). In control subjects, CD31 + /42b - endothelial microparticle levels were 6119 +/- 3592 counts/microL. CD62E + endothelial microparticle levels were 1930 +/- 966 counts/microL in women with preeclampsia versus 822 +/- 150 counts/microL in women with gestational hypertension ( P <.01). In control subjects, CD62E + endothelial microparticle levels were 712 +/- 160 counts/microL. Incubation of renal microvascular endothelial cells with plasma from women with preeclampsia resulted in a rise in CD31 + and CD62E + endothelial microparticle levels as compared with women with gestational hypertension and control subjects.Endothelial microparticle levels are higher in women with preeclampsia than in women with gestational hypertension and control subjects. The measurement of endothelial microparticles may be useful as a diagnostic tool for preeclampsia in pregnant women.

Multiple sclerosis (MS) has traditionally been viewed and researched as an immune-mediated disease with principal emphasis on the role of activated inflammatory cells, oligodendrocytes and astrocytes in its pathogenesis. Abnormalities of cerebral endothelial cells (CECs) is an under explored facet of MS pathogenesis and vascular abnormalities play a crucial role in formation of the MS lesions and disease progress, at least in the initial stages of disease. This review will focus on MS as a central nervous system (CNS) disease with a strong vascular constituent and examines abnormalities within CECs in MS and their role in the loss of blood–brain barrier and transendothelial migration of activated leukocytes into the CNS. One goal of this paper is to persuade and promote research on the endothelial abnormalities in pathogenesis of MS and to exploit existing knowledge on endothelial injury. A deeper understanding of endothelial pathophysiology in MS may help develop effective treatments through stabilization of endothelial function, translating into delay or arrest of MS disease onset and disability in MS patients.

This review summarizes recent developments in platelet biology relevant to neuroinflammatory disorders. Multiple sclerosis (MS) is taken as the "Poster Child" of these disorders but the implications are wide. The role of platelets in inflammation is well appreciated in the cardiovascular and cancer research communities but appears to be relatively neglected in neurological research.After a brief introduction to platelets, topics covered include the matrix metalloproteinases, platelet chemokines, cytokines and growth factors, the recent finding of platelet PPAR receptors and Toll-like receptors, complement, bioactive lipids, and other agents/functions likely to be relevant in neuroinflammatory diseases. Each section cites literature linking the topic to areas of active research in MS or other disorders, including especially Alzheimer's disease.The final section summarizes evidence of platelet involvement in MS. The general conclusion is that platelets may be key players in MS and related disorders, and warrant more attention in neurological research.

Elevation of free cytoplasmic calcium is the common pathway of platelet activation, leading to shape change, shedding of platelet microparticles (PMP), aggregation, and secretion of internal granules, including expression of CD62p on the surface. Platelet activation is well documented in unstable angina (UA) and acute myocardial infarction (MI). We investigated the following markers of platelet activation in 55 patients undergoing coronary angiography for suspected CAD: free cytoplasmic calcium, [Ca2+]cyt, PMP, CD62p expression, and platelet/leukocyte (P/L) interaction. [CA2+]cyt was measured by Fluo-3 and the other measurements were by flow cytometry. Patients were classified into three groups: unstable angina (UA, n = 11), recent myocardial infarction (MI, n = 11), and patient controls (CTL, n = 33). Blood was drawn before infusion of heparin through femoral lines at the time of catheterization for assays. Results: (1) PMP values were significantly higher in both UA and MI than in CTL, P < 0.05. There was no difference between UA and MI. (2) P/L interaction was significantly elevated only in UA, P < 0.05. (3) CD62p expression on free platelets did not differ significantly between any of the three groups. (4) The resting [Ca2+]cyt, thrombin-induced CA2+ influx, and release of CA2+ from internal stores were all significantly higher in platelets from the combined patient group (UA + MI) than in the patient group, P < 0.001 Conclusions: Results on calcium hemostasis and PMP were significantly different in patients with acute coronary syndromes than those with stable angina or no coronay ischemia; this may reflect underlying pathophysiology of acute coronary ischemia. P/L interaction was higher only in the UA group, suggesting a role of leukocytes in the UA. Am. J. Hematol. 54:95–101, 1997 © 1997 Wiley-Liss, Inc.

Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL(-1)), EMP induced up to 95% of platelets to aggregate. EMP-platelet interaction was 80% blocked by anti-CD42b, or by 0.1 microm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate-P) dissociated 50% within 15-25 min following 1:20 dilution. In contrast, aggregates formed with EMP persisted two- to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin-induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

The purpose of this study was to assess endothelial dysfunction in women with preeclampsia by measuring endothelial microparticles.A case-control study was conducted on 20 women with preeclampsia and 20 healthy pregnant women as control subjects. Endothelial microparticles were measured by flow cytometry with anti-CD31 and anti-CD42. CD31(+)/CD42(+) platelet microparticles were also quantified.Plasma endothelial microparticles levels were elevated significantly in women with preeclampsia as compared with control subjects (mean+/-SD and median [range]: 14,723+/-7,724 counts/microL and 12,378 counts/microL [1,442-33,772 counts/microL] vs 8406+/-2832 counts/microL and 9016 counts/microL [3,381-12,806 counts/microL]; P<.001). Plasma platelet microparticles levels were not different among cases compared to control subjects (10,751+/-6,114 counts/microL and 9463 counts/microL [3,000-23,895 counts/microL] vs 7871+/-4344 counts/microL and 6462 counts/microL [444-18,947 counts/microL]; P=.208). No significant correlation was found between plasma endothelial microparticles and mean arterial pressure in cases or control subjects.The elevation of endothelial microparticles in women with preeclampsia supports the endothelial injury theory in preeclampsia.

<h3>Objective</h3> To evaluate the efficacy, safety, and tolerability of combination therapy with intramuscular interferon beta-1a and oral doxycycline, a potent inhibitor of matrix metalloproteinases, in patients with relapsing-remitting multiple sclerosis (RRMS) having breakthrough disease activity. <h3>Design</h3> Open-label, 7-month trial. <h3>Setting</h3> Louisiana State University Health Sciences Center, Shreveport. <h3>Patients</h3> Fifteen patients with RRMS taking interferon beta-1a with breakthrough disease activity took doxycycline for 4 months. Patients underwent monthly neurologic examination, magnetic resonance imaging of the brain using triple-dose gadolinium, and safety blood work. <h3>Interventions</h3> Ongoing treatment with intramuscular interferon beta-1a plus oral doxycycline, 100 mg daily, for 4 months. <h3>Main Outcome Measures</h3> The primary end point was gadolinium-enhancing lesion number change, and the secondary end points were relapse rates, safety and tolerability of the combination of interferon beta-1a and doxycycline in patients with MS, Expanded Disability Status Scale score, serum matrix metalloproteinase-9 levels, and transendothelial migration of monocytes exposed to serum from patients with RRMS. <h3>Results</h3> Combination of doxycycline and interferon beta-1a treatment resulted in reductions in contrast-enhancing lesion numbers and posttreatment Expanded Disability Status Scale values (<i>P</i> &lt; .001 for both). Only 1 patient relapsed. Multivariate analyses indicated correlations between decreased serum matrix metalloproteinase-9 levels and enhancing lesion activity reduction. Transendothelial migration of monocytes incubated with serum from patients with RRMS undergoing combination therapy was suppressed. Adverse effects were mild; no adverse synergistic effects of combination therapy or unexpected adverse events were reported. <h3>Conclusions</h3> Combination of intramuscular interferon beta-1a and oral doxycycline treatment was effective, safe, and well tolerated. Controlled clinical trials in larger cohorts of patients with MS are needed to evaluate the efficacy and tolerability of this combination. <h3>Trial Registration</h3> clinicaltrials.gov Identifier:NCT00246324 Published online December 10, 2007 (doi:10.1001/archneurol.2007.41).

Summary. Background: Circulating cell-derived microparticles (MP) are important players in thrombogenesis, attributed in part to tissue factor (TF) carried on them. We developed MP-mediated thrombin generation assay (TGA) and measured a series of patients with thrombosis (TBS) and normal controls (NC). Methods: MP were isolated from plasma of 66 patients with TBS and 34 NC. The MP were resuspended in normal pooled particle-free plasma (PFP) containing corn trypsin inhibitor (to inhibit contact pathway). MP mediated TGA yields three parameters: lag time, peak and rate. This method is not influenced by anticoagulant therapy. Of the TBS patients, 41 had only a single thrombosis (S-TBS) and 25 had recurrences (R-TBS) within a 5-year period. In parallel, MP were quantitated by flow cytometry, and cell origin was determined: endothelial cells (EMP), leukocytes (LMP), red cells (RMP) and platelets (PMP). Results: MP from all TBS patients exhibited higher thrombin generation than NC by all three TGA parameters. R-TBS had significantly greater TGA values than S-TBS, reflected in higher peak and rate, and shorter lag time. MP numbers were also higher in TBS vs. NC, for all MP subtypes, and were significantly higher in R-TBS than S-TBS (except LMP). All MP levels correlated with thrombin generation (P < 0.0001), most closely between PMP and peak (R = 0.47) and rate (R = 0.43). Conclusions: MP-mediated TGA is a novel way to assess functional procoagulant activity of MP. Enhanced MP-mediated TGA was demonstrated in TBS patients, and significantly higher activity in R-TBS. These findings support a major role of MP in thrombogenesis.

Background: Splenectomy is frequently employed for therapeutic and diagnostic purposes in various clinical disorders. However its long-term safety is not well elucidated. Although risk of infection by encapsulated organisms is widely recognized, less well-known are risks of thrombosis and cardiovascular disease. Methods: We investigated levels of cell-derived microparticles (C-MP) in 23 splenectomized ITP (ITP-S) and 53 unsplenectomized ITP patients (ITP-nS). Assay of C-MP derived from platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) were performed by flow cytometry. Coagulation parameters included PT, aPTT and activities of FVIII, IX and XI. Results of all measures were compared between the two groups, ITP-S vs ITP-nS. Results: Levels of all C-MP were higher in ITP-S than ITP-nS but only RMP and LMP reached statistical significance (p=0.0035 and p<0.0001, respectively). The aPTT was significantly shorter in ITP-S (p=0.029). Interestingly, correlation analysis revealed that RMP, but not other C-MP, were associated with shortening of aPTT (p=0.024) as well as with increased activities of factors VIII (p=0.023), IX (p=0.021) and XI (p=0.0089). Conclusions: RMP and LMP were significantly elevated in splenectomized compared to non-splenectomized ITP patients. This suggests that the spleen functions to clear procoagulant C-MP, and that elevation of C-MP might contribute to increased risk of thrombosis, progression of atherosclerosis and cardiovascular disease following splenectomy.

Summary Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 μm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

Soluble CD40L (sCD40L) ELISA has emerged as a promising predictor of poor outcomes in acute coronary syndrome. Yet many blood processing techniques have been used with little consideration of their effect on the results.We measured sCD40L by ELISA in 10 patients with thrombocytopenia and 12 with normal or high platelet counts and 8 healthy controls using three sampling techniques: serum clotted on ice (serum-I) or at room temperature (serum-RT) and platelet poor plasma (PPP).Serum-RT samples, compared to serum-I, gave significantly higher CD40L values (p=0.003), demonstrating that ex vivo sCD40L release by activated platelets is inhibited by cold temperature. Although serum-I and PPP were comparable in patients with normal platelet counts, serum-I gave significantly higher values than PPP in the thrombocytosis group (p=0.01), suggesting that cold inhibition is insufficient in the latter group. To estimate the fraction of sCD40L that was microparticle-bound CD40L (mp-CD40L), 16 samples underwent 0.1-microm filtration. 50.6% of sCD40L was mp-CD40L in serum-RT, whereas 21.3% and 29.9% were observed in serum-I and PPP, respectively. Lastly, plasma sCD40L was assayed in 46 patients with and 35 without thrombosis. Plasma sCD40L did not correlate with platelet count in non-thrombotic, non-inflammatory patients but did (p<0.01) in those with thrombosis.Sample processing and temperature profoundly affect sCD40L assay. Serum-I and PPP minimize the release of sCD40L ex vivo and better represent sCD40L in vivo. However, PPP may be preferable particularly in patients with thrombocytosis. The existence of mp-CD40L highlights the importance of centrifuge conditions.

Monocyte migration through the disrupted cerebral endothelial cell (EC) junctions plays an essential role in formation of multiple sclerosis (MS) demyelinating lesions. During pathogenesis of MS, activated ECs release endothelial microparticles (EMP), which possibly facilitate transendothelial migration (TEMIG) of monocytes. To assess functional roles of EMP in MS, specifically, their (i) interaction with monocytes, (ii) effect on monocyte TEMIG in an in vitro model of the brain microvascular endothelial cells (BMVEC), (iii) phenotypic profiles of EMP elicited by MS plasma and (iv) the effects of IFN-beta 1b on release of EMP and on TEMIG of monocytes (mono) and monocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-beta 1b on the release of EMP and the TEMIG of mono and mono:EMP was assessed by preincubating BMVEC cultures of IFN-beta 1b prior to addition of plasma. Three EMP phenotypes, CD54, CD62E and CD31 were assayed. Plasma specimens from 20 patients with relapsing remitting MS (11 in exacerbation, MS-E, and 9 in remission, ME-R) and 10 healthy controls were studied. Incubation of BMVEC with MS-E plasma yielded elevated levels of EMPCD54, EMP62E and EMPCD31 relative to MS-R and control plasmas. MS-E but not MS-R or control plasma also augmented TEMIG of monocytes, respectively. Mono:EMP complexes further augmented TEMIG relative to mono alone, but only in the presence of MS-E plasma; there was no significant effect with MS-R or control plasmas. The presence of IFN-beta 1b inhibited TEMIG of mono and mono:EMP by 20% and 30%, respectively. MS-E but not MS-R plasma elicited release of activation-derived EMP and enhanced TEMIG of mono and mono:EMP. IFN-beta 1b inhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-beta 1b in MS.

Abstract Previously we reported that treating human fibroblasts in cell culture with high‐voltage, pulsed galvanic stimulation (HVPGS) can significantly increase cellular protein and DNA synthesis (Bourguignon and Bourguignon: FASEB J., 1:398–402, 1987). In this study we have identified two of the early cellular events which occur following exposure to HVPGS: (1) an increase in Ca 2+ uptake from the external medium and (2) an increase in the number of insulin receptors on the fibroblast cell surface. The increase in Ca 2+ uptake begins within the first minute of electric stimulation while increased insulin binding is not detected until the second minute of stimulation. The HVPGS‐induced increase in insulin binding can be inhibited by bepridil, a specific Ca 2+ channel blocker, suggesting that the Ca 2+ influx is required for the exposure of additional insulin receptors on the cell surface. Furthermore, we have determined that the addition of insulin to electrically stimulated cultures results in (1) an immediate, second increase in Ca 2+ uptake and (2) significant increases in both protein and DNA synthesis compared to cells which were not stimulated. All three of these insulin‐dependent effects are also inhibited by bepridil. Based on these results, we propose that HVPGS initially triggers the opening of voltage‐sensitive calcium channels in the fibroblast plasma membrane. The increased level of intracellular Ca 2+ then induces the exposure of additional insulin receptors on the cell surface. If insulin is available to bind the additional receptors, the fibroblasts will significantly increase both protein and DNA synthesis.

Background Anti-phospholipid antibodies (APLA) are often associated with thrombosis, defining the antiphospholipid syndrome (APS) but it remains unclear why many subjects who are positive for APLA chiefly anti-cardiolipin (aCL) or anti-β2GPI (aβ2GPI) do not develop thrombosis. A related question addressed in this study is whether the target of cellular injury in APS is predominately platelets or endothelial cells (EC). Methods aCL and aβ2GPI were determined by ELISA in 88 patients, 60 of whom were thrombotic and 28 non-thrombotic. Platelet activation was measured by CD62P and by concentration of platelet microparticles (PMP) and EC activation was assessed by endothelial microparticles (EMP), both by flow cytometry. Lupus anticoagulant (LAC) was measured in the hospital laboratory. Results There was no difference in frequency of aCL or aβ2GPI, neither IgG or IgM, between the thrombotic and non-thrombotic groups. Both groups showed elevated EMP compared to controls but this did not differ between thrombotic and non-thrombotic groups. In contrast, PMP were not significantly elevated in non-thrombotic but were elevated in thrombotic compared to non-thrombotic (p=0.03) and controls. CD62P, an independent marker of platelet activation, was also elevated in thrombotic vs. non-thrombotic. There was a trend for increased LAC in the thrombotic group but not significant. Conclusion Although all subjects had evidence of endothelial activation, only platelet activation differed between thrombotic and non-thrombotic. This supports the hypothesis that platelet activation predisposes to thrombosis in the presence of chronic EC activation. These data also raise the possibility of distinguishing risk-prone APLA-positive individuals.

All blood cells and the vascular endothelium shed microparticles (MP) from their plasma membranes when suitably stimulated, and assay of MP in patient blood has found increasing application to the monitoring of disease states. In addition, mounting evidence suggests that MP are not mere epiphenomena but play significant roles in the pathophysiology of thromboses, inflammation, and cancers. This chapter endeavors to summarize the limited number of studies thus far done on MP in neurological disorders such as multiple sclerosis (MS), transient ischemic attacks, and the neurological manifestations of antiphospholipid syndrome (APS). In addition, the chapter offers some plausible hypotheses on possible roles of MP in the pathophsyiology of these disorders, chiefly, the hypothesis that MP are indeed important participants in some neuropathologies, especially those which are ischemic in nature, but probably also inflammatory ones. The chapter also goes over the history and general principles of MP studies (e.g., assay methods and pitfalls), comparison with alternative methods (e.g., soluble markers of disease states), subclasses of MP (such as exosomes), and other topics aimed at helping readers to consider MP studies in their own clinical fields. Tables include a listing of bioactive agents known to be carried on MP, many of which were heretofore considered strictly soluble, and some of which can be transferred from cell to cell via MP vectors, for example certain cytokine receptors.

It is emerging that cell-derived microparticles (MP) have multiple functional activities in areas including hemostasis, thrombosis, inflammation, and as messengers in the transport of bioactive lipids, cytokines, complement, and immune signaling. Some of these activities may be performed by distinct phenotypic subsets of MP, even if derived from the same cell type. The focus of this article concerns the size classes of MP, covering methods of MP size measurement, differences in composition between size classes, and relation of size to functional (procoagulant) activity. Some of the issues considered remain to be resolved, such as whether the MP known as exosomes are truly a distinct class of MP, as well as the detailed mechanisms underlying the release of MP of different size ranges.

Platelet factor 3 (PF3) was assayed by Russell's viper venom (RVV) in three plasma fractions, platelet-rich plasma (PRP), platelet poor plasma (PPP), and 0.1 μm particlefiltered plasma (PFP), in 42 healthy controls, 34 patients with recent cerebrovascular accidents (CVA) and 28 with recent ischemic events from coronary artery disease (CAD). Platelet microparticles (PMP) were assayed in PPP by flow cytometry. Relative to controls, the RVV clotting times were shortened in all three plasma fractions in both patient groups, p<0.001. PMP were also elevated in both patient groups, p<0.001. Linear regression analysis showed that the RVV times of PPP are inversely correlated with PMP, p<0.005, in patient groups but not in controls. There was no correlation of RVV time with PT, APTT or FIB. After converting RVV times to units of PF3 activity, it could be shown that only about of the total PF3 activity was contributed by platelets. The major contribution to the PF3 activity in controls was from microparticles <0.1 μm but in patients was due mainly to microparticles >0.1 μm. The RVV time was superior to routine coagulation tests in discriminating thrombotic patients from healthy controls.

