Tissue Factor-Negative Cell-Derived Microparticles Play a Distinctive Role in Hemostasis: A Viewpoint Review.

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.

Microparticles in stored red blood cells as potential mediators of transfusion complications.

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.

Role of platelets in neuroinflammation: a wide-angle perspective.

OBJECTIVES: 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. ORGANIZATION: 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. CONCLUSION: 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.

Microparticle size and its relation to composition, functional activity, and clinical significance.

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.

Antiphospholipid antibodies: paradigm in transition.

OBJECTIVES: 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. ORGANIZATION: 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. CONCLUSION: 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.

Potential roles of cell-derived microparticles in ischemic brain disease.

PURPOSE: The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases. MATERIALS AND METHODS: 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. DISCUSSION: 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. CONCLUSION: If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

Preclinical Evaluation of Safety and Biodistribution of Red Cell Microparticles: A Novel Hemostatic Agent.

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.

Evidence of platelet activation in multiple sclerosis.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Interleukin-11 for treatment of hepatitis C-associated ITP.

BACKGROUND: Immune thrombocytopenic purpura (ITP) is frequently associated with chronic hepatitis C (HpC-ITP). METHODS: 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 microg/kg daily) while group 2 (5 patients) at low dose (15-35 microg/kg three/week). RESULTS: In group 1, mean platelet counts rose from initial 54 x 10(9)/l to 103 x 10(9)/l (p = 0.02) and in group 2, from an initial 51 x 10(9)/l to 74 x 10(9)/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 >2 years with low-dose maintenance. CONCLUSION: When used based on individual tolerance, rIL-11 appears useful in HpC-ITP.

Pharmacokinetics of Human Red Blood Cell Microparticles Prepared Using High-Pressure Extrusion Method.

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.

Platelet activation rather than endothelial injury identifies risk of thrombosis in subjects positive for antiphospholipid antibodies.

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-beta2GPI (abeta2GPI) 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 abeta2GPI 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 abeta2GPI, 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.

Clinical and neuroimaging correlates of antiphospholipid antibodies in multiple sclerosis: a preliminary study.

BACKGROUND: 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. METHODS: 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). RESULTS: 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

Phenotype analysis and clinical management in a large family with a novel truncating mutation in RASGRP2, the CalDAG-GEFI encoding gene.

BACKGROUND: Genetic 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 αIIbß3 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. OBJECTIVES: To better understand the phenotypical and clinical profiles of patients with CalDAG-GEFI deficiency. PATIENTS: We report a five-generation family with a novel truncating CalDAG-GEFI mutation detailing clinical management and phenotypic variability. RESULTS: Patients 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 ß3 in their platelets but decreased αIIbß3 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. CONCLUSIONS: A long-term prospective study is warranted for full clinical exploration of CalDAG-GEFI to understand the bleeding phenotyes and their management.

Elevation of endothelial microparticles, platelets, and leukocyte activation in patients with venous thromboembolism.

OBJECTIVES: 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). BACKGROUND: The details of interactions between endothelium, platelets, and leukocytes in VTE are not well understood. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Endothelial microparticles and platelet and leukocyte activation in patients with the metabolic syndrome.

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.

Postprandial hypertriglyceridemia increases circulating levels of endothelial cell microparticles.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Correlation between apoptotic endothelial microparticles and serum interleukin-6 and C-reactive protein in healthy men.

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.

Digoxin use is associated with increased platelet and endothelial cell activation in patients with nonvalvular atrial fibrillation.

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, beta-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.

Life-threatening hypercoagulable state following splenectomy in ITP: successful management with aggressive antithrombotic therapy and danazol.

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.