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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.

Objectives 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. Methods 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. Results 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). Conclusions Presurgical levels of CD41 + PMPs and CD235a + RMPs are the main risk factors for transfusion in CABG, followed by HGB and aPTT.

The roles of cell-derived microparticles (MP) in neuroinflammatory disorders are increasingly recognized as fundamental. Exosomes are now established as a well-defined subclass of MP. A major function is transport of many inflammatory mediators, now including gene-modulating RNA transcripts. Topics covered include traumatic brain injury, stroke, vascular dementia, infectious viral diseases, and the neurodegenerative/protein-misfolding diseases (ALS, MS, PD, etc.). It concludes with review of MP-based therapeutic strategies, including use of MP as vectors of gene therapy (via siRNA) and for drug delivery across the blood-brain barrier. Results to date are highly promising. It is expected that in the near future, this field will lead to improved understanding of neuroinflammatory conditions as well as more effective treatments for those condition.

BACKGROUND: It has been shown that platelet microparticles (PMP) exert pro-inflammatory activity. It has also been suggested that red cell MP (RMP) are similarly pro-inflammatory, but the evidence is less clear. This issue has clinical significance for several reasons. For example, there is evidence that both PMP and RMP have hemostatic activity [Horstman et al, Semin Thromb Hemost, 2019] and that infusion of RMP is a viable strategy for control of excessive bleeding [Jy et al,Thromb Haemost, 2013]. For these reasons we undertook assay of inflammatory potential of PMP and RMP by the method of CD11b expression on neutrophils, using N-Formylmethionyl-leucyl-phenylalanine (fMLP) as a positive control. METHODS: RMP were isolated from leukoreduced, type O+ packed RBCs obtained from three different donors at storage time points of 3 and 6 weeks (RMPStor). In addition, RMP were prepared by high-pressure extrusion from type O+ packed RBCs stored for less than one week (RMPxt). Platelets were isolated from healthy volunteers and sonicated to produce PMP. MP concentrations were determined using flow cytometry. RMP were identified by PE-labeled anti-glycophorin and PMP were identified by FITC-labeled anti-CD41. Each MP species was added to separate 500 µL aliquots of fresh whole blood collected from healthy volunteers for equal final concentrations of 20,000/µL, 40,000/µL, 80,000/µL, and 200,000/µL. To one of each pair, fMLP was added for a final concentration of 1 µM. Fresh, whole blood treated with sterile saline was used as a negative control. A positive control was obtained by adding fMLP to whole blood without MP treatment. The blood samples were then incubated for one hour under gentle shaking. Two-color flow cytometry with PE-Cy5-anti-CD11b and FITC-anti-CD45 MAbs were employed to measure the ratio of CD11b to CD45 expression on neutrophils (identified through FS, SS, and CD45+ FL gating). The differences in CD11b expression were compared using the paired t test. The mean values presented below were determined using the results from at least 5 replicate experiments in which the two groups were directly assayed against one another using neutrophils from a single donor, resulting in slightly different mean values for each group depending on the comparison being analyzed. RESULTS: For all RMP species, there was no significant difference between concentration and their effect on neutrophil CD11b expression. In contrast, the concentration of PMP positively correlated with an increase in CD11b expression (R2=0.78). At a MP concentration of 200,000/µL, there was no statistical difference when comparing neutrophil CD11b expression in the negative control vs. incubation with RMPxt (27.5 vs. 27, p=0.7), or the negative control vs. incubation with RMPStor Wk 6 (29.0 vs. 28.4, p=0.6). However, neutrophil CD11b expression was lower when incubated with RMPStor Wk 3 compared to the negative control (25.1 vs. 28.8, p CONCLUSION: In sum, PMP exhibited robust pro-inflammatory activity, but RMP did not. On the contrary, RMP exhibit modest but significant anti-inflammatory activity in this assay method. It remains necessary to elucidate the mechanism and to assess if these findings apply in vivo. Disclosures Jy: RxMP Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: US patents No. 8,150,632 and 9,155,764, Research Funding. Horstman:RxMP Therapeutics: Patents & Royalties: US patents No. 8,150,632 and 9,155,764. Ahn:RxMP Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: US patents No. 8,150,632 and 9,155,764, Research Funding.