Platelets have been implicated in memory disorders but this has not been investigated in patients with immune or idiopathic thrombocytopenia (ITP). ITP is an autoimmune disorder in which autoantibodies bring about platelet destruction. We previously reported a group of ITP patients who manifested TIA-like syndrome and gradual memory loss leading to dementia: platelet microparticles (PMP), a marker of platelet activation, were often elevated, suggesting that procoagulant PMP released from stimulated platelets contributed to thrombosis in small vessels. We have expanded on those studies to better define the clinical, laboratory, and radiologic characteristics of this syndrome.Twenty ITP patients with this syndrome were studied in comparison to twenty-three ITP patients without it (patient controls). Clinical and laboratory features were compared and radiologic images were analyzed. Factors influencing the rate of progression to advanced dementia were also investigated.Recurring dizzy or weak spells, TIA-like syndrome, recent memory loss, and cognitive impairment were common initial complaints. In some, these symptoms progressed rapidly to dementia but was indolent in others. Progression was faster in those with splenectomy and higher platelet counts. MRI showed enhanced signal in subcortical, periventricular areas, consistent with ischemic small vessel disease. Compared to patient controls, bleeding was less frequent and platelet activation (increased PMP, CD62p) was more frequent in the study group. Thrombotic complications may occur in ITP, manifested as TIA-like syndrome or memory loss due to ischemic small vessel disease, progressing to vascular dementia. Memory disturbances associated with platelet disorders warrants further investigation.

To the Editors: Thrombocytopenia and increased levels of platelet activation markers are common in untreated HIV-infected individuals.1,2 Although low platelet counts usually correct after initiation of highly active antiretroviral therapy (HAART),1 little is known about the functional state of platelets in this scenario. Cellular microparticles are small (<1.5 μm), cell membrane-derived vesicular fragments released upon cellular activation or apoptosis of parental cells.3 Abnormal levels of platelet microparticles (PMP) are associated with several prothrombotic conditions.4-6 Expression of P-selectin (CD62P) on platelets is a well-recognized indicator of platelet activation.7 We sought to evaluate these markers in HIV-infected patients with optimal response to stable HAART regimens. After proper approval and written informed consent, we enrolled adult (age ≥ 18 years), otherwise healthy HIV-infected patients receiving the same HAART for at least 6 months and who had HIV viral loads <50 copies/mL and CD4+ T-cell counts >200/mm3 in at least 2 separate measurements over that period of time. Patients were excluded if their platelet counts were <150,000 or >450,000/μL. A control group of healthy adults matched for age and sex was used for comparisons. The protocol for obtaining, processing, and measuring PMP has been described elsewhere.5 In brief, PMP were measured by flow cytometry using phycoerythrin-labeled anti-CD31 and fluorescein-isothiocyanate-labeled anti-CD42b (Pharmingen, San Diego, CA). Because CD31 occurs on platelets and endothelial cells but CD42b occurs only on platelets, PMP were defined as events CD31+/CD42b+. Values are reported as counts per microliter. CD31 also occurs on some leukocyte subsets, but they account for a negligible fraction of microparticles.5 We measured P-selectin expression as described previously.8 We used Mann-Whitney U and t test for comparisons of continuous variables, Fisher Exact test for comparisons of categorical variables, and Spearman rank correlation coefficients for assessment of correlations. All calculations were performed with Intercooled STATA 9.2 (College Station, TX). Twenty-eight HIV-infected patients and 27 healthy controls were enrolled in the study. Both groups had comparable ages (42 ± 6.7 and 41 ± 6.9, respectively; P = 0.59). The groups also demonstrated similar sex and race distributions (P = 0.72 and 0.41, respectively). The mean CD4 count in the HIV-infected group was 550 (±195). Fifteen (57%) of these patients had history of at least one other previous antiretroviral regimen. The mean time receiving the current HAART was 21 (±10.5) months; and the mean time with undetectable HIV viral load was 13 (±9) months. All patients were receiving nucleoside reverse transcriptase inhibitors (NRTIs) as the backbone of their HAART regimens [lamivudine: 19, abacavir (ABC): 13, TDF: 12, FTC: 4, zidovudine (AZT): 11, stavudine: 4, didanosine: 1]. Protease inhibitors (PIs) complemented the therapy in 13 (46%) patients (lopinavir (LPV): 5, nelfinavir (NFV): 2, indinavir (IDV): 2, atazanavir (ATV): 2, fosamprenavir (FPV): 2; all boosted with RTV except for NFV- and one FPV-containing regimens), and nonnucleoside reverse transcriptase inhibitors did so in 15 (54%) of them (efavirenz: 10, nevirapine: 5). PMP levels were significantly higher among HIV-infected subjects than controls [25.28 × 103 counts/μL, interquartile range (IQR): 17.89 × 103-36.18 × 103 versus 13.86 × 103 counts/μL, IQR: 99.84 × 103-20.74 × 103; P = 0.002; Fig. 1]. P-selectin (CD62P) levels, on the other hand, were similar among these groups (7.95 fluorescence intensity units (FIU), IQR: 4.05-15.92 versus 13.32 FIU, IQR: 4.91-23.02; P = 0.17; Fig. 1). We found no differences in the levels of PMP or CD62P when they were compared, within the HIV-infected group, between those receiving PI-based therapy or not; or those receiving ABC or not. Likewise, we found no relation between PMP levels and the duration of exposure to HAART or length of time with undetectable HIV viral load. Higher CD4+ T-cell counts, however, had a trend toward a negative correlation with PMP levels (rho = −0.32, P = 0.1).FIGURE 1: PMP levels (left side) and P-selectin (CD62P) levels (right side) in HIV-infected patients compared with controls.Earlier studies documented heightened levels of PMP and platelet activation in untreated HIV-infected patients.2,9 Platelet activity, but not PMP levels, could be reduced after a short period (3 months) of antiretroviral therapy.2 Our results now show that when compared with healthy controls, HIV-infected patients with good response to longer and stable courses of HAART continue to express higher levels of circulating PMP despite comparable levels of CD62P. In this setting, PMP could be more sensitive markers-than CD62P-of residual increased platelet activity due to unrelenting effects of the infection (eg, low-level viral replication and chronic immune stimulation). The trend toward a negative correlation between PMP levels and CD4+ T-cell counts favors this option. Alternatively, increased levels of PMP may persist from processes independent of platelet activation, such as, for instance, apoptosis. HIV virus infects megakaryocytes10 and cells of the monocyte/macrophage lineage11; with the latter being the main scavengers of apoptotic microvesicles.12 Persistent subclinical HIV viral activity (not detected with the currently available methods) may increase the levels of PMP by promoting apoptosis of thrombocytes or impairing the clearance of their apoptotic products. Another possibility is a direct toxic effect of HAART on platelets. Nucleoside reverse trancriptase inhibitors are known to cause mitochondrial toxicity13 and the potential for this effect to trigger apoptosis has been recognized.14 Under this scenario, a positive link between levels of PMP and longer periods of HAART exposure would be expected but this was not apparent in our analysis. PMP exert important procoagulant functions15 and their higher levels have been linked to the development of acute coronary syndromes (ACS).4,6,16 Hence, regardless of the mechanism involved in their release, PMP could contribute to the increased risk of ACS seen in the HIV-infected patients receiving HAART. PIs and ABC are the drugs more consistently associated with the occurrence of ACS in this population17 but we could not demonstrate any specific link between them and PMP levels. Although small, our groups were homogenous in terms of preexisting clinical characteristics and response to HAART. Our cross-sectional measurements, however, prevents us from drawing finer conclusions on the temporal effect of HAART on the measured variables. This study substantiates the concept of persistent platelet abnormalities in HIV-infected patients with good response to HAART. The causes of this phenomenon and its implications in the cardiovascular pathology of this population will require further investigations. ACKNOWLEDGMENT Supported by the Wallace H. Coulter Foundation. Vicente F. Corrales-Medina, MD* Jacques Simkins, MD* Julio A. Chirinos, MD* Jose A. Serpa, MD† Lawrence L. Horstman, BSc‡ Wenche Jy, PhD‡ Yeon-Soong Ahn, MD‡ *Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL †Department of Medicine, Baylor College of Medicine, Houston, TX ‡Wallace Coulter Platelet Laboratory, Miller School of Medicine, University of Miami, Miami, FL

This is a critical review of anti-phospholipid antibodies (aPL). Most prior reviews focus on the aPL syndrome (APS), a thrombotic condition often marked by neurological disturbance. We bring to attention recent evidence that aPL may be equally relevant to non-thrombotic autoimmune conditions, notably, multiple sclerosis and ITP.After a brief history, the recent proliferation of aPL target antigens is reviewed. The implication is that many more exist. Theories of aPL in thrombosis are then reviewed, concluding that all have merit but that aPL may have more diverse pathological consequences than now recognized. Next, conflicting results are explained by methodological differences. The lupus anticoagulant (LA) is then discussed. LA is the best predictor of thrombosis, but why this is true is not settled. Finally, aPL in non-thrombotic disorders is reviewed.The current paradigm of aPL holds that they are important in thrombosis, but they may have much wider clinical significance, possibly of special interest in neurology.

Desmopressin (DDAVP), an analog of vasopressin (AVP), has wide clinical application as an anti-hemorrhagic (AH) agent. DDAVP in vivo releases vWF from endothelial cells but is reported to have little action on platelets. However, DDAVP is often used to improve hemostasis in platelet dysfunctions. We examined the effect of DDAVP on platelet microparticle (PMP) formation and procoagulant activity in vitro using platelets from normal volunteers and in vivo in six patients receiving DDAVP therapy. In the former, platelets were incubated with DDAVP (0.5 to 25 nM) and PMP released were stained with FITC-labeled MAb alpha-GP IIb/IIIa for flow cytometry. Procoagulant activity was measured in a clot-based assay using Russel's viper venom (RVV) calibrated with cephalin. A mean increase of 2-3 fold was observed in both PMP and procoagulant activity. Parallel to these observations was a dose-dependent rise in organelle-associated Ca2+. The assays were also performed on six patients prior to and at one hour after infusion of DDAVP, and similar but lesser effects were observed. We conclude that DDAVP acts on platelets in vitro, and that these effects may contribute to the hemostatic action of DDAVP in platelet dysfunctions in vivo.

Endothelial cells (ECs) have long been implicated in the pathogenesis and pathophysiology of a myriad of vascular, thrombotic, and inflammatory disorders. The prevalence of endothelial perturbation in vascular disease has underscored the need for noninvasive sensitive and specific markers to monitor endothelial status. The popularity of endothelial microparticles (EMPs) as markers of perturbed has increased because of its promising clinical applications in the area of noninvasive EC monitoring. An overview of the main aspects of endothelial dysfunction as they relate to thrombogenesis and inflammation is important to provide a background of the factors affecting EMP release and their phenotype. General properties of microparticles such as mechanisms of microparticles generation and antigenic characterization of EMP are also discussed in this chapter. EMPs have emerged as a preferred direct method for assessing EC injury in different disorders. EMP analysis could provide insight into the actual status of the endothelium in vivo by a simple blood analysis. The main challenge remains in the selection of specific and sensitive monoclonal antibodies that may yield consistent results among different laboratories. Clinical endothelial research has been hampered by the difficulty of accessing and sampling ECs in a noninvasive manner. EMPs in clinical disease such as cardiovascular disease, diabetes mellitus, hypertension and preeclampsia, inflammatory and infectious disease, autoimmune disorders, hematological disorders, and neurological disease are also discussed in this chapter.

That L-arginine (L-Arg) augments the host response to acute bacterial sepsis suggests that this amino acid intervenes early in the immune response, perhaps via the nitric oxide synthetase (NOS) pathway. The effect of L-Arg supplementation on in vitro phagocytosis of fluorescein-labeled, heat-killed Staphylococcus aureus by peripheral blood neutrophils (PMNs) from 12 normal human volunteers was studied. Separated PMNs were incubated for 2 h with labeled bacteria, with and without supplemental L-Arg, D-arginine, glycine, and/or the NOS inhibitors L-canavanine, aminoguanidine, or L-NG-nitroarginine methyl ester. PMNs were fixed and extracellular fluorescence quenched with crystal violet. By flow cytometry and confocal microscopy, L-Arg supplementation was shown to result in a highly significant increase in PMN bacterial phagocytosis, the maximal effect being seen with L-Arg 380 μM and falling off with higher concentrations. This augmentation was completely abrogated by NOS inhibitors in molar excess, but inhibitors alone did not suppress phagocytosis below that of unsupplemented controls. Neither D-arginine nor glycine affected phagocytosis; the L-Arg effect was stereospecific and not related to utilization of L-Arg as an energy source. L-Arg supplementation significantly enhances bacterial phagocytosis in human neutrophils, perhaps by effects on cytoskeletal phenomena, and this appears to be mediated through NOS activity. Phagocytosis by nonspecific immune cells which intervene early in the response to sepsis is critically important, and beneficial effects of L-Arg on the clinical course of sepsis may be due at least in part to augmentation of phagocyte function. © 1996 Wiley-Liss, Inc.

Objectives The purpose of this study was to determine whether digoxin use is associated with increased flow cytometric markers of endothelial cell and platelet activation in patients with nonvalvular atrial fibrillation (AF). Background Increased intracellular calcium is a key event in platelet activation, and several studies have demonstrated that digitalis activates platelets in vitro. Intracellular calcium also is a key regulator of endothelial cell function, and endogenous digitalis-like substances have been shown to affect biologic processes in endothelial cells. Methods We studied 30 patients with nonvalvular AF. We measured the levels of (1) platelet expression of P-selectin (CD62P), (2) platelet microparticles (PMP); and (3) endothelial microparticles (EMP) identified by anti-CD31 (EMP31) and by anti-E-selectin antibodies (EMP62E). Results Patients who were taking digoxin (n = 16; mean digoxin level = 0.93 ng/dL) did not demonstrate any significant differences in clinical or echocardiographic characteristics compared with patients not taking digoxin (n = 14). Patients taking digoxin had significantly increased levels of CD62P expression in platelets and platelet-leukocyte conjugates and markedly increased markers of endothelial activation: EMP62E and EMP31. After adjusting for potential confounders (including age, congestive heart failure, coronary artery disease, ejection fraction, antiplatelet, β-blocker, and calcium channel blocker use), the differences persisted. Conclusions Digoxin use in patients with AF is associated with increased levels of endothelial and platelet activation. If digitalis activates endothelial cells and platelets at pharmacologic doses, use of digitalis in conditions such as AF could predispose to thrombosis and vascular events. The purpose of this study was to determine whether digoxin use is associated with increased flow cytometric markers of endothelial cell and platelet activation in patients with nonvalvular atrial fibrillation (AF). Increased intracellular calcium is a key event in platelet activation, and several studies have demonstrated that digitalis activates platelets in vitro. Intracellular calcium also is a key regulator of endothelial cell function, and endogenous digitalis-like substances have been shown to affect biologic processes in endothelial cells. We studied 30 patients with nonvalvular AF. We measured the levels of (1) platelet expression of P-selectin (CD62P), (2) platelet microparticles (PMP); and (3) endothelial microparticles (EMP) identified by anti-CD31 (EMP31) and by anti-E-selectin antibodies (EMP62E). Patients who were taking digoxin (n = 16; mean digoxin level = 0.93 ng/dL) did not demonstrate any significant differences in clinical or echocardiographic characteristics compared with patients not taking digoxin (n = 14). Patients taking digoxin had significantly increased levels of CD62P expression in platelets and platelet-leukocyte conjugates and markedly increased markers of endothelial activation: EMP62E and EMP31. After adjusting for potential confounders (including age, congestive heart failure, coronary artery disease, ejection fraction, antiplatelet, β-blocker, and calcium channel blocker use), the differences persisted. Digoxin use in patients with AF is associated with increased levels of endothelial and platelet activation. If digitalis activates endothelial cells and platelets at pharmacologic doses, use of digitalis in conditions such as AF could predispose to thrombosis and vascular events.

A correlation between plasma CD31+ endothelial microparticles (CD31+EMP) levels and clinical, as well as brain MRI activity, in multiple sclerosis (MS) patients has been previously reported. However, the effect(s) of treatment with interferon-β1a (IFN-β1a) on plasma levels of CD31+EMP has not been assessed. In a prospective study, we measured plasma CD31+EMP levels in 30 patients with relapsing-remitting MS. Using flow cytometry, in a blinded study, we measured plasma CD31+EMP in 30 consecutive patients with relapsing-remitting MS (RRMS) prior to and 4, 12, 24 and 52 weeks after initiation of intramuscular therapy with interferon-β1a (IFN-β1a), 30 micrograms weekly. At each visit, clinical examination was performed and expanded disability status scale (EDSS) scores were assessed. Plasma levels of CD31+EMP were significantly reduced from 24 through 52 weeks following initiation of treatment with IFN-β1a. Our data suggest that serial measurement of plasma CD31+EMP levels may be used as a surrogate marker of response to therapy with INF-β1a. In addition, the decline in plasma levels of CD31+EMP further supports the concept that IFN-β1a exerts stabilizing effect on the cerebral endothelial cells in pathogenesis of MS.

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration ([Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 μM, 2 μM and 50 μM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 μM). It is only 15–20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 μM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.

The presence of antiphospholipid antibodies (APLA) in multiple sclerosis (MS) patients has been reported frequently but no clear relationship between APLA and the clinical and neuroimaging features of MS have heretofore been shown. We assessed the clinical and neuroimaging features of MS patients with plasma APLA.A consecutive cohort of 24 subjects with relapsing-remitting (RR) MS were studied of whom 7 were in remission (Rem) and 17 in exacerbation (Exc). All subjects were examined and underwent MRI of brain. Patients' plasma was tested by standard ELISA for the presence of both IgM and IgG antibodies using a panel of 6 targets: cardiolipin (CL), beta2 glycoprotein I (beta2GPI), Factor VII/VIIa (FVIIa), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE).In exacerbation up to 80% of MS subjects had elevated titers of IgM antibodies directed against the above antigens. However, in remission, less than half of MS patients had elevated titers of IgM antibodies against one or more of the above antigens. This difference was significant, p < 0.01, for all 6 target antigens. Interestingly, none of the MS patients had elevated plasma titers of IgG against any of the target antigens tested. Correlation analysis between MRI enhancing lesions and plasma levels of APLA revealed high correlation for aPC, aPS and aFVIIa (p </= 0.0065), a trend for aPE and aCL (p = 0.056), and no correlation for abeta2GP1. The strongest correlation was for aFVIIa, p = 0.0002.The findings of this preliminary study show that increased APLA IgM is associated with exacerbations of MS. Currently, the significance of this association in pathogenesis of MS remains unknown. However, systematic longitudinal studies to measure APLA in larger cohorts of patients with relapsing-remitting MS, particularly before and after treatment with immunomodulatory agents, are needed to confirm these preliminary findings.

Heparin induced thrombocytopenia (HIT) is a well-known complication of heparin administration but usually resolves upon discontinuation without sequelae. However, a small proportion of HIT patients develop thrombosis associated with HIT, designated as HITT, which is often life-threatening and may lead to gangrene and amputations. Existing laboratory methods of confirming HIT/HITT do not distinguish between HIT and HITT. We report a flow cytometric assay of platelet activation marker CD62P to distinguish the effects of addition of HIT vs. HITT plasma to normal blood. Briefly, normal whole blood was incubated with platelet-poor plasma from 12 patients with HITT, 30 with HIT, and 65 controls, in presence and absence of heparin, and expression of CD62P was assayed by flow cytometry. When the ratios of fluorescent intensity of CD62P with heparin divided by that without heparin were compared, HITT plasma induced significantly higher ratios than HIT plasma (HITT ratios approximately 2.5 vs. HIT ratios approximately 1.2; p <0.001). Eleven of 12 HITT patients were positive by this test but only 5 of 30 HIT patients were positive (p <0.0005). In a case of HIT with silent thrombosis, this assay gave a positive results prior to clinically evident thrombosis. In conclusion, this method distinguishes HITT from HIT and may be clinically useful in the detection of HITT, allowing early intervention for preventing catastrophic thrombosis.