BACKGROUND: Immune Thrombocytopenic Purpura (ITP) is an autoimmune disorder in which the production of antiplatelet antibodies stimulates increased clearance of platelets by macrophages of the mononuclear phagocytic system, resulting in thrombocytopenia and bleeding diathesis. In contrast, Thrombotic Thrombocytopenic Purpura (TTP) is a disorder in which platelets are sequestered in microcirculation to form platelet-rich microthrombi in response to endothelial injury. This leads to thrombocytopenia, red cell fragmentation with hemolytic anemia, systemic microangiopathy, and multiorgan failure. Cell-derived microparticles (C-MP) are microvesicles released in cell activation or apoptosis. C-MP from endothelial cells (EMP), platelets (PMP), leukocytes (LMP), and erythrocytes (RMP) are of particular interest because they have emerged as useful biomarkers, reporting early stages of cellular injury prior to clinical manifestations of underlying cardiovascular, thrombotic, and inflammatory disorders. We investigated C-MP profiles in both ITP and TTP patients with the aim of gaining greater insight into the pathophysiology of these two distinct thrombocytopenic disorders. METHODS: A retrospective study was conducted comparing C-MP data of 29 ITP patients with those of 20 TTP patients. Both groups were stratified into active vs. remission phases. The ITP cohort consisted of 18 patients in active phase (7M/11F, mean age 49.3 yr, mean platelet count 48 x 103/µL) and 11 in remission (2M/9F, mean age 51.9 yr, mean platelet count 221 x 103/µL). The TTP cohort consisted of 8 patients in active phase (1M/7F, mean age 43.1 yr, mean platelet count 54 x 103/µL) and 12 in remission (2M/10F, mean age 43.7 yr, mean platelet count 223 x 103/µL). For ITP, the active phase was defined by platelet counts 3 months and the remission phase by counts >150 x 103/µL >3 months. None were splenectomized. For TTP, the active phase was defined as thrombocytopenia, clinical evidence of microangiopathy, elevated lactate dehydrogenase (LDH) and low ADAMTS13 activity prior to initiation of plasmapheresis. The remission phase was defined as sustained normalization of the laboratory parameters in the absence of further microangiopathy for at least one month. Flow Cytometry was employed to identify LMP by anti-CD45, RMP by anti-glycophorin, PMP by CD42+/CD31-, and EMP by anti-CD62E. The differences in C-MP patterns between ITP and TTP were evaluated. All C-MP data are presented in units of x103/µL. Statistical analysis was performed using the unpaired t test. RESULTS: When C-MP were compared between active ITP and TTP, TTP had far higher EMP (37 vs. 540, p CONCLUSION/DISCUSSION: PMP were much higher in active ITP than TTP, presumably reflecting the antibody-mediated platelet fragmentation inherent to ITP. This is further corroborated by the higher LMP. Interestingly, PMP in remission ITP was higher than in active ITP, although the PMP/platelet ratio remained constant. In contrast, the PMP/platelet ratio in TTP decreased from the active to remission phase, despite the increase in PMP. This suggests that the immunologic milieu in ITP favors a consistent generation of PMP throughout the different disease phases. EMP and RMP were much higher in active TTP than ITP, reflecting the endothelial injury and red cell fragmentation with hemolysis present in TTP. In addition, the elevated RMP in active TTP may contribute to the formation of microthrombi due to their inherent procoagulant activity. This study demonstrates that C-MP profiles can provide valuable insight on immune-mediated disorders which may lead to improvements in their diagnosis and management. Disclosures Ahn: RxMP Therapeutics, Inc.: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