The present paper describes a flow cytometric method for assay of platelet aggregates (PAg) in blood. This method combines and simplifies previously reported techniques, simultaneously enumerating PAg formed upon platelet activation, their expression of activation marker CD62P (P-selectin), and their content of bound leukocytes (LPAg). The sensitivity of this method to low levels of agonists (ADP, collagen) is compared to conventional aggregometry and some features of platelet-leukocyte interaction are explored. The results were: (1) ADP or collagen induced a dose-dependent increase in PAg number and corresponding decline in free platelets. The ED50 for ADP (0.15 microM) and for collagen (0.2 microg/mL) was about 1/20 the ED50 found by aggregometry, indicating 20-fold greater sensitivity. (2) At higher concentrations, the fraction of PAg with bound leukocytes (LPAg) increased to 60-70%. This rise correlated with PAg size and CD62P expression, but not with the number of PAg formed. (3) The response of whole blood (WBD) to agonists was qualitatively different from that of platelet-rich plasma (PRP): in WBD the population of CD62P+ PAg was much higher than in PRP and the population of CD62P+ free platelets was much lower. This implies that leukocytes rapidly recruit activated platelets. (4) Desmopressin (DDAVP) at 5 nmol/L induced a significant rise in activated (CD62P+) PAg and platelets, even though no effect of DDAVP could be detected by conventional aggregometry; this further confirms that DDAVP acts directly on platelets. (5) Plasma samples from TTP patients induced a rise in PAg when added to normal PRP, though little or no effect could be detected by aggregometry. In summary, the flow cytometric method described here appears useful for detecting low levels of platelet activation and provides information on platelet leukocyte interaction, potentially important in identifying and differentiating thrombogenic states. Since it is rapid and economical, it is well suited for clinical implementation.

We investigated antibodies to factor VII/VIIa (FVII/VIIa) and five other common target antigens in 33 patients with a history of anti-phospholipid syndrome (APS) and 50 healthy controls using an enzyme-linked immunosorbent assay (ELISA) technique. We found that antibody to FVII/VIIa, a previously unrecognized and common antigen in APS, was present in 67% of patients. Frequencies of antibodies to other target antigens were: anti-beta-2 glycoprotein 1 (anti-beta 2GP1), 88%; anti-cardiolipin (anti-CL), 76%; anti-phosphatidylethanolamine (anti-PE), 67%; anti-phosphatidylserine (anti-PS), 64%; and anti-phosphatidylcholine (anti-PC), 59%. Most patients had antibodies against multiple antigens, but a few were positive for only anti-beta 2GP1 (12%) or anti-CL (3%). Positivity for anti-FVII/VIIa was significantly associated with positivity for anti-PE, anti-PS and/or anti-PC (P < 0.05) but not anti-beta 2GP1. When frequencies of immunoglobulin G (IgG) versus immunoglobulin M (IgM) antibodies were compared, anti-beta 2GP1 IgG correlated with the lupus anticoagulant (P < 0.05) and was significantly more prevalent than IgM, but the reverse was seen for all other antigens. In arterial thrombosis, IgM was more prevalent for all antigens, and was significantly associated with FVII/VIIa, PE and PS, whereas in venous thrombosis, IgG was frequently prevalent, especially in association with FVII/VIIa, beta 2GP1 and CL. In summary, FVII/VIIa is a new and common antigen in APS. Anti-FVII/VIIa is often associated with anti-PE, anti-PS and anti-PC. The IgM class is more frequently associated with arterial thrombosis and the IgG class with venous thrombosis.

Inflammation has been associated with increased cardiovascular risk, and endothelial cell (EC) apoptosis has been implicated in atherogenesis. The correlation between circulating concentrations of interleukin-6 (IL-6), C-reactive protein (CRP), and endothelial microparticles (EMPs) expressing an apoptotic (EMP31) or activation (EMP62E) phenotype in 20 middle-aged healthy men was investigated. IL-6 was significantly correlated with EMP31 (r = 0.6, p = 0.004), which persisted after adjusting for body mass index and CRP. CRP was significantly correlated with body mass index (r = 0.49, p = 0.02) but not with EMP31 or EMP62E. EC apoptosis is associated with IL-6 levels in men and might be partially responsible for the increased cardiovascular risk associated with subclinical inflammation. Inflammation has been associated with increased cardiovascular risk, and endothelial cell (EC) apoptosis has been implicated in atherogenesis. The correlation between circulating concentrations of interleukin-6 (IL-6), C-reactive protein (CRP), and endothelial microparticles (EMPs) expressing an apoptotic (EMP31) or activation (EMP62E) phenotype in 20 middle-aged healthy men was investigated. IL-6 was significantly correlated with EMP31 (r = 0.6, p = 0.004), which persisted after adjusting for body mass index and CRP. CRP was significantly correlated with body mass index (r = 0.49, p = 0.02) but not with EMP31 or EMP62E. EC apoptosis is associated with IL-6 levels in men and might be partially responsible for the increased cardiovascular risk associated with subclinical inflammation.

Rituximab, a chimeric monoclonal CD20 antibody, is useful in the treatment of B-cell lymphomas and certain autoimmune diseases. We report a successful outcome of rituximab for life threatening hypercoagulable state associated with lupus anticoagulant (LA). A 30-year-old woman initially presented 10 years ago with DVT and positive serology for SLE and LA. While on Coumadin, she suffered from recurrent DVT in the legs and arms, pulmonary emboli, Budd-Chiari syndrome, mesenteric vein thrombosis, bone infarcts, recurrent strokes, and chronic ITP. All measures including plasmapheresis and monthly IV cyclophosphamide were of no benefit. She was recently admitted with spontaneous subdural hematoma with INR of 3.8. Upon discontinuation of anticoagulation for surgical drainage, she developed acute abdomen from thrombosis and recurrent DVT. Because she had failed prior standard measures, 4 weekly infusions of rituximab (375 mg/m2) were given following 2 rounds of plasmapheresis. Subsequently, she made a remarkable recovery over the next month and has been free of thrombosis on Coumadin for over 15 months. LA, IgM antibodies to cardiolipin, and B2GP1 were consistently positive. After rituximab therapy, LA became negative and IgM antibodies to cardiolipin decreased and ITP went into remission. Rituximab induced a lasting remission in a woman suffering from life-threatening hypercoagulable state associated with LA. Her clinical remission was associated with disappearance of LA.

It has been suggested that Helicobacter pylori eradication often increases platelet counts in patients with chronic idiopathic thrombocytopenic purpura (ITP). In addition, H. pylori has been shown to induce platelet activation (CD62p or P-selectin expression) in previous studies. We assessed the response of platelet count and CD62p expression after eradication therapy in patients with ITP and H. pylori infection.We prospectively studied 15 ITP patients diagnosed with H. pylori infection by serology and breath test. A follow-up breath test was used to document eradication. Two out of 15 patients showed improvement in platelet counts after 6 months, 1 of which may have had drug-induced thrombocytopenia. Overall, certain platelet response rate in our series was 6.7% (1/15). We found that platelet CD62p expression by flow cytometry was elevated in 10/15 (66.7%) H. pylori-infected patients, which is a statistically significant difference when compared with 3/33 (9.1%) control ITP patients seronegative for H. pylori (p = 0.002). In addition, eradication therapy decreased CD62p expression (p = 0.04). However, reduction in platelet activation was not associated with an increase in platelet counts (mean 72.4 x 10(9)/l before and 68.7 after therapy; p = 0.4).In our series, platelet activation was common in ITP patients with H. pylori, and eradication therapy decreased platelet activation but seldom increased platelet counts. Increased platelet CD62p expression is a putative link between chronic infections and atherosclerosis, but further study is needed to clarify the implications of our observation.

Although the presence of antiphospholipid antibodies (APLA) in immune thrombocytopenic purpura (ITP) has been reported, their clinical significance is not clear. The present study investigated APLA profiles in relation to the clinical stages of ITP. We studied APLA in 40 patients in three stages of ITP: exacerbation/relapse (n=7), stable (n=14) and remission (n=19). Both IgG and IgM APLA to six target antigens were measured by enzyme-linked immunosorbent assay: beta2-glycoprotein 1 (beta2GP1), cardiolipin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and factor VII/VIIa. The central finding was that APLA were common in ITP but differed significantly in disease stages, being highest in exacerbation (86% positive), intermediate in stable disease (57%) and lowest in remission (42%). In exacerbations, APLA were predominantly of IgG class, while in stable disease, IgM predominated. During remission, APLA often became undetectable. Both the frequency and titres of APLA were significantly higher during exacerbation than remission. An inverse correlation was found between platelet count and nearly all APLA (except beta2GP1). Sequential study of six patients revealed that APLA tended to emerge and rise with exacerbation, concurrently with new episodes of bleeding and became undetectable during remission. These findings raise the possibility that APLA may play a role in the exacerbation and remission of ITP or they may be a consequence of platelet destruction.

The calcium-sensitive fluorescent probe chlorotetracycline was used to monitor calcium movement in human platelets. The chlorotetracycline fluorescence signal is a linear measure of the level of free calcium in the dense tubules and in the mitochondria, with probe sensitivity in the millimolar range. Experiments perturbing the system with the calcium ionophore A23187 shows that the level of free internal calcium in the organelle depends upon the cytoplasmic level, which, in turn, depends upon the passive permeability of the plasma membrane. Chlorotetracycline in the cytoplasmic compartment does not respond to changes in the cytoplasmic calcium concentration, which is held in the micromolar to submicromolar range by an extrusion system. The calcium concentration in the cytoplasmic compartment can be directly manipulated by the calcium ionophore A23187 and is measured in parallel experiments with Quin 2, a high-affinity indicator. The calcium transport systems of the organelles are shown to be less susceptible to short circuit by A23187. Analysis shows that mitochondrial uptake is slow (t 1/2 = 20 minutes), produces a large increase in chlorotetracycline fluorescence, and is inhibited by sodium azide plus oligomycin. Uptake by the dense tubules is more rapid (t 1/2 = 2 minutes), produces a smaller increase in chlorotetracycline fluorescence, is inhibited by trifluoperazine, and is less sensitive to A23187. The Km is estimated as 1 microM or lower. Studies show that the chlorotetracycline technique is useful for the monitoring of calcium uptake and release by the platelet organelles, and suggests that the Quin 2/chlorotetracycline technique will be useful as a diagnostic of both physiological and pathological activation mechanisms.

The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases.An extensive PubMed search of literature pertaining to this study was performed in April 2009 using specific keyword search terms related to cell-derived microparticles and ischemic stroke. Some references are not cited here as it is not possible to be all inclusive or due to space limitation.Cell-derived microparticles are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. This review outlines evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. More specifically, cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions.If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

The presence of anti-CD36 antibodies in plasma of patients with thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), and heparin-induced thrombocytopenia without/with thrombosis (HIT/HITT) has been examined by immunoblots, and a monoclonal antibody capture assay, the platelet-associated IgG characterization assay (PAICA). Results with PAICA showed that 73% (8/11) of patients with TTP were positive, and 71% (10/14) by immunoblots. With ITP, 20% (6/30) were positive by PAICA and 19% (3/16) by immunoblots; HIT, 30% (3/10) were positive by PAICA and 60% (6/10) by immunoblot; HITT, 50% (2/4) by PAICA and 100% (4/4) by immunoblot. Purification of CD36 by fast protein liquid chromatography (FPLC) from Triton X-100 extracts of normal platelet membranes resulted in the isolation of two different forms: the classic 88 kD form, and a second, lighter 85 kD form. Our data indicated that the patients' plasma autoantibodies reacted strongly with the 85 kD form. Conventional monoclonal and polyclonal antisera produced to the 88 kD form reacted strongly with the 88 kD form but weakly with the 85 kD form. These results confirm the possible importance of anti-CD36 antibodies in the pathophysiology of TTP and other thrombocytopenias and demonstrate the presence of a previously unrecognized target antigen for these antibodies.

American Journal of HematologyVolume 81, Issue 6 p. 391-396 Original ArticleFree Access Antiphospholipid antibodies (APLA) in immune thrombocytopenic purpura (ITP) and antiphospholipid syndrome (APS) Carlos J. Bidot, Carlos J. Bidot Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorWenche Jy, Wenche Jy Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorLawrence L. Horstman, Lawrence L. Horstman Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorEugene R. Ahn, Eugene R. Ahn Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorMiriam Yaniz, Miriam Yaniz Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorYeon S. Ahn, Corresponding Author Yeon S. Ahn [email protected] Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FL 33136Search for more papers by this author Carlos J. Bidot, Carlos J. Bidot Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorWenche Jy, Wenche Jy Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorLawrence L. Horstman, Lawrence L. Horstman Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorEugene R. Ahn, Eugene R. Ahn Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorMiriam Yaniz, Miriam Yaniz Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaSearch for more papers by this authorYeon S. Ahn, Corresponding Author Yeon S. Ahn [email protected] Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FloridaWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, Miami, FL 33136Search for more papers by this author First published: 05 May 2006 https://doi.org/10.1002/ajh.20571Citations: 28AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Antiphospholipid antibodies (APLA) are associated with anti-phospholipid syndrome (APS), a thrombotic disorder, but they are also frequently detected in immune thrombocytopenic purpura (ITP), a bleeding disorder. To investigate possible differences of APLA between these two disorders, we assayed IgG and IgM APLA by ELISA in 21 patients with ITP and 33 with APS. The APLA reacting against two protein target antigens, β2-glycoprotein 1 (β2GP1) and FVII/VIIa, and four phospholipids [cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)] as well as lupus anticoagulant (LA) were analyzed. We made the following observations: (i) IgG and IgM antibodies to β2GP1 and IgM antibodies to FVII/VIIa were more common in APS than ITP, P < 0.05, while IgG antibodies against the phospholipids (aCL, aPC, aPS, aPE) were more common in ITP than APS, P < 0.05; (ii) multiple APLA ≥3 antigens) were more frequent in APS than ITP, P < 0.05; (iii) LA was frequently associated with APS but was absent in ITP; (iv) APLA is quite common in ITP: two-thirds were positive for at least one APLA. In summary, APLA are prevalent in ITP but their profile differs from APS. In APS, antibodies were predominantly against β2GP1 and 80% had positive LA, while in ITP the APLA reacted most often with the phospholipids without LA. The difference in APLA may result in opposite clinical manifestations in two disorders. Am. J. Hematol. 81:391–396, 2006. © 2006 Wiley-Liss, Inc. REFERENCES 1 Wilson WA, Gharavi AE, Koike T, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum 1999; 42: 1309– 1311. 2 Levine JS, Branch DW, Rauch J. The antiphospholipid syndrome. N Engl J Med 2002; 346: 752– 763. 3 Chi HS. Recent advances in the diagnosis of antiphospholipid syndrome. Int J Hematol 2002; 76( Suppl II): 47– 51. 4 Thrombosis and thrombocytopenia in antiphospholipid syndrome (idiopathic and secondary to SLE): first report from the Italian Registry. Haematologica 1993; 78(5): 313– 318. 5 Galli M, Finazzi G, Barbui T. Thrombocytopenia in the antiphospholipid syndrome. Br J Haematol 1996; 93: 1– 5. 6 Ahn YS, Horstman LL. Idiopathic thrombocytopenic purpura: pathophysiology and management (review). Int J Hematol 2002; 76: 123– 131. 7 McMillan RM, Imbach P. Immune thrombocytopenic purpura. In: J Loscalzo, AI Schafer, editors. Thrombosis and hemorrhage. 2nd edition. Baltimore: Williams & Wilkins; 1998. p 643– 664. 8 Harris EN, Charavi AE, Hedge U, et al. Anticardiolipin antibodies in autoimmune thrombocytopenic purpura. Br J Haematol 1985; 59: 231– 234. 9 Bidot CJ, Jy W, Horstman LL, et al. Antiphospholipid antibodies in immune thrombocytopenic purpura tend to emerge in exacerbation and decline in remission. Br J Haematol 2005; 128: 366– 372. 10 Bidot CJ, Jy W, Horstman LL, et al. Factor VII/VIIa: a new antigen in the antiphospholipid antibody syndrome. Br J Haematol 2003; 120: 618– 626. 11 Tincani A, Allegri F, Sanmarco M, et al. Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations. A Cooperative Project of the European Antiphospholipid Forum. Thromb Haemost 2001; 86: 575– 583. 12 Arnout W, Meijer P, Vermylen J. Lupus anticoagulant testing in Europe: an analysis of results from the First European Concerted Action on Thrombophilia (ECAT) Survey using plasmas spiked with monoclonal antibodies against human β2-glycoprotein 1. Thromb Haemost 1999; 81: 929– 934. 13 Reber G, Schousboe I, Tincani A, et al. Inter-laboratory variability of anti-β2-glycoprotein I measurement: collaborative study in the frame of the European Forum on Antiphospholipid Antibodies Standardization Group. Thromb Haemost 2002; 88: 66– 73. 14 Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update (on behalf of the SSC of ISTH). Thromb Haemost 1995; 74: 1185– 1190. 15 Ahn YS, Horstman LL, Jy W, Jimenez JJ, Bowen B. Vascular dementia in patients with immune thrombocytopenic purpura (ITP). Thromb Res 2002; 107: 337– 344. 16 Hughes GRV, Harris EN, Gharavi AE. The anticardiolipin syndrome. J Rheumatol 1985; 13: 486– 489. 17 Greaves M. Antiphospholipid antibodies and thrombosis. Lancet 1999; 353: 1348– 1353; see also four letters in response: Greaves M. Antiphospholipid antibodies and thrombosis. Lancet 1999; 354: 71. 18 Arnout J, Vermylen J. Current status and implications of autoimmune antiphospholipid antibodies in relation to thrombotic disease (with Editorial, 878–880). J Thromb Haemost 2003; 1: 931– 942. 19 Arnout J, Wittevrongel C, Vanrusselt M, Hoylaerts M, Vermylen J. Beta-2-glycoprotein 1 dependent lupus anticoagulants form stable bivalent antibody beta-2-glycoprotein 1 complexes on phospholipid surfaces. Thromb Haemost 1998; 79: 79– 86. 20 Ichikawa K, Khamashta MA, Koike T, Matsuura E, Hughes GRV. β2-Glycoprotein 1 reactivity of monoclonal antibodies from patients with the antiphospholipid syndrome. Arthritis Rheum 1994; 37: 1453– 1461. 21 Day HM, Thiagarajan P, Ahn C, Reveille JD, Tinker KF, Arnett FC. Autoantibodies to β2-glycoprotein 1 in systemic lupus erythematosus and primary antiphospholipid antibody syndrome: clinical correlations in comparison with other antiphospholipid antibody tests. J Rheumatol 1999; 25: 667– 674. 22 Arfors L, Winiarski J, Lefvert AK. Prevalence of antibodies to cardiolipin in chronic ITP and reactivity with platelet membranes. Eur J Haematol 1996; 56: 230– 234. 23 Bevers EM, Smeets EF, Confurius P, Zwaal RFA. Physiology of membrane lipid asymmetry. Lupus 1994; 3: 235– 240. 24 Horstman LL, Ahn YS. Platelet microparticles: a wide-angle perspective (review). Crit Rev Oncol/Hematol 1999; 30: 111– 142. 25 Shan H, Goldman J, Cunto G, et al. Heterogeneity of anti-phospholipid and anti-endothelial cell antibodies. J Autoimmun 1998; 11: 651– 660. 26 Gilman-Sachs A, Lubinski J, Beer AE, Brend S, Beaman KD. Patterns of anti-phospholipid antibody specificities. J Clin Lab Immunol 1991; 35: 83– 88. 27 Papa ND, Rascho E, Moroni G, et al. Anti-endothelial cell IgG fractions from systemic lupus erythematosus patients bind to human endothelial cells and induce a proadhesive and proinflammatory phenotype in vivo. Lupus 1999; 8: 423– 429. 28 Williams FMK, Parmar K, Hughes GRV, Hunt BJ. Systemic endothelial cell markers in primary antiphospholipid syndrome. Thromb Haemost 2000; 84: 742– 746. 29 Simantov R, LaSala J, Lo S, Gharavi A, Sammaritano L, Salmon J. Activation of cultured vascular endothelial cells by antiphospholipid antibodies. J Clin Invest 1995; 96: 2211– 2219. 30 Hill MB, Phipps JL, Hughes P, Greaves M. Anti-endothelial cell antibodies in primary antiphospholipid syndrome and SLE: patterns of reactivity with membrane antigens on microvascular and umbilical venous cell membranes. Br J Haematol 1998; 103: 416– 421. 31 Lipp E, von Felten A, Sax H, Muller D, Berchtold P. Antibodies against platelet glycoproteins and antiphospholipid antibodies in autoimmune thrombocytopenia. Eur J Haematol 1998; 60: 283– 288. 32 George JN, Woolf SH, Raskob GE, et al. Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology. Blood 1996; 92: 1556– 1564. 33 Stasi R, Stipa E, Masi M, et al. Long-term observation of 208 adults with chronic idiopathic thrombocytopenic purpura. Am J Med 1995; 98: 436– 442. 34 Stasi R, Stipa E, Masi M, et al. Prevalence and clinical significance of elevated antiphospholipid antibodies in patients with idiopathic thrombocytopenic purpura. Blood 1994; 84: 4203– 4207. 35 Diz-Kucukkaya R, Hachihanefioglu A, Yenerel M, et al. Antiphospholipid antibodies and antiphospholipid syndrome in patients presenting with immune thrombocytopenic purpura: a prospective cohort study. Blood 2001; 98: 1760– 1764. 36 Forastiero R, Martinuzzo M, Carreras LO, Maclouf J. Anti-β2 glycoprotein 1 antibodies and platelet activation in patients with antiphospholipid antibodies: association with increased excretion of platelet-derived thromboxane urinary metabolites. Thromb Haemost 1998; 79: 42– 45. 37 Galli M, Bevers EM, Comfurius P, Barbui T, Zwaal RFA. Effects of antiphospholipid antibodies on procoagulant activity of activated platelets and platelet-derived microvesicles. Br J Haematol 1993; 83: 466– 472. 38 Nomura S, Komiyama Y, Matsuura E, Kokawa T, Takahashi H, Koike T. Binding of beta 2-glycoprotein 1 to platelet-derived microparticles (Letter, with critique). Br J Haematol 1993; 85: 639– 640. 39 Nomura S, Ohtani T, Kagawa H, et al. Relationship between cytokines and platelet-derived microparticles. Thromb Haemost 1999;( Suppl): 363 (abstract 1152). 40 Godeau B, Piette JC, Fromont P, Intrator L, Schaeffer A, Bierling A. Specific anti-platelet glycoprotein autoantibodies are associated with the thrombocytopenia of primary antiphospholipid syndrome. Br J Haematol 1997; 98: 873– 879. 41 Macchi L, Rispal P, Clofent-Sanchez G, et al. Anti-platelet antibodies in patients with systemic lupus erythematosus and the primary antiphospholipid antibody syndrome: their relationship with the observed thrombocytopenia. Br J Haematol 1997; 98: 336– 341. 42 Angles-Cano E, Guillin MC. Antiphospholipid antibodies and the coagulation cascade. Rheum Dis Clin 2001; 27: 573– 586. 43 Jy W, Horstman LL, Arce M, Ahn YS. Clinical significance of platelet microparticles in autoimmune thrombocytopenias. J Lab Clin Med 1992; 119: 334– 345. Citing Literature Volume81, Issue6June 2006Pages 391-396 ReferencesRelatedInformation

The leukocyte CD44 and CD45 cell surface receptors are associated via the linker proteins ankyrin and fodrin with the cytoskeleton, which itself is important in immune cell functions such as adherence, chemotaxis, and phagocytosis. The effects of rat antihuman CD44 and CD45 monoclonal antibodies on phagocytosis of fluoresceinated heat-killed Staphylococcus aureus 502A by normal human neutrophils (PMNs) during 2 hr incubation in RPMI-1640 was studied via flow cytometry and confocal microscopy. Flow cytometry was performed using an excitation wavelength of 488 nm, fluorescence being measured at 515–560 nm on 50,000 PMNs per sample. Confocal microscopy was performed on samples after further incubation with rhodamine-conjugated antiankyrin. Anti-CD44 resulted in an increase of 27–31% compared to control (P = 0.004) in the proportion of PMNs fluorescing, an increase of 17–24% (P = 0.001) in mean intracellular fluorescence per PMN, and an increase in total PMN fluorescence of 50–58% compared to control (P < 0.001). In contrast, anti-CD45 had little effect on phagocytosis. Colchicine (a microtubule-disrupting agent) enhanced, whereas cytochalasin-D (a microfilament inhibitor) inhibited bacterial phagocytosis; cytochalasin-D completely abrogated the effect of anti-CD44 on this PMN function. Hyaluronic acid augmented phagocytosis by an increment similar to that observed with anti-CD44. Two-color flow cytometry and confocal microscopy demonstrated that ankyrin always colocalized with ingested fluorescein isothiocyanate (FITC)-labeled bacteria. These data strongly suggest that CD44 is involved in bacterial phagocytosis, provide further evidence of CD44 receptor linkage to cytoskeletal elements in human leukocytes, and suggest that ankyrin has a significant role in the transport of phagosomes. © 1996 Wiley-Liss, Inc.

Abstract In this study both a Ligand‐dependent treatment [concanavalin A (Con A)] and a ligand‐independent treatment [high‐voltage pulsed galvanic stimulation (HVPGS)] have been used to initiate lymphocyte activation via a transmembrane signaling process. Our results show that both treatments cause the exposure of two different hormone [insulin and interIeukin‐2 (IL‐2)] receptors within the first 5 min of stimulation. When either insulin or IL‐2 is present in the culture medium, the stimulated lymphocytes undergo the following responses: (1) increased free intra‐cellular Ca 2+ activity; (2) aggregation of insulin or IL‐2 receptors into patch/cap structures; (3) tyrosine‐kinase‐specific phosphorylation of a 32‐kd membrane protein; and finally (4) induction of DNA synthesis. Further analysis indicates that hormone receptor capping is inhibited by (1) cytochalasin D, suggesting the involvement of microfilaments; (2) sodium azide, indicating a requirement for ATP production; and (3) W‐5, W‐7, and W‐12 drugs, implying a need for Ca 2 + /calmodulin activity. Treatment with these metabolic or cytoskeletal inhibitors also prevents both the tyrosine‐kinase‐specific protein phosphorylation and DNA synthesis which normally follow hormone receptor capping. Double immunofluorescence staining shows that actomyosin, Ca 2+ /calmodulin, and myosin light‐chain kinase are all closely associated with the insulin and IL‐2 receptor cap structures. These findings strongly suggest that an actomyosin‐mediated contractile system (regulated by Ca 2+ , calmodulin, and myosin light‐chain kinase in an energy‐dependent manner) is required not only for the collection of insulin and IL‐2 receptors into patch and cap structures but also for the subsequent activation of tyrosine kinase and the initiation of DNA synthesis. We, therefore, propose that the exposure and subsequent patching/capping of at least one hormone receptor are required for the activation of mouse splenic T‐Iymphocytes.

Using chlorotetracycline (CTC) as a probe we studied calcium homeostasis of platelets in various disorders. Studied were healthy subjects and patients with disorders where platelets play an important role. These included thromboses, hypertension, diabetes mellitus, vasculitis, immune thrombocytopenia, thrombotic thrombocytopenic purpura, myelofibrosis, hemolytic anemias and uremia. Significant elevation of calcium levels were observed in all of these disorders except uremia. Nifedipine reduced or normalized the increased levels in most patients and its discontinuation resulted in a return of the abnormality. We propose that platelets in thromboses and related disorders are exposed to subcritical concentrations of activating factors, leading to enhanced calcium influx and elevated free cytoplasmic calcium followed by elevated resting dense tubular calcium. Nifedipine appears to protect platelets from these stimuli and coupled with their known action on vessel walls, calcium channel blockers show promise as antiatherogenic as well as antithrombotic agents.

Essentials•Mutations in the RASGRP2 gene represent a new inherited platelet function disorder.•Report a five generation family with a novel frameshift mutation in RASGRP2 (p.F497Sfs*22).•Partial platelet activation defect and serious bleeding complications in homozygous patients.•Patients respond to recombinant Factor VIIa infusion but not platelet transfusions.BackgroundGenetic variants in the RASGRP2 gene encoding calcium and diacylglycerol‐regulated guanine nucleotide exchange factor I (CalDAG‐GEFI) represent a new inherited bleeding disorder linked to major defects of platelet aggregation and activation of αIIbβ3 integrin. They are of major interest as CalDAG‐GEFI is receiving attention as a potential target for antiplatelet therapy for prevention and treatment of cardiovascular disorders including arterial thrombosis and atherosclerosis.ObjectivesTo better understand the phenotypical and clinical profiles of patients with CalDAG‐GEFI deficiency.PatientsWe report a five‐generation family with a novel truncating CalDAG‐GEFI mutation detailing clinical management and phenotypic variability.ResultsPatients IV.6 & IV.4 manifested with episodes of serious mucocutanous bleeding or bleeding after surgery not responding to platelet transfusion but responding well to recombinant Factor VIIa infusions. Their blood counts and coagulation parameters were normal but platelet aggregation to ADP and collagen was defective. Further work‐up confirmed normal levels of αIIb and β3 in their platelets but decreased αIIbβ3 function. DNA analysis by whole exome sequencing within the BRIDGE‐BPD consortium (Cambridge, UK), allowed us to highlight a homozygous c.1490delT predicted to give rise to a p.F497Sfs*22 truncating mutation near to the C‐terminal domain of CalDAG‐GEFI. Sanger sequencing confirmed that both patients were homozygous for the c.1490delT and 3 out of 4 close family members were heterozygous.ConclusionsA long‐term prospective study is warranted for full clinical exploration of CalDAG‐GEFI to understand the bleeding phenotyes and their management.

Spontaneous intracerebral hemorrhage (sICH) is the deadliest stroke subtype with no effective therapies. Limiting hematoma expansion is a promising therapeutic approach. Red blood cell-derived microparticles (RMPs) are novel hemostatic agents. Therefore, we studied the potential of RMPs in limiting hematoma growth and improving outcomes post-sICH.sICH was induced in rats by intrastriatal injection of collagenase. RMPs were prepared from human RBCs by high-pressure extrusion. Behavioral and hematoma/lesion volume assessment were done post-sICH. The optimal dose, dosing regimen, and therapeutic time window of RMP therapy required to limit hematoma growth post-sICH were determined. We also evaluated the effect of RMPs on long-term behavioral and histopathologic outcomes post-sICH.RMP treatment limited hematoma growth following sICH. Hematoma volume (mm3) for vehicle- and RMP- (2.66×1010 particles/kg) treated group was 143±8 and 86±4, respectively. The optimal RMP dosing regimen that limits hematoma expansion was identified. RMPs limit hematoma volume when administered up to 4.5-hour post-sICH. Hematoma volume in the 4.5-hour post-sICH RMP treatment group was lower by 24% when compared with the control group. RMP treatment also improved long-term histopathologic and behavioral outcomes post-sICH.Our results demonstrate that RMP therapy limits hematoma growth and improves outcomes post-sICH in a rodent model. Therefore, RMPs have the potential to limit hematoma growth in sICH patients.

Case Reports| July 11 2003 Life-Threatening Bleeding from Refractory Acquired FVIII Inhibitor Successfully Treated with Rituximab Subject Area: Hematology , Oncology Wenche Jy; Wenche Jy aWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, Search for other works by this author on: This Site PubMed Google Scholar Teresa Gagliano-DeCesare; Teresa Gagliano-DeCesare cDepartment of Internal Medicine, University of Miami School of Medicine, Miami, Fla., USA Search for other works by this author on: This Site PubMed Google Scholar Daniel H. Kett; Daniel H. Kett bDivision of Critical Care, Search for other works by this author on: This Site PubMed Google Scholar Lawrence L. Horstman; Lawrence L. Horstman aWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, Search for other works by this author on: This Site PubMed Google Scholar Joaquin J. Jimenez; Joaquin J. Jimenez aWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, Search for other works by this author on: This Site PubMed Google Scholar Zoneddy Ruiz-Dayao; Zoneddy Ruiz-Dayao aWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, Search for other works by this author on: This Site PubMed Google Scholar Edgardop S. Santos; Edgardop S. Santos aWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, Search for other works by this author on: This Site PubMed Google Scholar Yeon S. Ahn Yeon S. Ahn aWallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology, Search for other works by this author on: This Site PubMed Google Scholar Acta Haematol (2003) 109 (4): 206–208. https://doi.org/10.1159/000070973 Article history Received: February 05 2003 Accepted: March 12 2003 Published Online: July 11 2003 Content Tools Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Wenche Jy, Teresa Gagliano-DeCesare, Daniel H. Kett, Lawrence L. Horstman, Joaquin J. Jimenez, Zoneddy Ruiz-Dayao, Edgardop S. Santos, Yeon S. Ahn; Life-Threatening Bleeding from Refractory Acquired FVIII Inhibitor Successfully Treated with Rituximab. Acta Haematol 1 June 2003; 109 (4): 206–208. https://doi.org/10.1159/000070973 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsActa Haematologica Search Advanced Search Article PDF first page preview Close Modal This content is only available via PDF. 2003Copyright / Drug Dosage / DisclaimerCopyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. You do not currently have access to this content.

We read with great interest the review article, ‘Cellular microparticles: what are they bad or good for?’, by Freyssinet in the Journal [1Freyssinet J.M. Cellular microparticles: what are they bad or good for?.J Thromb Haemost. 2003; 1: 1655-62Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar]. It opens with a succinct review of the hypothesis that a necessary prelude to vesiculation and shedding of microparticles (MP) is migration of phosphatidylserine (PS) from the inner to the outer leaflet of the membrane bilayer, implying by the illustration that surface exposure of PS is a defining feature or signature of MP. Freyssinet supported the concept that the role of MP is not limited to procoagulant activities with thrombotic potential but extends to inflammation, and in addition they can behave as true diffusible vectors in transcelluar signaling. He discusses at some length evidence that MP can bind to and influence leukocyte activation. It should be added that our laboratory was the first to demonstrate clearly that platelet microparticles (PMP) bind to neutrophils, activating them and causing formation of grape‐like clusters of PMP–neutrophil complexes [2Jy W. Mao W.W. Horstman L.L. Tao J. Ahn Y.S. Platelet microparticles bind, activate and aggregate neutrophils in vitro.Blood Cells Molecules Dis. 1995; 21: 217-31Crossref PubMed Scopus (0) Google Scholar]. In that paper we advanced the concept that PMP can play an important role as a messenger linking thrombosis and inflammation through interaction with neutrophils [2Jy W. Mao W.W. Horstman L.L. Tao J. Ahn Y.S. Platelet microparticles bind, activate and aggregate neutrophils in vitro.Blood Cells Molecules Dis. 1995; 21: 217-31Crossref PubMed Scopus (0) Google Scholar]. That work was not cited in the review. He then concisely reviews some of the pertinent clinical publications, citing two of ours, namely, our original observation on PMP in patients with idiopathic thrombocytopenic purpura (ITP) in 1992 [3Jy W. Horstman L.L. Arce M. Ahn Y.S. Clinical significance of platelet microparticles in autoimmune thrombocytopenias.J Lab Clin Med. 1992; 119: 334-45PubMed Google Scholar] and our comprehensive review of PMP in 1999 [4Horstman L.L. Ahn Y.S. Platelet microparticles: a wide‐angle perspective.Crit Rev Oncol/Hematol. 1999; 30: 111-42Crossref PubMed Scopus (0) Google Scholar]. In our 1992 report, we demonstrated that PMP are hemostatically functional because ITP patients with high PMP do not bleed in spite of severe thrombocytopenia—certainly an example of ‘good’ MP—but we also found that those with unusually high MP suffered from ischemic small vessel diseases of the central nervous system, manifesting as recurrent transient ischemic attack, confirmed by magnetic resonance imaging [3Jy W. Horstman L.L. Arce M. Ahn Y.S. Clinical significance of platelet microparticles in autoimmune thrombocytopenias.J Lab Clin Med. 1992; 119: 334-45PubMed Google Scholar], some patients progressing to advanced vascular dementia [3Jy W. Horstman L.L. Arce M. Ahn Y.S. Clinical significance of platelet microparticles in autoimmune thrombocytopenias.J Lab Clin Med. 1992; 119: 334-45PubMed Google Scholar, 5Ahn Y.S. Horstman L.L. Jy W. Jimenez J.J. Bowen B. Vascular dementia in patients with immune thrombocytopenic purpura (ITP).Thromb Res. 2003; 107: 337-44Abstract Full Text Full Text PDF Scopus (47) Google Scholar]; hence in this setting, they are certainly ‘bad’ MP. Our reports certainly fit into the dichotomy of MP into ‘good’ and ‘bad’ as Freyssinet proposed. However, good or bad may not be qualitative but quantitative in some clinical settings. Among the most exciting recent developments in MP studies is the burgeoning field of endothelial microparticle (EMP) analysis, not covered in his review. We have recently reviewed this field [6Horstman L.L. Jy W. Jimenez J.J. Ahn Y.S. Endothelial microparticles as markers of endothelial dysfunction.Frontiers Bioscience. 2004; 9: 1118-35Crossref PubMed Google Scholar]. An important issue arising in our EMP studies is the distinction between MP‐bound and true soluble circulating markers of endothelial activation: we have shown that many of the markers assumed to be ‘soluble’ are in reality MP‐bound, at least in part (as discussed in [6Horstman L.L. Jy W. Jimenez J.J. Ahn Y.S. Endothelial microparticles as markers of endothelial dysfunction.Frontiers Bioscience. 2004; 9: 1118-35Crossref PubMed Google Scholar]). The pathophysiological mechanisms of release of true soluble and MP‐bound species are quite different and the two forms may be functionally distinct as well (because true soluble forms generally lack transmembrane domains). It remains to be seen which form is the better marker of disease activity. Meanwhile, assays by ELISA methods measure the sum of both forms. Finally, it is important to add our observation that not all MP are positive for PS. For example, the majority of EMP from activated endothelial cells were not positive for PS as judged by annexin V (AnV) binding, since many more EMP were counted by CD62E than by AnV. Even EMP from apoptotic cells, which are much richer in AnV binding, still gave only about half as many positives for AnV as for CD31 [6Horstman L.L. Jy W. Jimenez J.J. Ahn Y.S. Endothelial microparticles as markers of endothelial dysfunction.Frontiers Bioscience. 2004; 9: 1118-35Crossref PubMed Google Scholar, 7Jimenez J.J. Jy W. Mauro L.M. Soderland C. Horstman L.L. Ahn Y.S. Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis.Thromb Res. 2003; 109: 175-80Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar]. Therefore, assay methods which define total MP in terms of positivity for PS, as employed by others [8Mallat Z. Benamer H. Hugel B. Benessiano J. Steg P.G. Freyssinet J.M. Tedgui A. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes.Circulation. 2000; 101: 841-3Crossref PubMed Google Scholar] and illustrated in the review [1Freyssinet J.M. Cellular microparticles: what are they bad or good for?.J Thromb Haemost. 2003; 1: 1655-62Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar], can give grossly misleading results; for example, will tend to underestimate MP arising from cellular activation as distinct from apoptosis. Thus the critical question of how best to measure MP deserves further investigation. Our recent study of the mechanism of EMP generation indicated that capping precedes release of EMP from the membrane in a well‐orchestrated manner, as opposed to random release from the membrane [9Jy W. Jimenez J.J. Mauro L.M. Ahn Y.S. Newton K.R. Mendez A.J. Arnold P.L. Schultz D.R. Agonist‐induced capping of adhesion proteins and microparticle shedding in culture of human renal vascular endothelial cells.Endothelium. 2002; 9: 179-89Crossref PubMed Scopus (0) Google Scholar]. There may be many diverse routes governing release of MP, the floppase hypothesis being only one of them. The review of Freyssinet is timely and informative, bringing to attention a number of interesting papers and raising many questions. We hope it will initiate momentum towards a forum to refine and standardize MP assay methods, and to further elucidate the true role(s) of MP in health and disease. Methods to identify ‘good’ and ‘bad’ MP would certainly be welcome, as that could substantially improve the early diagnosis of thrombotic and inflammatory disorders, which are by far the most common and devastating illnesses in the modern world.