BACKGROUND: Cell derived MPs are small membrane vesicles released during cell activation or apoptosis. They express an inside-out membrane, which exposes the negatively charged phospholipid layer outside, allowing clotting factors to be anchored and generate thrombin. Among various species of MPs, RMP (red cell MPs), PMP (platelet MPs), LMP (leukocytes), EMP (endothelial cells) are of special interest. They play an important role in hemostasis, thromboses, and inflammation. MPs mirror early injury of parent cells and are sensitive early biomarkers of underlying disorders. TTP is a microangiopathy mediated by antibody-induced depletion of ADAMTS13, a von Willebrand factor-cleaving protease. Endothelial injury promotes platelet clumping and formation of platelet rich microthrombi in the microcirculation. Subsequent platelet sequestration leads to impaired microcirculation, thrombocytopenia and red cell fragmentation with a microangiopathic hemolytic anemia. Exchange plasmapheresis (EPP) is the standard therapy. It removes antibodies to ADAMTS13, replacing it with ADAMTS13 rich plasma. It is possible that EPP removes thrombogenic MPs to improve the clinical course of TTP. In this study, we investigated MP profiles in active and remission phase of TTP and the effect of EPP on MP profiles. We also aimed to determine if MP profiles may be a useful measure for monitoring clinical course and tracking progress of therapy. METHODS: A retrospective study was conducted evaluating MP assays in patients with TTP. MP profiles were reviewed in acute and remission phases of TTP. Acute phase was defined as thrombocytopenia, clinical evidence of microangiopathy and hemolytic anemia and low ADAMTS13 activity. Remission was defined as sustained normalization of laboratory parameters and no further microangiopathy for at least one month. Patients were studied longitudinally, with MP assays before and after EPP. EMP were measured by CD31+/CD42b− (EMP31), CD62E+ (EMP62); PMP by CD31+/CD42b+ (PMP42) and CD41+ (PMP41). All were measured in platelet-poor plasma by flow cytometry. All MP data are presented in units of x105/µL. The differences in MP patterns among TTP patients in active and remission phases of disease, as well as the effect of EPP on MP profiles were assessed. RESULTS: Among 20 patients with TTP, 8 (40%) were in acute phase and 12 (60%) in remission. An average of 10.7 EPP were performed. The average platelet count prior to EPP was 50.6x103/µL, which increased to 248 x103/µL following the last EPP. ADAMTS13 activity was generally For patients in the acute phase, PMPs were low: PMP41 0.32 (±0.11), PMP42 3.11 (±2.56), but consistently increased following EPP to the point of statistical significance at the last treatment: PMP41 1.26 (±0.71), p=0.005 and PMP42 7.65 (±5.03), p=0.039. Moreover, levels were higher in the remission phase for both PMPs, but only statistically significant for PMP41: 1.39, p=0.034. Conversely, EMP62E was initially elevated on presentation, but declined with successive EPP: 6.07 (±3.02) prior to initiation of EPP to 4.5 (±3.1) upon the last day of EPP. Furthermore, this pattern continued into remission, with EMP62E of 2.72 (±2.11), p=0.009. Similar to the trend in EMP62E, RMP was elevated in the acute phase (25.1 [±18.5]) before steadily declining with EPP (19.65 [±12.53]). This progressive drop in RMP persisted for those in remission phase of TTP 10 (±5.42), p=0.015. DISCUSSION/CONCLUSION: Taken collectively, cell derived MP's were found to reflect disease activity and response to therapy. There was a linear correlation between PMP levels and platelet count. Low PMPs in the acute phase reflects the initial thrombocytopenia characteristic of TTP. When EPP was initiated, the disease improved and there was a rise in PMP which mirrored the rise in platelets. This rise was sustained in the remission phase. Conversely, EMP62E and RMP are increased in acute phase TTP due to endothelial activation, microthrombi deposition and red cell fragmentation. After EPP the disease is quiescent, hence the progressive decline of EMP62E and RMP. These results show promising utility of cell derived MP profiles as a clinical tool to surveil chronic TTP patients and to predict disease relapse at an early stage. After EPP is initiated, change in MP Profiles may be used to monitor response to therapy and determine the appropriate time to wean EPP. Disclosures No relevant conflicts of interest to declare.

Secondary malignancy frequently develops among recipients of organ transplants, most commonly malignancies of the lymphoid system and skin. However, chronic myeloid leukemia (CML) is rare following transplant, with only a handful of cases reported, all of whom had kidney transplant and received azathioprine for immunosuppression. We report three cases of post-transplant CML seen at a single institution within a two-yr period. Two had received liver and one a kidney transplant. None were on azathioprine but all had tacrolimus. CML is a rare hematological malignancy, usually presenting with high white counts and splenomegaly. In all three of our subjects, presentation of CML post-transplant was so atypical that their diagnosis could easily be missed. All had rapid and excellent response to imatinib, and underwent clinical remission. This is the first report of CML developing in the course of tacrolimus therapy among liver transplant recipients. Presentations of CML were highly atypical and easy to miss in early stage. Awareness of atypical CML developing post-transplant is important since early and timely therapeutic intervention with imatinib is critical for improving quality of life and overall prognosis.

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.

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.