Summary The resting levels of cytoplasmic Ca2+ (measured by Quin 2 fluorescence) and dense tubular Ca2+ (measured by chlorotetracycline, CTC, fluorescence) are shown to be higher in platelets from patients with arterial thrombosis than from normal donors. Turbidmetric studies of aggregation of diluted platelet-rich plasma (PRP) at 135 μM Ca2+ showed increased rates of aggregation for patients relative to normal controls. For ADP-stimulated aggregation, increased maximal rates (Vmax) and decreased doses for half-maximal rates were observed. With collagen-stimulated aggregation, patient samples showed only decreased ED50 values relative to normal controls. The changes in these values are linearly correlated with the elevation of resting dense tubular Ca2+ level determined by the calcium-CTC test carried out at 2 mM external Ca2+. For ADP-stimulated aggregation this relationship can be mimicked by pre-incubating normal platelets with subcritical concentrations of the Ca2+ ionophore A23187. These results suggest that elevated cytoplasmic and dense tubular Ca2+ in the “resting state” is a major factor in arterial thrombosis, rendering the platelet more sensitive to the stimulation by physiologic agents.

Improved understanding of presurgical risk factors for transfusions will lead to reduction in their number and related complications. The goal of this study is to identify these factors in coronary artery bypass graft (CABG) surgery.Presented herein are results of analyses of data from an ongoing study of transfusion in CABG surgery. Of 122 patients, 81 received transfusion (Tx) and 41 did not (NoTx). In addition to routine tests, presurgical levels of microparticles from platelets (PMPs), red cells (RMPs), and other lineages were assayed.The Tx and NoTx groups were similar with respect to most presurgical variables but differed in distribution of gender, blood type, diabetes prevalence, activated partial thromboplastin time (aPTT), hemoglobin (HGB), and microparticle levels. Stepwise multiple logistic regression was used to evaluate presurgical variables and to develop a model to assess risk factors for transfusion. CD41(+) PMP and CD235(+) RMP levels were found to be the main risk factors for transfusion. The Model's discriminating ability was assessed using receiver operating characteristic curve analysis, which showed that the area under the model curve (± standard error) was 0.86 ± 0.04 (95% confidence interval, 0.77-0.94). According to the model, patients with higher presurgical levels of circulating CD41(+) PMP, CD235a(+) RMP, and HGB, as well as a shorter aPTT, are less likely to receive transfusion(s).Presurgical levels of CD41(+) PMPs and CD235a(+) RMPs are the main risk factors for transfusion in CABG, followed by HGB and aPTT.

Circulating activated platelet aggregates (aPA) were assayed by flow cytometry employing mAb alpha-CD62p in eight patients with thrombotic thrombocytopenic purpura (TTP). Elevation of aPA was observed in all patients in active stages of TTP; aPA normalized in remission. Plasma infusions with plasmapheresis decreased aPA in responding patients. The rise and fall of aPA preceded relapses and improvements, respectively. These changes were seen prior to the traditional indicators, LDH, haematocrit, and platelet count. Incubation of plasma from TTP patients with normal whole blood induced formation of aPA; this effect was significantly greater than that of plasmas from ITP patient controls (P < 0.01), suggesting the presence of an aPA-promoting factor in TTP plasma. Parallel experiments using a platelet aggregometer failed to detect effect of TTP plasma on normal blood. In summary, aPA appear to be a marker of disease activity, rising with relapse, falling with plasma therapy, and normalizing in remission. The flow cytometric assay of aPA is more sensitive than aggregometry in detecting the putative aPA-promoting factor in TTP.

Capping and release of membranous, small (< 1.5 microm) endothelial microparticles were quantified by immunofluorescence microscopy and flow cytometry after treatment of cultures of human renal microvascular endothelial cells with agonists tumor necrosis factor-alpha (TNF-alpha) or mitomycin C. For constitutive marker CD31, both agonist-treated attached, monolayer, and detached, free endothelial cells formed caps and released microparticles. TNF-alpha and mitomycin C induced dissimilar appearing CD31-containing caps after 3 h, followed by endothelial microparticle release after 6 h. The degree of capping correlated with increasing counts of released microparticles. For lymphokine-inducible CD54, TNF-alpha also induced CD54-containing caps and microparticle release, but mitomycin C failed to induce the expression of either entity. Neither capping nor microparticle release caused by TNF-alpha was part of an apoptotic pathway that involved caspase 3. Mitomycin C treatment of endothelial cells caused capping and microparticle release with a time course similar to TNF-alpha induction for 15 to 24 h, but assays for caspase 3 were positive, confirming the apoptotic action of mitomycin C. Membrane capping and microparticle release from endothelial cells are a convenient experimental model for studying protein movement, release of microparticles, and their possible biological significance.

This study presents a quantitative comparison of the free cytoplasmic calcium concentration ([Ca2+]cyt) and the free concentration in the lumen of the dense tubules of the human platelet. The former was measured by the fluorescence of the high affinity indicator quin2 and latter by the fluorescence of chlorotetracycline (CTC). The CTC technique monitors calcium-CTC complex accumulation in the lumen of dense tubules and mitochondria when washed platelets were incubated in 2 mM Ca2+. Resting cytoplasmic and dense tubular Ca2+ concentrations were studied in platelets from patients suffering from venous and arterial thrombosis. Compared with normal controls (0.40 +/- 0.10, n = 54), the values of the calcium-CTC ratios were 0.68 +/- 0.19 (n = 16, p less than 0.005) in venous thrombosis; 0.75 +/- 0.18 (n = 14, p less than 0.005) in cardiovascular accident; 0.84 +/- 0.18 (n = 6, p less than 0.005) in occlusive peripheral vascular diseases; and 0.42 +/- 0.10 (n = 21, p greater than 0.1) in patient controls. The dense tubular Ca2+ levels for both patients and controls were perfectly correlated with the cytoplasmic levels using an equation that assumes that the dense tubular free calcium concentration ([Ca2+]dt) has a second power dependence on [Ca2+]cyt. The abnormal Ca2+ handling of platelets obtained from thrombotic patients could be completely reversed by preincubation with the calcium channel blocker verapamil. These observations suggest that the primary Ca2+ handling defect is the leakage through activated channels in the plasma membrane. The defect and the elevated resting [Ca2+]cyt and [Ca2+]dt are adequate to explain the observation of increased rates of collagen-activated aggregation in the above-mentioned group of patients. The results can be explained by platelets from thrombosis patients being exposed to activating factors in the circulation, resulting in Ca2+ channel activation. Channel activation persists through the process of platelet isolation and washing and is manifested in higher measured values of [Ca2+]cyt and [Ca2+]dt in the "resting state." This would bring the platelet closer to its aggregation when aggregation-inducing agents are added. The CTC test is shown to be a useful and convenient means of detecting this abnormality.

A life-threatening hypercoagulable state (HCS) is reported that developed after splenectomy in idiopathic thrombocytopenic purpura (ITP). A 50-year-old active male was rejected for blood donation because of an incidental finding of low platelet counts, 40,000/uL. The diagnosis was ITP. Although asymptomatic, he underwent splenectomy because of poor response to steroids and intravenous (IV) gamma globulin. One month after splenectomy, he suffered pulmonary emboli without deep venous embolism (DVT), followed by bilateral DVT, threatening amputation of the legs. Emergency thrombolysis, insertion of stent, and IV heparin saved his legs. Extensive workup for HCS was negative. IV heparin was withheld for colonoscopy for possible gastrointestinal neoplasm, at which time DVT recurred, necessitating another thrombolysis and heparin infusion. He was discharged on enoxaparin, antiplatelet therapy, and danazol. Platelet hyperactivation, characterized by high platelet microparticles (PMP) and CD62P, was present throughout his course of active ITP, resolving when ITP went into remission with danazol therapy. ITP has remained in remission for 4 years after stopping enoxaparin and danazol. In vitro, his plasma in active ITP induced activation of normal platelets, generating PMP and inducing CD62p-positive platelets and platelet aggregates; his plasma from remission had no effect. This indicates the presence of a platelet activating factor, possibly anti-platelet antibodies. Splenectomy may have allowed procoagulant PMP to accumulate to high levels resulting in HCS. We advise awareness of thrombotic complications post-splenectomy in the subset of ITP patients who are largely asymptomatic and exhibit persisting platelet activation.

Circulating cell-derived microparticles (MPs) exhibit procoagulant activity and have been investigated for a possible role in some human pathologies. However, their potential role in hemostasis has been neglected and often denied. This review brings to attention a specific body of direct clinical evidence supporting an important but distinctive role of MPs in hemostasis. Evidence for a role of MPs in hemostasis includes: (1) two congenital bleeding disorders attributed to impaired release of MPs; (2) two recent studies of trauma patients relating naturally elevated endogenous MPs at admission to reduced transfusion requirements and better outcomes; (3) a study of coronary surgery patients showing that elevated MP before surgery reduces transfusion requirements during surgery; and (4) a clinical study of patients with immune thrombocytopenia demonstrating that those with high circulating MP have reduced bleeding compared to patients with similar platelet counts but lower MP levels. Mechanisms involving potentiating the contact factor pathway are thought to play a key role and are probably synergistic with polyphosphate released from activated platelets at sites of endothelial injury. Hemostatic defect of patients with deficient MP-mediated coagulation resembles deficiency of FXI (hemophilia C), distinct from hemophilia A or B, so can be termed type C hemostasis. A better understanding of this proposed hemostatic pathway may lead to improved methods for controlling excessive bleeding in surgery, trauma, and other clinical settings.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal stem-cell disorder in which blood cells lack complement inhibiting membrane proteins, and become susceptible to complement-mediated injury, leading to chronic intravascular hemolysis and pancytopenia. Glucocorticoids have been a mainstay of therapy. For patients refractory to glucocorticoids and requiring blood transfusions, an alternative therapy is needed. We studied danazol therapy in 5 patients refractory to other treatments. Four of the 5 benefited, showing rise in hematocrit and eventual cessation of transfusion requirements. Remissions lasted > or =2 years in 3 and 10 years in 1 patient. Danazol was well-tolerated without serious side effects. Danazol appears to be a good alternative treatment in PNH.

Introduction: Risk factors for thrombosis (TB) in thrombocythaemia (TC) associated with myeloproliferative disorder (MPD) are not well defined. Methods: We measured antiphospholipid antibodies (APLA) in 35 patients with TC associated with MPD. Fourteen had TB and 21 did not. We assayed IgG and IgM APLA by ELISA for 6 antigens: β2GP1, cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and FVII/VIIa, together with markers of activation of platelets (CD62P) and endothelium [endothelial microparticles (EMP)]. Results: At least one positive APLA was detected in 66% of TC patients overall. The incidence was significantly higher in the TB subgroup (92.8%) than non-TB (47.6%, p < 0.05). Multiple APLA (positive for more than one antigen) were also more frequent in TB, for both IgG and IgM, for all 6 antigens tested (p < 0.05). However, IgM APLA predominated, being about 2-fold more frequently positive than IgG for all 6 antigens. Platelet CD62P was significantly higher in the TB group (p < 0.05). EMP did not differ between TB and non-TB. The most frequent thrombotic complication was recurring ischemic cerebral vascular accidents (ICVA), leading to progressive cognitive impairment. Venous TB often developed at unusual sites. Recurring and reversible TB were common features in TC. Summary: This study suggests that APLA and platelet activation are risk factors for TB in TC. APLA are prevalent in TC, and IgM APLA predominated over IgG. Activation of platelets but not of endothelium may be consistent with the reversible and recurrent features of TB in TC.

&lt;i&gt;Background:&lt;/i&gt; Immune thrombocytopenic purpura (ITP) is frequently associated with chronic hepatitis C (HpC-ITP). &lt;i&gt;Methods:&lt;/i&gt; Recombinant interleukin-11 (rIL-11), which has both thrombopoietic and anti-inflammatory properties, was evaluated in 12 patients with HpC-ITP in this pilot study. Group 1 (7 patients) was treated at high dose (50 µg/kg daily) while group 2 (5 patients) at low dose (15–35 µg/kg three/week). &lt;i&gt;Results:&lt;/i&gt; In group 1, mean platelet counts rose from initial 54 × 10&lt;sup&gt;9&lt;/sup&gt;/l to 103 × 10&lt;sup&gt;9&lt;/sup&gt;/l (p = 0.02) and in group 2, from an initial 51 × 10&lt;sup&gt;9&lt;/sup&gt;/l to 74 × 10&lt;sup&gt;9&lt;/sup&gt;/l (p = 0.04). Antiplatelet antibody (aPlt-Ab) decreased in group 1. LFT improved in both groups. The mean HCV-RNA decreased in group 1 (p = 0.04), not in group 2. Side effects were common and troublesome, but were minimized with individualized dosing. One patient achieved good remission of both ITP and HpC lasting &gt;2 years with low-dose maintenance. &lt;i&gt;Conclusion:&lt;/i&gt; When used based on individual tolerance, rIL-11 appears useful in HpC-ITP.

We report on clinical, laboratory, and angiographic findings that appear to characterize a group of 13 middle-aged patients who suffered acute coronary syndromes (ACS) despite little angiographic evidence of atherosclerotic heart disease (ASHD) or other risk factors. Nine of the 13 were ≤46 years of age and the rest ranged to 59 years. All had evidence of platelet disorders (PD): seven had chronic immune thrombocytopenia (ITP), one had familial thrombocytopenia, and five had other disorders affecting platelets. Evidence of long-standing chronic platelet activation was the common feature of the group, as found by (i) elevated platelet microparticles (PMP), (ii) thrombocytopenia, and (iii) enhanced procoagulant activity of plasma. Data on the 7 with ITP were compared to 20 ITP without ACS: the former had higher PMP (p &lt; 0.01) and platelet-associated IgM (p &lt; 0.05) relative to the ITP patient controls. Another set of patient controls consisted of 20 ACS with documented ASHD: although activation indicators were abnormal also in this group relative to normal controls (p &lt; 0.01), the PD group of 13 had more marked abnormalities in all tests (p &lt; 0.03), particularly in PMP and thrombocytopenia (p &lt; 0.01). The seven youngest in the PD group appeared to respond to antiplatelet therapy since no recurrence of coronary ischemia was seen in up to 3 years of observation. It is suggested that chronic platelet activation by antibodies or immune complex may predispose those in the PD group to ACS (e.g., when under stress) despite the absence of ASHD and few other known risk factors. The true incidence of this syndrome is unknown but may be substantial. Key Words: Chronic platelet activation-Platelet microparticles-Platelet.associated IgM-Immune thrombocytopenia-Acute coronary syndromes-Middle age.

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet‐rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro . The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan‐leucocyte marker). Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg; only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti‐CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet‐rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN‐depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN–platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet‐rich microthrombi in TTP.

The kinetics of ATP-driven Ca2+ uptake by the dense tubules were studied in digitonin-permeablized human blood platelets. Digitonin at 3 μg/ml was shown capable of permeablizing the plasma membrane to lactate dehydrogenase and the cytoplasmic Ca2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca2+. Experimentation was carried out with platelets treated with 3 μg/ml digitonin reisolated and resuspended in detergent-free medium (‘digitonin-permeablized’ platelets). Active Ca2+ accumulation, which occurs over a period of minutes, was monitored by the increase in the fluorescence of chlorotetracycline after the addition of Mg-ATP (37°C). The active uptake is inhibited by 15 μM trifluoperazine. The process is saturable with respect to external [Ca2+], with a Km of 180 ± 5 nM and a Hill coefficient (n) of 1.40 ± 0.05. Analysis of the maximal uptake in steady state gave similar results (Km = 160 ± 5 nM, n = 1.50 ± 0.05). The rate of uptake at [Ca2+] ≈ Km is increased when the digitonin-permeablized platelets are preincubated with 100 nM phorbol 12-myristate 13-acetate. Actively accumulated Ca2+ is rapidly released (less than 1 min) by addition of d-myo-inositol triphosphate (IP3). The maximal extent of release is 50%; the EC50 for IP3 is approx. 12 μM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.

The measurement of endothelial microparticles (EMP) is as challenging as it is promising. We have been working to optimize our flow cytometric method based on years of prior work on platelet MP (PMP) [1Jy W. Horstman L.L. Arce M. Ahn Y.S. Clinical significance of platelet microparticles in autoimmune thrombocytopenias.J Laboratory Clin Med. 1992; 119: 334-45PubMed Google Scholar]. The distinctive feature of our recent work on EMP is its emphasis on phenotypic subsets of EMP, based on evidence that not all EMP are alike [2Jimenez J.J. Jy W. Mauro L. Soderland C. Horstman L.L. Ahn Y.S. Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis.Thromb Res. 2003; 109: 175-80Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar]. In vitro studies showed that EMP from apoptotic endothelial cells (EC) were distinct from those from activated EC, in that activated EC release predominantly CD62E+ and CD54+ EMP whereas apoptotic EC are rich in CD31+ EMP [2Jimenez J.J. Jy W. Mauro L. Soderland C. Horstman L.L. Ahn Y.S. Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis.Thromb Res. 2003; 109: 175-80Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar]. This criterion was applied in vivo to demonstrate that the vascular endothelium in TTP is activated, not apoptotic [3Jimenez J.J. Jy W. Mauro L.M. Horstman L.L. Soderland C. Ahn Y.S. Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation.Br J Haematol. 2003; 123: 896-902Crossref PubMed Scopus (122) Google Scholar]. It is possible that selective shedding of certain lipid rafts are responsible for the multiple phenotypes of EMP; we have shown that capping of CD31 on endothelial cells (EC) appears to precede EMP release [4Jy W. Jimenez J.J. Mauro L.M. Ahn Y.S. Newton K.R. Mendez A.J. Arnold P.L. Schultz D.R. Agonist‐induced capping of adhesion proteins and microparticle shedding in culture of human renal vascular endothelial cells.Endothelium. 2002; 9: 179-89Crossref PubMed Scopus (0) Google Scholar]. The following describes our basic flow cytometric methods, given in more detail in the references and discussed in our recent review [5Horstman L.L. Jy W. Jimenez J.J. Ahn Y.S. Endothelial microparticles as markers of endothelial dysfunction (Review).Frontiers Biosci. 2004; 9: 1118-35Crossref PubMed Google Scholar]. All monoclonal antibodies are commercially available, as listed in references. The principle of this method is that both EMP and PMP are often CD31+, but only PMP are CD42b+, thus both may be enumerated in a single run. We mixed PMP and EMP at different ratios and verified that there was no significant overlap between these two types of MP under our experimental conditions. Blood is collected in citrate vacutainers using 21‐G needle, the first tube is discarded, and light tourniquet removed. Within 4 h of collection, the blood is centrifuged at 200 × g for 10 min to prepare platelet‐rich plasma (PRP) and the PRP is further centrifuged for 7 min at 1500 × g to obtain platelet‐poor plasma (PPP). This speed preserves the majority of MP; residual contaminating platelets are gated out in flow cytometry. Then 25 µL of the PPP is incubated with 4 µL of anti‐human FITC‐CD42b and 4 µL of PE‐CD31. Samples are incubated at room temperature for 20 min with gentle shaking, then 0.5 mL PBS is added and the sample is ready for flow cytometry in a Coulter EPICS XL. Detection of particles is by triggering on a fluorescent signal greater than that of matched isotype control from the same supplier. The rationality of this method is that, although we observed in vitro (tissue culture) that both CD54+ and CD62E+ EMP are abundantly released by stimulated EC, those which are CD54+ avidly bind to leukocytes in vivo, sharply reducing their apparent presence [6Jy W, Minagar A, Jimenez JJ, Sheremata WA, Mauro L, Horstman LL, Bidot CJ, Ahn YS. Endothelial microparticles (EMP) bind to monocytes to activate and enhance transmigration: elevated circulating EMP‐monocyte conjugates in multiple sclerosis. Frontiers Biosci 2004; 9: 3137–44.Google Scholar]. To 25 µL PPP prepared as above is added 4 µL of fluorochrome‐labeled anti‐CD62E. The remaining steps are as above. (i)CD31 is also expressed on some leukocytes, therefore we tested coexpression of CD31 and CD45 in several samples and found about 5–10% of MP to be of leukocyte origin. This was considered negligible but it is possible that some patient samples are selectively richer in leukocyte MP.(ii)Our protocol requires the use of fresh plasma samples (centrifuged within 4 h), as freezing of plasma was found to alter counts and some antigen properties of the MP. This is labor‐intensive and precludes side‐by‐side comparison of samples in larger batches.(iii)Standard clinical flow cytometry is limited in its ability to detect weakly expressed antigens, limiting the lower size range of detectability. The above methods are rapid, economical, and well‐suited to clinical laboratory applications, and have been fruitfully applied to many thrombotic and inflammatory disorders such as those mentioned above and others in our recent review [5Horstman L.L. Jy W. Jimenez J.J. Ahn Y.S. Endothelial microparticles as markers of endothelial dysfunction (Review).Frontiers Biosci. 2004; 9: 1118-35Crossref PubMed Google Scholar]. Some of our findings are in reasonably good agreement with those of others, such as ours on CAD [7Bernal‐Mizrachi L. Jy W. Jimenez J.J. Pastor J. Mauro L. Horstman L.L. DeMarchena E. Ahn Y.S. High levels of circulating endothelial microparticles in patients with acute coronary syndromes.Am Heart J. 2003; 145: 962-70Crossref PubMed Scopus (0) Google Scholar], consistent with others [8Mallat Z. Benamer H. Hugel B. Benessiano J. Steg P.G. Freyssinet J.M. Tedgui A. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary symptoms.Circulation. 2000; 101: 841-3Crossref PubMed Google Scholar]. On the other hand, our findings in pre‐eclampsia [8Mallat Z. Benamer H. Hugel B. Benessiano J. Steg P.G. Freyssinet J.M. Tedgui A. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary symptoms.Circulation. 2000; 101: 841-3Crossref PubMed Google Scholar, 9Gonzalez‐Quintero V. Jimenez J.J. Jy W. Mauro L.M. Horstman L. O'Sullivan M. Ahn Y.S. Elevated plasma endothelial microparticles in pre‐eclampsia.Am J Obstet Gynecol. 2003; 189: 589-93Abstract Full Text Full Text PDF PubMed Google Scholar], for example, are disparate with those of VanWijk et al.[10VanWijk M.J. Nieuwland R. Boer K. VanDerPost J.A.M. VanBavel E. Sturk A. Microparticle subpopulations are increased in pre‐eclampsia: possible involvement in vascular dysfunction?.Am J Obstet Gynecol. 2002; 187: 450-6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Accordingly, it is important for all workers in this field to be able to compare their results. We propose that a practical first step in that direction would be the adoption of standard microparticle preparations made available to all, such as standard lyophilized microparticles based on methods such as those given by Arnout et al.[11Arnout J. Huybrechts E. Vanrusselt M. Vermylen J. A new lupus anticoagulant test based on platelet‐derived vesicles.Br J Haemat. 1992; 80: 341-6Crossref PubMed Google Scholar]. In addition to numeration of MP by different markers, future work on methodologies might benefit from considering functional assays such as prothrombinase [8Mallat Z. Benamer H. Hugel B. Benessiano J. Steg P.G. Freyssinet J.M. Tedgui A. Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary symptoms.Circulation. 2000; 101: 841-3Crossref PubMed Google Scholar], tissue factor, promotion of cell–cell interactions, cytokines, and possibly MP‐associated von Willebrand factor activity [12Jy W. Jimenez J.J. Mauro L.M. Horstman L.L. Bidot C.J. Tiede M.P. Ahn E. Ahn Y.S. Endothelial microparticles (EMP) interact with platelets via a vWF dependent pathway to form platelet aggregates, more resistant to dissociation than those induced by soluble vWF.Blood. 2003; 102: 783aGoogle Scholar], as this may help differentiate functionally distinct MP phenotypes.

Abstract We previously described a subgroup of immune thrombocytopenic purpura (ITP) patients presenting with recurring transient ischemic attack‐like symptoms and progressive cognitive impairment due to small vessel disease (SVD) seen in the brain. They presented minimal bleeding despite thrombocytopenia, and platelet activation was elevated compared to classic ITP. On the hypothesis that the blood–brain barrier (BBB) is compromised in this subgroup, we investigated the effect of plasma from SVD‐ITP patients on the transendothelial migration of leukocytes (TEML). Brain microvascular endothelial cells (BMVEC) were grown to confluence on 6.5‐μm pore filters and plasma from 10 healthy controls, 20 classic ITP, and 5 SVD‐ITP were added and incubated 24 hr. Then 1 × 10 5 monocytes (U937) were added and the number migrated through the EC monolayer after 6 hr was measured by flow cytometry. The effect on TEML of danazol was also assessed. We found that plasma from SVD‐ITP but not classic ITP induced 10‐fold rise in EC activation marker CD62E and a sevenfold increase in TEML, to 38.5% ± 12.5% of cells migrated, compared to normal controls (5.6% ± 1.2%) or classic ITP (6.1% ± 0.2%), P &lt; 0.001. Preincubation of U937 with endothelial microparticles (EMP) increased TEML by 20.0% ± 6.4% with SVD‐ITP plasma, significantly more than with classic ITP or control plasmas, P = 0.003. Pretreatment of cultures with danazol (100 μg/mL) inhibited TEML by 25% in all wells tested, whether or not EMP were added. In summary, SVD‐ITP plasma activates EC and augments TEML, suggesting plasma‐mediated BBB dysfunction in this syndrome. Danazol modestly but significantly inhibited TEML. Am. J. Hematol., 2008. © 2007 Wiley‐Liss, Inc.

Abstract Concentrations of danazol in patient plasma and red blood cells (RBC) were assayed over a 6‐month period in 75 patients on danazol therapy using a high‐pressure liquid chromatography (HPLC) method more reliable than previous radioimmunoassay (RIA) methods. It was found that plasma danazol rose regularly for 15 days after the beginning of treatment, reaching a steady state plateau of 175 ± 76 ng/ml in 20 patients on normal dose, and less for lower dose schedules. After stopping danazol, concentrations declined to near zero in a similar time frame. RBC concentrations on a packed volume basis were similar to plasma levels. However, the membrane ghosts of RBC contained about 50% of the total RBC danazol, implying about 100‐fold higher concentration in membranes than in plasma. Similar distributions were obtained in vitro with both RBC and platelets, and were confirmed by 14‐C‐labeled danazol. These findings tend to support the hypothesis that the benefits of danazol in immune disorders may be attributable in part to its intercalation in the lipid bilayer of the plasma membrane, altering antigen/receptor expression to modulate immune reactions. This hypothesis was first suggested when it was observed that the RBC of patients on danazol therapy showed morphological changes and increased resistance to osmotic lysis. It was later shown that danazol in vitro reduces binding of autoantibodies, and protects against complement‐mediated lysis, suggesting direct action of danazol on the membranes. This hypothesis is discussed, and danazol's effect in protecting against complement‐mediated lysis is described.

It was reported that elevated levels of platelet microparticles (PMPs) in patients with immune thrombocytopenic purpura (ITP) were associated with decreased bleeding, and in some cases with small vessel thromboses (J Lab Clin Med 1992; 119:334). To investigate the possible role of complement in PMP production in ITP, an in vitro assay was developed to simulate ITP: platelets were opsonized with well-defined monoclonal antibodies against glycoprotein IIb/IIIa, of immunoglobulin G (alpha-CD41), and of immunoglobulin M (alpha-Plt-1) class, then exposed to serum as a source of complement. PMP generation and lysis were monitored by flow cytometer, by release of lactic dehydrogenase, and by generation of procoagulant activity. These effects were largely abolished by heating the serum (30 minutes, 65 degrees) or by incubation with alpha-C1q, confirming the role of complement. At low concentrations of serum, both monoclonal antibodies promoted PMP shedding in a concentration-dependent manner without loss of platelet population; at higher concentrations, extensive lysis occurred, but marked variations in resistance to lysis were observed in platelets from different individuals. The PMPs produced were associated with increased procoagulant activity, as measured by the Russell's viper venom test. The immunoglobulin M antibody was more potent than the immunoglobulin G antibody in promoting lysis, and the resulting PMPs had greater procoagulant activity. To clarify the variation seen in platelets from different donors, data was sorted on the basis of gender, with the finding that women's platelets are significantly more sensitive to complement-mediated damage than men's. This may explain in part why ITP is three to four times more prevalent in women than in men. We conclude that complement activation is the most likely explanation for the elevated level of PMPs often seen in patients with ITP and sometimes associated with thrombosis and that the determining factors are the concentration and nature of antibody as well as individual differences in sensitivity to complement-mediated damage. Because complement activation can occur without participation of antibody, complement activation may also be the cause of elevated PMP levels seen in other thrombotic disorders not involving platelet-specific antibodies.

Red blood cell microparticles (RMPs) is a high potency hemostatic agent, which may serve as a viable therapeutic approach. They generate thrombin in vitro and effective in arresting bleeding in animal bleeding models. However, prior to ascertaining the clinical efficacy of RMPs, detailed preclinical evaluation is necessary. Therefore, we aimed to characterize RMPs, ascertain their stability, and determine their pharmacokinetics in rats. RMPs were prepared from human RBCs by a high-pressure extrusion method. Pharmacokinetic parameters were computed from groups receiving various RMPs dosing regimens. Volume of distribution, elimination rate constant, and clearance for RMPs were also assessed. Major portion of prepared microparticles were RMPs and a very small portion of particles were from platelets and leukocytes. RMPs were stable when stored at 5 and -20°C for at least 12 months. In vivo half-life was found to vary for each paradigm, but in general, was less than 2 min for most of the paradigms evaluated. Our results demonstrate that RMPs are stable during prolonged storage and have a short half-life. Therefore, the clinical use of RMPs as a hemostatic agent, within a tailored treatment paradigm, may be advantageous in achieving prolonged systemic therapeutic benefit without provoking any thrombotic complications.

Background: Uncontrollable bleeding is a major cause of mortality and morbidity worldwide. Effective hemostatic agents are urgently needed. Red cell microparticles (RMPs) are a highly promising hemostatic agent. This study evaluated the safety profile of RMPs preliminary to clinical trial. Methods and Results: RMPs were prepared from type O+ human red blood cell by high-pressure extrusion. Male rats were treated with RMPs either a 1 × bolus, or 4 × or 20 × administered over 60 minutes. The vehicle-treated group was used as a control. Effects on physiological parameters were evaluated; namely, blood pressure, body and head temperature, hematocrit, and blood gases. We did not observe any adverse effects of RMPs on these physiological parameters. In addition, brain, heart, and lungs of rats treated with 4 × dose (bolus followed by infusion over 60 minutes) or vehicle were examined histologically for signs of thrombosis or other indications of toxicity. No thrombosis or indications of toxicity in brain, heart, or lungs were observed. Studies revealed that RMPs were distributed mainly in liver, spleen, and lymph nodes, and were potentially excreted through the kidneys. Conclusions: Our study indicates that RMP administration appears not to have any negative impact on the parameters studied and did not produce thrombosis in heart, brain, and lungs. However, more detailed long-term studies confirming the safety of RMP as a hemostatic agent are warranted.

Abstract Background Lymphotropism of HCV induces chronic immune stimulation, facilitating production of wide variety of autoantibodies, leading to HCV driven autoimmunity. Immune thrombocytopenic purpura (ITP) is one of extra hepatic manifestations of autoimmune diseases in HCV infection. The treatment landscape of hepatitis C virus infection has been dramatically altered in 2013 with the approvals of the second-generation protease inhibitor simeprevir and the nucleotide polymerase inhibitor sofosbuvir. In most patients treated with the direct-acting antiviral agent (DAA), HCV RNA became undetectable. However a little is known about how DAA therapy affects autoimmune diseases associated with HCV, especially in ITP. Cessation of immune stimulation by HCV following the treatment may alter immune responses and may modify autoimmune diseases. We report here a case of acute exacerbation of ITP following DAA treatment. DAA therapy appears to precipitate acute exacerbation of thrombocytopenia and ITP became refractory to all treatments including the measures that used to induce remission prior to DAA treatment. Case report A 67-year-old man was diagnosed chronic HCV infection, genotype 1b, in 1997. He failed treatment with Interferons. Chronic active hepatitis subsequently progressed into cirrhosis with portal hypertension. His ITP required frequent treatments with Prednisone and IVIG. It became severe in early 2013. His platelet counts were low at 10,000. He responded well to high dose Prednisone but could not continue because of worsening diabetes. He also failed Rituxan. He received multiple treatments including eltrombopag and romiplostin which he did not respond well to. His ITP finally stabilized with monthly infusion of WinRho as maintenance therapy with platelet counts hovering around 40.000 for 6 months. In March 2014, he was started on HCV treatment with simeprevir, sofosbuvir and ribavirin. His baseline platelet count prior to the new treatment was in the 50000’s. It plummeted to below 10.000 two weeks into treatment. He developed multiple ecchymosis and blood blisters in the mouth. He completed his HCV treatment on June 3, 2014. His viral load was no longer detectable. However, ITP remained refractory and severe with platelet counts around 10,000. High dose Prednisone or WinRho which had been effective in raising platelet counts previously were no longer effective after DAA therapy. During this acute exacerbation of ITP, he was treated with IVIG 3 times a week and low dose Prednisone and NPlate weekly with increasing dosage. He developed frequent blood blisters in mouth and gum bleeding requiring emergency room visits and hospitalization where he received platelet transfusions along with IVIGG and high dose Prednisone. His hemoglobin was stable throughout. IVIGG was reduced to 2 times a week. At 8 weeks following completion of DAAs, his platelet count improved to 40.000s range without clinical sign of bleeding. Discussion It has been shown that the frequency of B-cell production of anti- GPIIb-IIIa antibody, an antibody against the major platelet surface autoantigen (BP IIb-IIIa) recognized by anti-platelet antibodies in patients with idiopathic thrombocytopenic purpura (ITP), was greater in HCV-ITP (9). An inverse correlation was found between platelet count and B-cell anti–GPIIb-IIIa Ab production in 51 patients with liver cirrhosis (73% with hepatitis C). In our patient, ITP was stable with maintenance infusion of WinRho for 6 months. Acute exacerbation of ITP 2 weeks into DAA therapy suggests that it was triggered by the DAAs. Thrombocytopenia is a well-recognized complication of interferon therapy for treatment of viral hepatitis. Simeprevir and sofosbuvir however, are not well known to affect the platelet count. A little is known on effect of DAAs on thrombocytopenia. Immune alteration following eradication of HCV and subsequent modification of autoimmunity could affect course of ITP associated with HCV infection. Meanwhile, it is important for physicians and patients to be aware of potentially dangerous complications of these second generation DAAs. Disclosures No relevant conflicts of interest to declare.

Ca2+ is known to be required for mitogen-mediated lymphocyte activation. In order to further define the regulatory role of Ca2+, we have examined the activation events which occur following treatment with ionomycin (a Ca2+ ionophore), as compared to those occurring following concanavalin A (Con A) treatment of mouse splenic T-lymphocytes. Our results indicate that ionomycin and Con A induce the exposure of both interleukin-2 (IL-2) and insulin receptors on the surface of the lymphocytes within the first 5 min of treatment. The exposed insulin and IL-2 receptors have the following properties: (1) they consists of both high- and low-affinity receptors; and (2) they appear on the cell surface in small clusters (i.e., patches) or, occasionally, a large aggregate (i.e., cap). c-myc gene expression and DNA synthesis occur in both the ionomycin and Con A-treated lymphocytes when either IL-2 or insulin is present in the culture medium. Furthermore, the exposure of both hormone receptors can be inhibited by either EGTA (a Ca2+ chelator), bepridil (a Ca2+ channel blocker), W-7 (a calmodulin antagonist) or cytochalasin D (a microfilament inhibitor). Treatment with these inhibitors also blocks the expression of c-myc gene and DNA synthesis which occur at later times during IL-2 and insulin-induced activation of ionomycin- and Con A-treated lymphocytes. These findings suggest that a Ca2+ and calmodulin-mediated contractile system is involved in the exposure of certain hormone receptors which appear to be required for complete lymphocyte activation.

Abstract Introduction. Rituximab, humanized anti-CD20, was initially approved for the treatment of CD20-positive B cell lymphoma, but has recently been shown to be effective in the treatment of a wide range of autoimmune disorders. In a cohort of 25 patients, Stasi et al reported a 52% overall response rate in patients with chronic idiopathic thrombocytopenia (ITP) (Blood2001; 98:952–957). Overall response rates reported since then have been concordant, about 50%. Although rituximab has been increasingly employed for the treatment of ITP, the mechanism of immunological response is not clear. We investigated the effect of rituximab on titers of anti-platelet glycoprotein-specific antibodies in patients with refractory ITP. Methods. We studied 12 patients with chronic ITP (9 females, 3 males) of ages ranging from 22–87 years, mean age 43 years. All had been treated with glucocorticoids and failed to sustain remission. Three patients failed splenectomy. Their previous medications were continued without major alteration of the dosages during the period of followup. Rituximab was given 375 mg/m2 weekly for 4 courses. Once remission was achieved, previous medications were gradually tapered and stopped completely if remission was sustained. Complete remission (CR) was defined as normalization of platelet counts that lasted longer than 4 months. Partial remission (PR) was defined as a doubling of platelet count if initial counts were ≥20,000 or an increase to ≥30,000 if counts were 20,000 or below. Non-responders (NR) showed neither. Lab studies included CBC, platelets, and blood chemistries. IgM and IgG antibodies against platelet glycoproteins (GP IIb/IIIa, GP Ib/IX, GP IV) were assayed by PAICA prior to and following rituximab therapy. Results. Ten of 12 patients showed improvement of platelet counts following rituximab therapy. There were 6 CR, 4 PR and 2 NR. Among the 6 CR, remissions lasted for a range of 6–29 months with a mean of 17.0 months. Four of the 6 were able to stop previous therapy for ITP, and the other two were able to reduce their dosages by at least half. Among the 4 PR, remissions were maintained for 4–16 months (mean, 7.3 months). Anti-platelet antibodies became negative in 6 of 6 CR, in 3 of 4 PR and in 0 of 2 NR. Although on average, all anti-platelet antibodies tested were reduced after rituximab therapy, IgM anti-GP IIb/IIIa, IgM and IgG anti-GP Ib/IX and IgM anti-GP IV reached statistical significance (p&amp;lt;0.05). All patients tolerated rituximab well without adverse side effects. Conclusions. Rituximab frequently induces long lasting clinical remissions that do not require further therapy for ITP and reduction or disappearance of autoantibodies in ITP, unusual features compared to other measures. This finding indicates that a putative mechanism of action of rituximab is inhibition or destruction of autoantibody-producing B cells in ITP. Rituximab is highly effective with minimal side effects and should be considered as primary therapy in patients with chronic ITP.