Endothelial microparticles (EMPs) are sub-microscopic membrane vesicles that are shed from the surface of endothelial cells during activation, injury and/or apoptosis. Endothelial cells release phenotypically and quantitatively distinct endothelial microparticles (EMPs) in activation and apoptosis. Therefore, the phenotypic assessment of EMPs can provide useful information reflecting the nature of endothelial injury. We tested the hypothesis that heart transplantation (HT) modifies the pattern of endothelial injury seen in patients with congestive heart failure (CHF).Flow cytometry was used to measure EMPs identified by E-selectin (CD62) and platelet-endothelial cell adhesion molecule 1 (CD31) in 23 patients with advanced heart failure and in 23 HT recipients. A cohort of 23 healthy individuals served as controls.Heart failure patients were found to have significantly higher levels of EMP62E (577 counts/microl) than controls (192 counts/microl) and post-transplant patients (152 counts/microl) (p0.0001). Levels of endothelial microparticles expressing CD31 were significantly different among study groups (analysis of variance [ANOVA], p = 0.001). Heart failure patients had significantly higher levels (1,526 counts/microl) than controls (395 counts/microl) (p0.01). Levels of EMP31 remained elevated after heart transplant (935 counts/microl) (p = non-significant). The EMP62/EMP31 ratio, an index of activation (high ratio) or apoptosis (low ratio), was significantly different between the groups (ANOVA, p = 0.01). Post-transplant patients had significantly lower ratios (0.16) than CHF patients (0.38) and controls (0.49).Cardiac transplantation is associated with a different pattern of endothelial cell injury than that seen in heart failure. The phenotypic assessment of EMPs in post-transplant patients is consistent with increased apoptotic activity.

Risk factors for thrombosis (TB) in thrombocythaemia (TC) associated with myeloproliferative disorder (MPD) are not well defined.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: beta2GP1, cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and FVII/VIIa, together with markers of activation of platelets (CD62P) and endothelium [endothelial microparticles (EMP)].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%, p0.05). Multiple APLA (positive for more than one antigen) were also more frequent in TB, for both IgG and IgM, for all 6 antigens tested (p0.05). 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 (p0.05). 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.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.

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 witheld 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.

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-b1b on release of EMP and on TEMIG of monocytes (mono) and monocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-b1b on the release of EMP and the TEMIG of mono and mono:EMP was assessed by preincubating BMVEC cultures of IFN-b1b 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-b1b 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-b1b inhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-β1b in MS.

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; p0.0001) 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; p0.0001).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.

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.

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.

Introduction: 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. Methods: 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). Results: 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-μm 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

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.

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.

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.

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-α 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. Results: 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. Conclusion: 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.

Our understanding of the pathophysiology of ITP owes to pioneering work of W J Harrington in 1951, delineating the immunologic nature of platelet destruction. In ITP, antibody-coated platelets are destroyed by macrophages of RES. However, other mechanisms are also implicated: C-mediated platelet lysis and newly described C-independent peroxide injury. Both induce platelet fragmentation and lysis, generating procoagulant platelet microparticles (PMP). A third mechanism of platelet consumption in the microvasculature is proposed, based on overlapping syndromes of ITP and TTP in some patients. In assessing hemostasis in ITP, platelet counts alone is not sufficient. Evaluation of platelet clumping, giant platelets, and platelet activation, marked by increased PMP is useful. Patients with platelet activation or giant platelets bleed less and detection of clumping prevents unwarranted therapy. Thrombotic complications may develop in ITP. A syndrome, characterized by recurrent TIA-like symptoms, progressive memory loss due to ischemic small vessel disease is described. The management of ITP should include the search for and elimination of underlying causes and careful evaluation of hemostasis. Therapy is divided into definitive vs symptomatic measures. The former including splenectomy, danazol, chemotherapy offers lasting remission after therapy was stopped, while the later including glucocorticoids, gammaglobuin, antiD antibodies and others increases platelet counts but seldom sustains remission upon withdrawal. Danazol therapy is up-dated since it is an effective and safe definite measure in ITP.

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 microparticlesapproximately 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.

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-alpha) 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-alpha, 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.

SummaryHeparin 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 ~2.5 vs. HIT ratios ~1.2; p

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 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 μM) and for collagen (0.2 μg/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. Am. J. Hematol. 57:33–42, 1998. © 1998 Wiley-Liss, Inc.

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.

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.

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 < 0.01) and platelet-associated IgM (p < 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 < 0.01), the PD group of 13 had more marked abnormalities in all tests (p < 0.03), particularly in PMP and thrombocytopenia (p < 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.