Abstract BACKGROUND: Blood transfusion (Tx) carries greater risks of adverse events (AEs) than previously appreciated. These adverse effects include higher incidence of post-surgical infections, longer hospital stay, higher mortality, more frequent serious adverse events (SAE’s), and generally poorer surgical outcomes. Accordingly, ameliorating these adverse effects constitutes an urgent challenge to medical science. Factors responsible for Tx-related adverse events (AE’s) are not well understood. Many potentially toxic substances are released during blood storage, and many of them have been implicated or postulated as culprits. Washing of packed RBC remove these products and may ameliorate transfusion-related AE’s. Benefits of washed RBC are well established for pediatric surgical patients, chiefly by preventing hyperkalemia, but use of washed RBC in adult surgical patients has not heretofore been systematically investigated. We here report results of a prospective randomized study directly comparing surgical outcomes, in terms of mortality and AE’s, between groups of adult CABG patients transfused with either washed or unwashed (conventional) RBC. METHODS: A prospective randomized study of 148 patients undergoing coronary artery bypass graft (CABG) was conducted. Fifty-eight patients were randomized to receive unwashed (conventional) RBC (UW group) and 41 to washed RBC (W group). The remaining 49 did not require Tx. The main in-hospital outcomes recorded included mortality, serious adverse events (SAE’s), non-serious adverse events (AE’s), and SOFA scores pre- and post-surgery. A telephone interview was conducted at day 30 post-discharge, and mortality at one-year was also assessed. The statistical techniques used for the comparison of the UW and W RBC groups included: independent sample t-tests for variables with normal or approximately normal distribution; Mann-Whitney tests for variables with skewed distributions and for ordinal variables; chi-squared tests or Fisher’s exact tests for discrete variables; and logistic regression model for assessing different factors as predictors of the occurrence of each kind of event. RESULTS: Between the 2 groups, demographic, clinical, and comorbidity data were similar and there was no statistically significant difference in number of serious AE’s (SAE’s). However, 4 of 6 patients died from SAE’s in the UW group but all 7of 7 with SAE in the W group survived. The in-hospital mortality was greater in the UW group (4 vs. 0, p = 0.149) but 1-year post-op mortality was significantly higher in UW group (7 vs. 0, p=0.036). Frequency of less serious AE’s was higher in UW group in every category. Negative binomial regression analyses showed that, after adjusting for comorbidities, UW-group are likely to experience 64% more AEs (p= 0.027). The 30-day follow-up showed similar trends of higher AE’s in UW-group, but only CNS-related AE’s were significant (30 vs. 5, p&lt;0.01). CONCLUSIONS / DISCUSSION: These data suggest major benefits to patient outcomes by use of washed RBC in CABG. Most important is significant reduction of mortality. Less serious AE’s were also lower in the W group in nearly every category, but only CNS-related AE’s were statistically significant in this comparatively small patient population. To our knowledge, this is the first prospective randomized study in adults to assess possible benefits of washing RBC prior to cardiac surgery. At present, washed RBCs are seldom used in adults but the present study clearly demonstrates major advantages. It may be possible to reduce costs of washing by using on-site cell call-salvage equipment but this needs to be evaluated. This study was undertaken with the hypothesis that cell-derived microparticles (MP) are major culprits in Tx-associated AE’s. Further study is needed to determine if that hypothesis is correct. Other evidence has led us to conjecture that MP are largely responsible for post-surgical adverse outcomes; the present study is consistent with that conjecture but does not prove it. A major shortcoming of this study is the comparatively small patient population. A much larger study, including other types of surgery, is certainly warranted by these findings, and should be designed to include more quantitative evaluation of post-surgical cognitive impairment. Disclosures No relevant conflicts of interest to declare.

The effect of pulsatile blood flow on platelet thrombogenicity and platelet fragmentation (PF) in a hollow fiber hemodialyzer (HFD) was quantified with 111In labeled platelets and 125I labeled fibrinogen; 150 ml of blood was collected from Beagle dogs, Yorkshire pigs, and a human volunteer (non-smoker). Platelets were labeled with 111In tropolone (300 microCi) and fibrinogen was labeled with 125I. Sham dialysis (SHD) was performed with 120 HFDs (0.9 meter2) at 37 degrees C, with flow-rates of 150, 250, 500, and 950 ml/min.; after SHD, the washed HD radioactivity was measured with an ionization chamber. PF was measured by flow cytometry with GP IIb-IIIa murine monoclonal antibody. Platelet deposition decreased significantly for 3 species at higher flow; fibrinogen deposition (10-12%, 55-65 mg/m2), was not affected by flow. Adherent platelet thrombus decreased from (8.2 +/- 3.4) to (3.1 +/- 1.0) with human blood as flow rate increased from 150 to 950 ml/min; platelet thrombus level also decreased significantly (p < 0.005) from (20.3 +/- 6.2) to (4.5 +/- 1.9) with canine blood. Higher values were obtained for canine than human and porcine platelets. Platelet fragmentation, on the other hand, increased from 2.1-2.2% to 10.2-11.3% with increase of flow. Like platelets, deposition of canine fibrinogen was slightly higher than that of pig and human. The studies of adherent thrombus and platelet fragmentation identified an important flow-window of reduced thrombogenicity and acceptable fragmentation, encouraging extracorporeal circulation at higher blood flow.

Abstract BACKGROUND. Cell derived microparticles (MP), are small vesicle (&lt;1um) released in cell activation or apoptosis. Depending on stimulus, MPs can be heterogeneous in phenotype and functional activities such as hemostatic vs thrombogenic vs proinflammatory. In this study, we compared RMP produced in three ways: (1) induced by calcium ionophore (RMP-CaI), (2) released from packed cells (PC) during storage up to 42 days (RMP-Stor) and (3)by high-pressure extrusion of washed RBC (RMP-HPE). The aim of the study was to elucidate how the functional property of these species vary with mode of production. METHODS. For RMP-Stor, samples were taken at intervals up to 42 days of PC stored at 4oC, then centrifuged to recover the RMP released. For RMP-CaI, supernatants of fresh RBC exposed to Ca2+/ionophore (A23187) for 30 min were centrifuged. For RMP-HPE, washed RBC were subjected to high-pressure extrusion, and the resulting RMP were collected by centrifugation and washed. Flow cytometric counts were by CD235a (glycophorin A) and annexinV (AnV) binding. Thromboelastography (TEG) compared equal counts of each sample type, based on CD235a, diluted as needed with particle-free pooled plasma (PFP). RESULTS: (1) Yield per mL of initial packed RBC: The highest yield of RMP was seen in RMP-HPE, as expected since ≈100% of RBC convert to RMP-HPE. This was followed by RMP-CaI, which was about 3x more than RMP-Stor at 42 days. The RMP-Stor increased linearly from day 21 to 42. (2) AnV binding was expressed as ratio AnV+/CD235+, and is a marker of procoagulant phospholipid. This ratio was least in RMP-HPE (0.38) and more than doubled in both RMP-Stor (0.92) and RMP-CaI (1.0). (3) Size of RMP by forward scatter (FS) increased steadily with time in RMP-Stor, from day 21 (0.56 um) to day 42 (0.79 um). These values are somewhat larger than RMP-CaI (0.47 um) or RMP-HPE (0.52 um). The side-scatter signal correlated with the FS values. (4) Acetylcholinesterase (AChE) activity was by far highest in RMP-Stor (56 nM/min, per 106 particles) vs. 4.5 for RMP-CaI, and 1.5 for RMP-HPE. (5) Procoagulant activity: In TEG, the increase in MA (maximum amplitude) by addition of RMP-Stor was 64 mm, but was not increased by RMP-HPE or RMP-CaI. The R-time (lag) showed greatest shortening with RMP-Stor (10.2 min), followed by RMP-CaI (6.7 min), and RMP-HPE (3.6 min) (6)Anti-fibrinolytic activity: In presence of 0.4 ug/mL of tissue plasminogen activator (tPA), for the purpose of making fibrinolytic activity more detectable in a short time, all 3 species of RMP effectively inhibit t-PA-mediated fibrinolysis measured by TEG parameters, including MA, Ly30, and TTG (total thrombus formation). The RMP-HPE was most effective followed by RMP-Stor, and RMP-CaI. CONCLUSION/DISCUSSION. Results demonstrate clear and distinctive differences in functional and phenotypic properties of the 3 species compared. RMP-Stor and RMP-CaI are lager in size, higher in PS and AChE expression, and higher in procoagulant activity, compared to RMP-HPE. On the other hand, RMP-HPE are higher in number generated per mL packed RBC and exhibit stronger inhibition of tPA-mediated fibrinolysis. It is well known that RMP generated by storage and calcium ionophore are enriched in lipid rafts. In contrast, the RMP-HPE are not enriched in lipid rafts, as reflected in low AChE. We conjecture that the contents of lipid raft on RMP may play an important role in determining the functional properties of RMP. Disclosures No relevant conflicts of interest to declare.

Abstract BACKGROUND. RMP have been developed as novel hemostatic agents for treatment of bleeding disorders [Thromb Haemost 110:751,2013]. RMP generate thrombin in vitro through activation of contact pathway independent of tissue factor. They correct coagulopathy associated with most factor deficient plasma, indicating promotion of secondary hemostasis. In addition, they promote primary hemostasis by enhancing platelet adhesion and aggregation. Recent literature suggests that excessive levels of tPA are responsible for the uncontrollable bleeding seen in trauma-related coagulopathies [J Trauma and Acute Care Surg 80:1,2016]. In this study, we used thromboelastography (TEG) to investigate the effects of RMP on tPA, a relation hitherto unexplored. METHODS. The RMP were made by high-pressure extrusion of washed red cells. TEG was used to assess coagulation and fibrinolysis in fresh whole blood (diluted to 80%) in presence vs. absence of RMP (2.78x107/mL final conc. based on flow cytometry of PE-labeled mAb to CD235a). To hasten fibrinolysis for ease of measurement, tPA was added in all runs at final conc. 0.4 ug/mL. Calcium chloride (0.2 M) was added to each trial to induce coagulation. Additions to cup were: 325 uL of 80% blood + 10 uL tPA + 10 uL saline or RMP + 20 uL CaCl2. The tPA was stored in 50% glycerol at -20°C. RESULTS. Representative TEG curves of tPA-induced fibrinolysis in the presence or absence of RMP are shown in Figure 1 below. In the presence of tPA without RMP, the MA is small and the amplitude will decline to the baseline in 30 - 60 min, reflecting ongoing fibrinolysis. The MA was increased by about 50% by addition of RMP, from 25mm to 37mm (p=0.016). Judging from fibrinolysis measured by the-built-in LY30 parameter (% lysed at 30 min after MA was achieved), the mean of n=6 paired t-tests was 61.3% lysed in absence of RMP vs. 36.3% with RMP, indicating 40% inhibition of fibrinolysis (p=0.046). To assess the overall anti-fibrinolytic effect of RMP, we used the integral of the 1st derivative of the TEG curve, called TG (thrombus generation), which amounts to the total or net hemostatic-like activity. By this measure, addition of RMP gave TG = 457 vs. 319mm/min in absence of RMP (p=0.0044 by paired t test, P=0.037 by unpaired t test). DISCUSSION. These findings suggest that RMP exert significant anti-fibrinolytic activity, enhancing its hemostatic activity. Uncontrollable bleeding due to fibrinolysis is a major component of many bleeding disorders, including acute coagulopathy of trauma which is seen in about 20% of cases and is associated with poor outcome. The mechanistic basis of this activity is not fully understood. It was suggested that RMP are incorporated into the fibrin network to consolidate its structure thus stabilizing the network and impeding fibrinolysis. Full exploration of this activity is warranted since treatment for excessive fibrinolysis would have great impact in clinical management of many bleeding disorders. Disclosures No relevant conflicts of interest to declare.

We studied the effect of incubating murine lymphocytes with cis-unsaturated fatty acids on expression and capping of CD44 and CD45. Lymphocytes were incubated with stearic (18:0) or oleic (18:1 omega-9) acid bound to bovine serum albumin (BSA). After incubation with rat anti-CD44 or anti-CD45 monoclonal antibodies and then with fluorescent-labeled anti-rat antibody, mean fluorescence intensity (FI) was measured by using flow cytometry. Capping was measured after warning and fixation in paraformaldehyde. Steady-state fluorescence anisotropy (rs) was measured after the cells had been incubated with trimethylammoniumdiphenylhexatriene. Incubation with oleic acid, but not stearic acid or BSA alone, was associated with an increase in FI of CD44. Expression of CD45, however, was increased by both stearic and oleic acids to the same degree over BSA controls. CD44 and CD45 capping were both increased by incubation with oleic acid. Rs was decreased in cells incubated with oleic acid, suggesting an increase in membrane fluidity. We conclude that incubation with oleic acid increases expression of CD44 and increases capping of both CD44 and CD45. These findings were confirmed in feeding experiments, in which rs was reduced and CD44 capping increased by polyunsaturated fatty acid diets.

Abstract INTRODUCTION: Excessive bleeding is a life-threatening challenge in many areas of medical practice, especially in surgical procedures and trauma care. Few treatment options are available to meet the challenge of preventing or treating excessive bleeding, and none of them is satisfactory. The efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models, as summarized in a recent publication by Jy et al. [Thromb. &amp; Haemost., 2013;110:751-60]. No adverse effects were noticed in short-term observation of either model with the effective dose used. The rate of clearance of RMP in vivo has not been analyzed systemically. The purpose of this study is to characterize the pharmacokinetics / rate of clearance of RMP in rabbit model by different infusion regimens and to establish the relationship between blood concentration and hemostatic efficacy of RMP. METHODS: (i) RMP were produced by high pressure extrusion method [Thromb. &amp; Haemost., 2013; 110:751-60]. The resulting product was washed twice with isotonic saline, lyophilized, and stored at -80°C. (ii) Pharmacokinetics of RMP were measured using either bolus infusion of RMP (3x109 counts/kg) during 1 min., or a combination of bolus (1/3 of total RMP) followed by continuous infusion (2/3 of total RMP) for 30 min to the sedated non-bleeding rabbits. Blood samples (1 mL each) were collected at intervals: 5 min pre-injection, and at 1, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 5 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with dual labeling: anti-CD235a-PE and Annexin V-FITC. The former is specific for human RMP and will not label rabbit RMP. The latter is not species sensitive and labels both human and rabbit MP. (iii)The procoagulant activity of RMP in rabbit blood was assayed by thromboelastogram (TEG). RESULTS:(1) BolusInfusion of RMP (3 x109 counts/kg) resulted in a rapid rise of RMP levels peaking at 1 min post-infusion followed by rapid decline to baseline by 10 – 15 min. The half-life (T1/2) in circulation was estimated to be ≈ 4 – 7 min. The peak RMP concentration reached 2.5 – 3.4 x107 counts/mL. (2) The 2 markers used yielded small but significantly different rates of clearance after reaching peak concentrations: the T1/2 by anti-CD235a was 6.2 ±1.0 min, and by annexin V was 4.4 ±0.7 min (p = 0.01). These results indicate that these two phenotypes of RMP were cleared by different mechanisms. RMP expressing phosphatidylserine (annexin V positive) seem to be cleared faster than those expressing CD235a. (3) Bolus followed by continuous infusion resulted in a smaller initial spike (0.7 – 1.0 x107 counts/mL) followed by a rapid decline to ≈25-30%, then steady rise over the course of 30 min. infusion, finally reaching a steady-state level of 0.4 -0.6 x107 counts/mL. RMP levels returned to baseline within 15 min after cessation of infusion. (4) The ex vivo TEG data revealed good correlation between rise and fall of circulating RMP and procoagulant activity. However, T1/2 for procoagulant activity was longer (7.4 ±1.2 min.) compared to T1/2of circulating RMP, whether measured by anti-CD235a or annexin V. On the other hand, bolus followed by continuous infusion resulted in steady elevation of procoagulant activity, with little fluctuation during the course of infusion. CONCLUSIONS: These data demonstrate that bolus followed by continuous infusion of RMP is capable of maintaining almost steady-state levels for extended periods, with concomitantly increased procoagulant activity. Accordingly, this regimen is expected to be the optimum clinical treatment for excessive bleeding. This work also demonstrates the existence of different rate of clearance for different phenotypes of RMP, suggesting that multiple mechanisms are involved in the clearance of RMP. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 2277 Introduction: Thromboelastography (TEG) reports several viscoelatstic changes during clot formation, giving useful information on multiple parameters, and can give information on effects of therapeutic measures. We previously demonstrated that RMP can improve clotting parameters in selected bleeding disorders, judging by TEG. In this report we characterize the abnormalities seen in TEG in several disease states, and how RMP can modify or correct the abnormal parameters. Methods: (i) The following patient groups were studied by TEG: Patients with ITP (n=10), other non immune TP (n=10), thrombocytopenia from aplastic anemia (AA, n=3), antiphospholipid syndrome (APS, n=10), systemic lupus erythmatosus (SLE, n=8), hemophilia (n=3), hemolytic anemia (HA, n=4) and healthy controls (n=84). (ii) RMP were produced from washed, packed RBC by high-pressure extrusion and were added at 1×10E8/mL f.c. to the test samples. (iii) TEG was performed on whole citrated blood, each in presence and absence (+/−) of added RMP. Parameters measured were R (lag time), k (clot formation time), A, angle (initial rate of clot growth), MA (maximum amplitude, or clot strength), G (shear elastic modulus, another measure of clot strength) and CI (coagulation index), a composite measure. Results: (1) Baseline findings (absence of RMP). All disease groups except HA had longer R (lag) time than controls (p≤0.03), but only AA, ITP, and SLE had longer k times (p≤0.03). HA had a significantly shorter k time and higher angle A (initial rate) compared to controls, both at p&lt;0.01. Angle A was lower in Hemophilia, TP, ITP, AA and SLE (all p≤0.05 or better). APS did not differ from controls. Finally, MA (amplitude) and G (elastic modulus) were lower in TP, ITP, and AA than controls (p≤0.04), while the other diseases did not differ from controls in this measure. (2) Effect of RMP. The R and k parameters of all disease groups except HA were significantly shortened upon addition of RMP (p≤0.05 or better). RMP also shortened R and k of healthy controls (p&lt;0.01, n=84). TP, ITP, AA, hemophilia APS, and controls also had shorter R+k time, and steeper angle A (p≤0.05 or better for all measures). HA had a shorter R+k time (p&lt;0.05) but angle A remained unaffected. Additionally, modest increases in MA and G were seen in hemophilia and APS in the presence of RMP (p&lt;0.04 or better). (3) Global measures. The CI (coagulation index) was significantly improved (p&lt;0.05 or better) by addition of RMP in TP, ITP, APS, and SLE, indicating a better global coagulation profile in those diseases. (4) Combined groups. When all of the hematologic disorders were combined, significant differences in R, k, R+k, A, and G were observed in the presence of RMP (p≤0.02), but no difference in MA was seen, although it was very close to significance (p=0.051). CI was also markedly improved by RMP (p=0.02). (5) General observation. It is important to add that in nearly all groups, some patients responded strongly to RMP but others weakly or not at all, indicating different status of the disorder, possibly reflecting a state of platelet activation, inflammation, or disease activity. Conclusions/Discussion: RMP appear to have broad efficacy in correcting abnormal coagulation profiles in several disorders with differing pathologies. The parameter most strongly affected was time for initial fibrin formation, R. Effect on clot strength (G, MA) was statistically significant but varied among individual patients. RMP could correct abnormal clotting parameters in blood from differing pathologies, although there were individual variations. These data support the hypothesis that RMP can serve as a useful hemostatic agent to reduce bleeding in a variety of conditions of differing etiology. However, the factors responsible for such variations remain unknown. Elucidation of underlying factors influencing the effect of RMP will further improve and refine its therapeutic benefit and indications of its application. Disclosures: No relevant conflicts of interest to declare.

HomeStrokeVol. 54, No. 4Red Blood Cell Microparticles Limit Hemorrhage Following Intracerebral Hemorrhage in Spontaneously Hypertensive Rats Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessResearch ArticlePDF/EPUBRed Blood Cell Microparticles Limit Hemorrhage Following Intracerebral Hemorrhage in Spontaneously Hypertensive Rats Ashish K. Rehni, Sunjoo Cho, Hever Navarro Quero, Zhexuan Zhang, Chuanhui Dong, Weizhao Zhao, Miguel A. Perez-Pinzon, Sebastian Koch, Wenche Jy and Kunjan R. Dave Ashish K. RehniAshish K. Rehni https://orcid.org/0000-0002-9107-3277 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. *A.K. Rehni and S. Cho contributed equally. Search for more papers by this author , Sunjoo ChoSunjoo Cho Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. *A.K. Rehni and S. Cho contributed equally. Search for more papers by this author , Hever Navarro QueroHever Navarro Quero Department of Medicine, University of Miami Miller School of Medicine (H.N.Q., W.J.), University of Miami, Coral Gables, FL. Search for more papers by this author , Zhexuan ZhangZhexuan Zhang https://orcid.org/0000-0002-8149-9186 Department of Biomedical Engineering (Z.Z., W.Z.), University of Miami, Coral Gables, FL. Search for more papers by this author , Chuanhui DongChuanhui Dong Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. Search for more papers by this author , Weizhao ZhaoWeizhao Zhao https://orcid.org/0000-0002-9890-5785 Department of Biomedical Engineering (Z.Z., W.Z.), University of Miami, Coral Gables, FL. Search for more papers by this author , Miguel A. Perez-PinzonMiguel A. Perez-Pinzon https://orcid.org/0000-0001-6555-8935 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. Neuroscience Program (M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Search for more papers by this author , Sebastian KochSebastian Koch https://orcid.org/0000-0003-0244-6033 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Search for more papers by this author , Wenche JyWenche Jy Department of Medicine, University of Miami Miller School of Medicine (H.N.Q., W.J.), University of Miami, Coral Gables, FL. Search for more papers by this author and Kunjan R. DaveKunjan R. Dave Correspondence to: Kunjan R. Dave, PhD, Department of Neurology, University of Miami Miller School of Medicine, 1600 NW 10th Ave, RMSB No. 7046, Miami, FL 33136. Email E-mail Address: [email protected] https://orcid.org/0000-0002-0173-5338 Peritz Scheinberg Cerebral Vascular Disease Research Laboratories (A.K.R., S.C., M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Department of Neurology (A.K.R., S.C., C.D., M.A.P.-P., S.K., K.R.D.), University of Miami, Coral Gables, FL. Neuroscience Program (M.A.P.-P., K.R.D.), University of Miami, Coral Gables, FL. Search for more papers by this author Originally published2 Mar 2023https://doi.org/10.1161/STROKEAHA.122.042152Stroke. 2023;54:e152–e154Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 2, 2023: Ahead of Print Hematoma expands over time in spontaneous intracerebral hemorrhage (sICH) and correlates with post-sICH neurological impairment.1 Hypertension is a major risk factor for sICH, resulting in increased hematoma volume, worse outcomes, and increased mortality following sICH.2 We previously demonstrated that red blood cell–derived microparticles (RMPs) enhance both primary and secondary hemostasis and limit hematoma expansion in naive rats following collagenase-induced sICH.3,4 Considering the importance of validating the efficacy of new drugs in animals suffering from risk factors or comorbidities to improve the translational value of preclinical studies, we evaluated RMP efficacy in limiting hematoma growth and related neurological deficits following sICH in male SHR (spontaneously hypertensive) rats, compared with their respective control, male WKY (Wistar-Kyoto) rats. Experimental details are provided in the Supplemental Material.Animal experiments were performed as per the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Miami Institutional Animal Care and Use Committee. Physiological parameters (Table S1) and blood pressure (Figure S1) were all within normal ranges. The hematoma volume in RMP-treated WKY rats was significantly lower (87±9 mm3; n=10; P<0.001) than in vehicle-treated WKY rats (141±10; n=10; Figure [B]). The hematoma volume in RMP-treated SHR rats was also significantly lower (85±9 mm3; n=10; P<0.01) than in vehicle-treated SHR rats (130±10; n=10; Figure [B]). The hematoma frequency maps showed that the hematoma volumes in RMP-treated WKY and SHR rat groups were smaller at multiple coronal levels when compared with their respective vehicle group (Figure [D]). The neurological scores in RMP-treated WKY and SHR rats were significantly lower than in their respective vehicle-treated rats (Figure [C]). These results demonstrate that RMPs can limit hematoma growth and lower neurological deficits following post-collagenase injection in SHR rats.Download figureDownload PowerPointFigure. The effect of red blood cell-derived microparticle (RMP) treatment on hematoma expansion postcollagenase-induced intracerebral hemorrhage in spontaneously hypertensive (SHR) rats. Experimental design (A); hematoma volume (B); neurological score determined 24 hours post-spontaneous intracerebral hemorrhage (C); and hematoma frequency maps at 7 coronal levels (D). BP indicates blood pressure; and WKY, Wistar-Kyoto. ⁂P<0.001 vs vehicle-treated WKY rats. ††P<0.01 vs vehicle-treated SHR rats.No significant difference in the hematoma volume was observed between vehicle-treated SHR rats and normotensive vehicle-treated WKY rats and between RMP-treated SHR rats and WKY rats (Supplemental Results). These results are in line with a previous study that did not find differences in hematoma size between WKY and SHR rats.5 We previously observed that RMPs exert a substantial ameliorative effect on hematoma growth and neurological impairment in naive rats 24 hours after sICH induction and RMP treatment resulted in significantly improved long-term behavioral and histological outcomes.4 Independent replication and evaluating the effects in female and aged animals and in a different species remain to be addressed. We conclude that RMP treatment is able to limit hematoma growth and attenuate neurological impairment post-sICH in an animal model of hypertension—a prominent risk factor for sICH.Data AvailabilityAll data generated or analyzed during this study are included in this article and the Supplemental Material.Article InformationAcknowledgmentsWe thank Dr Brant Watson for critical reading of this article.Sources of FundingThis work was supported by the National Institutes of Health (NS094896) and the James and Esther King Biomedical Research Program (9JK08). The funding agencies were not involved in the collection, analysis, and interpretation of data; in writing of the report; or in the decision to submit the manuscript for publication.Supplemental MaterialSupplemental Materials and MethodsSupplemental ResultsTable S1Figure S1Nonstandard Abbreviations and AcronymsRMPred blood cell–derived microparticleSHRspontaneously hypertensivesICHspontaneous intracerebral hemorrhageWKYWistar-KyotoDisclosures RxMP Therapeutics provided the testing material. Dr Jy and the University of Miami have partial ownership in RxMP Therapeutics. Dr Jy received grant support for nonrelated work from RxMP Therapeutics and is the inventor of US patents related to red cell microparticles and serves as a scientific advisor/consultant to RxMP Therapeutics.Footnotes*A.K. Rehni and S. Cho contributed equally.This manuscript was sent to Jean-Claude Baron, Guest Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page e154.Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.122.042152.Correspondence to: Kunjan R. Dave, PhD, Department of Neurology, University of Miami Miller School of Medicine, 1600 NW 10th Ave, RMSB No. 7046, Miami, FL 33136. Email kdave@med.miami.eduReferences1. Lord AS, Gilmore E, Choi HA, Mayer SA; VISTA-ICH Collaboration. Time course and predictors of neurological deterioration after intracerebral hemorrhage.Stroke. 2015; 46:647–65210.1161/STROKEAHA.114.007704LinkGoogle Scholar2. Francoeur CL, Mayer SA; VISTA-ICH Collaborators. Acute blood pressure and outcome after intracerebral hemorrhage: the VISTA-ICH cohort.J Stroke Cerebrovasc Dis. 2021; 30:105456. 10.1016/j.jstrokecerebrovasdis.2020.105456CrossrefGoogle Scholar3. Jy W, Johansen ME, Bidot C, Horstman LL, Ahn YS. Red cell-derived microparticles (rmp) as haemostatic agent.Thromb Haemost. 2013; 110:751–760. doi: 10.1160/TH12-12-0941CrossrefGoogle Scholar4. Rehni AK, Cho S, Quero HN, Shukla V, Zhang Z, Dong C, Zhao W, Perez-Pinzon MA, Koch S, Jy W, et al. Red blood cell microparticles limit hematoma growth in intracerebral hemorrhage.Stroke. 2022; 53:3182–3191. doi: 10.1161/STROKEAHA.122.039641LinkGoogle Scholar5. Wu G, Bao X, Xi G, Keep RF, Thompson BG, Hua Y. Brain injury after intracerebral hemorrhage in spontaneously hypertensive rats.J Neurosurg. 2011; 114:1805–1811. doi: 10.3171/2011.1.JNS101530CrossrefGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetails April 2023Vol 54, Issue 4 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/STROKEAHA.122.042152PMID: 36861474 Originally publishedMarch 2, 2023 Keywordsrats, inbred SHRcell-derived microparticlesratsrats, inbred WKYcerebral hemorrhagePDF download Advertisement SubjectsIntracranial Hemorrhage

Abstract The cytoplasmic regions of the CD3 complex are presumably involved in signal transduction following ligand—receptor binding. We investigated the effects of incubating either stearic or oleic acid on the association of murine lymphocyte CD3 complex with the cytoskeleton. Both cytochalasin D, an inhibitor of microfilament formation, and W7, an inhibitor of calmodulin, inhibited capping of CD3. The association of CD3 with the cytoskeleton was confirmed by confocal laser scanning microscopy studies, which showed co‐localization of the cross‐linked CD3 receptors and the membrane attachment proteins ankyrin and fodrin. Although exogenous oleic acid increased plasma membrane fluidity, neither expression nor capping of CD3 receptors was increased. Nonetheless, oleic acid did increase uptake of tritiated thymidine after binding of anti‐CD3 antibodies. Lymphoproliferation was progressively inhibited by both cytochalasin D and W7, confirming the importance of intact cytoskeleton for cellular activation.

Abstract BACKGROUND: Cell-derived microparticles (C-MP) are microvesicles released during activation and apoptosis from platelets (PMP), leukocytes (LMP), endothelial cells (EMP) and red cells (RMP). They commonly express phosphatidylserine (PS) judged by binding annexin V (AnV) and carry markers of parent cells. C-MP are involved in thrombosis, inflammation and angiogenesis. Little is known how C-MP are cleared from circulation. To test that the spleen might be involved, we investigated C-MP profiles in patients who underwent splenectomy for various causes (group A), those with hypersplenism (group B), and healthy controls with spleen (group C). MATERIAL AND METHODS: Group A (Gr A) consisted of 44 patients (18 M and 26 F, mean age 57.2 yr) with splenectomy; group B (Gr B) consisted of 25 patients (12 M and 13 F, mean age 61.6 yr) with hypersplenism. Gr A had 25 ITP, 7 myeloproliferative disorders, 4 lymphoma, 3 hemolytic anemias, 4 trauma related splenectomy, 1 unknown reason. Group B had 19 chronic hepatitis C, 2 alcoholic, 2 idiopathic and 1 autoimmune hepatitis, 1 portal vein thrombosis. Group C (Gr C) were healthy controls with spleen (n=109). Flow cytometry was used to assay C-MP. PMP were identified by CD41+, LMP by CD45+, EMP by CD31+/CD41−, and RMP by glycophorin+. Profile of C-MP and blood counts were compared among the 3 groups. RESULTS: Table 1 summarizes C-MP data among the 3 groups. Gr A and B were well matched for ages and sex. Mean values of WBC and Hgb were normal in all groups while the platelet count was low in Gr B. Mean values of LMP, EMP and RMP were significantly higher in Gr A compared to Gr B and C, but PMP were not. All species of C-MP (PMP, LMP, EMP, RMP) in Gr B were significantly lower than Gr C (healthy control) and were markedly depressed compared to Gr A (see Table 1). Correlation analysis revealed that PMP correlated with platelet count (p&lt;0.0001) but no correlation between LMP and WBC was seen, or between RMP and Hgb. Accordingly, high LMP and RMP in Gr A cannot explain the relatively high Hgb and WBC levels in this group. DISCUSSION/COMCLUSIONS: Our data suggest that spleen sequesters and/or clears C-MP of all types (PMP, LMP, EMP, RMP). C-MP express PS (bind AnV), which is known to promote clearance from circulation. Splenectomy is known to be associated with thrombosis but its mechanism in not well elucidated. Elevated C-MP in splenectomized patients may contribute to their increased risk of thrombosis. Table 1 Gr A (Splntomy) Gr B (Hyperspln) Gr C (Control) p value (A-C) p value (B-C) Patient no. 44 25 109 PMP 13858 7073 15992 n. s. &lt;0.0001 LMP 2043 1180 1385 &lt;0.0001 &lt;0.001 EMP 857 238 436 &lt;0.001 &lt;0.0001 RMP 2964 926 1138 &lt;0.0001 &lt;0.005

Abstract BACKGROUND . EMP have emerged as sensitive biomarkers of endothelial dysfunction. Little is known of their function. EMP interact with leukocytes and platelets and exert prothrombotic and proinflammatory effects. Recently we reported that vWf is expressed in EMP. The present study extends this observation to characterize EMP-bound vWf (EMP/vWf), specifically their multimer size and proaggregatory activity in ristocetin-induced platelet aggregation. METHODS . EMP, released from human renal microvascular endothelial cells 24hr following stimulation with TNF-α, were isolated by centrifugation (10,000g x 30min) and washed twice for vWf multimer analysis and proaggregatory activity assay. vWf multimer size was analyzed by 0.8% argarose gel electrophoresis and Western blotting. The proaggregatory activity of vWf was assessed flow ctyometrically by a two-step method: first, washed platelets were incubated with either EMP or soluble vWf such as plasma or Humate P (a commercial vWf multimers) in the presence of ristocetin for 10 min, then disappearance of free platelets as they formed small aggregates was measured. Second, the formed platelet aggregates were diluted 20-fold with buffer and degree of disaggregation was determined at intervals to assess the stability of PltAg. RESULTS . (i) Electrophoresis showed that vWf bands from normal plasma do not exceed 1,000 kDa and Humate P about 2,000 kDa while vWf bands on EMP extends much higher than 2,000 kDa. The protein banding pattern of EMP/vWf also differed from free soluble vWf (plasma) or humate P, the latter both showing clear ladder-like banding while EMP/vWf often appeared as a continuous smear of sizes in the high molecular weight regions, probably due to covalent bonds to transmembrane structures. (ii) Incubation of EMP (1 x 107 in 100 μL) with humate P (0.3 U) for 2hr (rotary shaker, 100 rpm) followed by washing the EMP revealed that significant additional vWf was adsorbed to the EMP: the vWf in the supernatant was decreased by 30% – 40%, and Western blotting showed additional high MW band on the treated EMP. This demonstrates that EMP express unoccupied sites capable of binding free vWf. (iii) Incubating EMP/vWf with washed platelets in the presence of ristocetin resulted in formation of platelet aggregates (PltAg). When PltAg were diluted 1:20, the PltAg began dissociating into free platelets in a time-dependent manner. The time for 50% dissociation of PltAg was 15–20min with soluble vWf (plasma or Humate P), but was prolonged to 45–60min with EMP/vWf. We attribute this mainly to the presence of ULvWf on EMP. (iv) Plasma from 4 acute TTP patients showed elevated EMP and enhanced formation of PltAg resistant to disaggregate. While 5 vWD plasma showed the opposite effect and their deficiency was corrected by added EMP. CONCLUSIONS . A new role of EMP is indicated in the present study. EMP-bound vWf is functionally active, inducing platelet aggregates of greater stability than free vWf in a ristocetin-based assay. EMP are capable of adsorbing additional vWf from plasma, indicating presence of extra binding sites. EMP enhance the stability of aggregates probably through ULvWf on the EMP, or to additional platelet-EMP-platelet adhesion molecules, or both. Through their high-affinity interaction with ULvWf, EMP likely play a role in vWf-dependent thrombotic disorders such as TTP and other platelet mediated thrombosis.

Abstract The use of new generation sequencing technology has led to the recent identification of disease-causing mutations in the RASGRP2gene that encodes CalDAG-GEFI, a small GTPase-activating nucleotide exchange factor essential for integrin activation in platelets and megakaryocytes. Abrogation of CalDAG-GEFI function leads to a Glanzmann thrombasthenia (GT)-like platelet defect with a major loss in the platelet aggregation response. It is important to know the clinical profiles of patients with CalDAG-GEFI deficiency. Here we present variable phenotypes encountered in a large family with Jamaican ancestry associated with a truncating CalDAG-GEFI mutation. The initial propositus (case 1) was a 51 yr-old Jamaican man with a GT-like platelet function phenotype and absent platelet aggregation to a range of physiologic agonists (including ADP and collagen) but a normal response to ristocetin. His platelets contained a full complement of the αIIbβ3 integrin and Sanger sequencing failed to reveal any mutations in the ITGA2B and ITGB3genes. His sister presented a similar profile and flow cytometry showed that platelets of both patients bound 5-30% of the normal levels of PAC-1 after platelet stimulation with ADP thereby confirming a much decreased αIIbβ3 function. Yet western blotting confirmed normal levels of αIIb and β3 in their platelets that also contained an abundant storage pool of fibrinogen (Fg). Clot retraction occurred but was reduced and platelets bound to a Fg-covered surface (result provided by the late Professor JG White, Minneapolis, MN). DNA from case 1 was analyzed by whole exome sequencing within the BRIDGE - Bleeding and Platelet Disorders consortium (Cambridge, UK) and a homozygous c.1490delT predicted to give rise to a p.F497fs*22 truncating mutation near to the C-terminal domain of CalDAG-GEFI was highlighted in Bordeaux. Consanguinity is present within the family. Sanger sequencing performed in Marseille showed that case 1 and his sister were homozygous for the c.1490delT and that 3 (mother, aunt and niece) out of 4 close family members were heterozygous for the mutation. Transfection of human embryonic kidney (HEK) cells with the mutated CalDAG-GEFI confirmed the expression of the truncated protein. The propositus and his sister have experienced spontaneous bleeding and both have had excessive bleeding after surgery. In particular, the sister had episodes of severe epistaxis and heavy menstruation requiring frequent hospitalizations as a child with no benefit from platelet transfusions; she underwent hysterectomy and cholecystectomy under platelet transfusions and rFVIIa infusion. The propositus had episodes of gingival bleeding as a child and needed hospitalization for a tooth extraction. In 2006 he presented with headaches and was found to have a meningioma that was removed surgically (6 hours) under the cover first of platelet transfusions that again failed to prevent bleeding. This led to the use of rFVIIa that reduced bleeding despite a transient hypoxemia that led to a reduced dose and the adjunction of aminocaproic acid. The patient made a full recovery. Of interest, he has previously reported problems with wound repair namely abnormal scarring with excessive keloid. Also of note is the occurrence of ITP within 2 family members (aunt and her son who is otherwise asymptomatic) and his father died from bleeding complications following a major accident. We recommend the assembly of as much data as possible on the range of phenotypes and treatment of bleeding in families with inherited RASGRP2 defects. Disclosures No relevant conflicts of interest to declare.

Dietary lipids enhance immune function and improve outcome from injury or infection in animal models. We tested the hypothesis that amount, type, or both, of dietary lipid increases intracellular calcium concentration, a surrogate for lymphocyte activation.Mice were fed 2 weeks on semipurified diets with 5% (by weight [w/w]), 10% (w/w), or 20% (w/w) dietary fat consisting of coconut, olive, safflower, or linseed oil. Changes in intracellular calcium concentration after mitogen stimulation of splenic lymphocytes was estimated by using flow cytometry.Olive oil diets increase intracellular calcium concentration after concanavalin A, lipopolysaccharide, and CD3 stimulation. On the other hand, linseed oil (which is high in omega-3 fatty acids, which have been shown in other studies to enhance immune function) depresses intracellular calcium levels. The amount of dietary fat had no effect on intracellular calcium.Olive oil merits further study in the application of nutritional pharmacology to immunomodulation of the critically injured, because it may enhance lymphocyte function.