Severe pruritus is frequently associated with myeloproliferative and other systemic illnesses, and often fails to respond to conventional measures. We used danazol (Danocrine), a synthetic attenuated androgen, in the treatment of severe pruritus refractory to conventional therapy. Eight patients had myeloproliferative disorders (MPD), seven had autoimmune disorders, and seven had skin diseases. Danazol at 400-800 mg/day was administered, and previous medications were tapered off. When itching was controlled with danazol alone, the dosage was reduced or discontinued, and resumed if itching recurred. Clinical responses were graded, and side effects were monitored. Overall, in 12 of 22 patients refractory to other measures, itching was controlled with danazol alone. In 10 patients itching returned when danazol was discontinued or dosage was continued for up to 5 years in responders. No serious side effects were observed. Our experience indicates that danazol is a good alternative for patients with severe pruritus associated with myeloproliferative and other systemic disorders.

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 isolated from stored platelets or thrombin activated platelets was incubated with leukocytes and binding assessed by flow cytometry. FITC-labeled alpha-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 form interaction with platelets. In these clusters the adherent PMP appeared to serve as bridges between the neutrophil. Addition of EGTA after brief incubation (5-10 min) released most of the bound PMP but if added after > 10 min, only approximately 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 significantly higher percentage of CD41a(+)neutrophils than did platelets incubated at the same numerical ratio. PMP association with neutrophils was less markedly inhibited by alpha-CD62P (AC1.2) than platelets, but binding of both PMP and activated platelets was inhibited approximately 90% by antisialyl 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.

BACKGROUND. Cell derived microparticles (MP), are small vesicle ( 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.

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.

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.

There is no good alternative therapy available for elderly patients with advanced myeloproliferative disorders (MPD) who failed on conventional therapies and are not candidates for bone marrow transplant. We report here an effective therapy that induced exceptionally long-lasting remissions and improved quality of life. Eighteen elderly patients (mean age: 70·6 years) (16 myelofibrosis and 2 thrombocythemia) who had failed on conventional therapies were treated. Danazol was administered daily at 200-800 mg throughout the study. Chemotherapy was applied intermittently as needed to reduce spleen size and blood counts. Busulfan (2-4 mg/day) was used most often and 6-mercaptopurine (6-MP) (50-100 mg/day) and/or cytarabine (100-200 mg/m(2)) if the white blood cell (WBC) count rose rapidly. When MPD stabilized, chemotherapy was discontinued and dosage of danazol was reduced. Therapy was well tolerated. Overall, 61% of patients responded with unexpectedly long-lasting remissions and improved quality of life. Three (17%) had excellent (E) response, defined by normalization of blood counts and non-palpable spleen, while eight (44%) had good (G) response, defined by rise of Hct by ≥7% and ≥50% reduction of spleen. Mean duration of remission was 45 months (10-78 months) in E responders and 11 months in G responders (2-22 months). This regimen offers a safe and effective alternative for advanced MPD in the elderly.

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 x 10(7)/mL). PMP values in all groups except AD were significantly above normal (p0.01). However, the elevation in SCVA was more marked than in LCVA (p0.01). Administration of the calcium channel blocker, nifedipine, to 11 TIA patients reduced PMP significantly.

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.

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.

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.

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.

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

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 × 105 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 < 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.

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.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.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 p0.0001, 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).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.

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.

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.

Background : Circulating cell-derived microparticles (MP) are currently of interest in the fields of hemostasis, thrombosis, and inflammation. Administration of MP has been proposed for use as a hemostatic agent. Efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models [Thromb. & Haemost., 2013;110:751-60]. One of the challenges in developing MP as a hemostatic agent is uncertainty of half-life (T1/2) in circulation, reportedly ranging from several minutes to hours. The purpose of this study was to assess the pharmacokinetics of RMP in rabbits vs. rats, and comparing alternative dosing regimens and assay markers. Although this work was aimed at optimizing the dosage regimen of RMP as a hemostatic agent, it has wider interest because very few studies have addressed the question of MP half-life in circulation. Methods . (i) HumanRMP were produced by high-pressure extrusion [Thromb. & 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 anesthetized rabbits or rats, via cannulated jugular vein. Blood samples (0.4 mL each) were collected from the cannulated carotid artery at intervals: 5 min pre-injection, and at 1, 2, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 8 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with 2 separated labels: anti-CD235a-PE (anti-glycophorin) and annexin V-FITC (AnV). The former is specific for human RMP and does not label rabbit or rat RMP. The latter is not species-sensitive, and labels both human and host MP. (iii) The procoagulant activity (PCA) of RMP was assayed by thromboelastogram (TEG) ex vivo. Results : (1) BolusInfusion of RMP (3 x109 MP/kg) resulted in a rapid rise of RMP levels peaking at 1.0 min post-infusion, followed by slower decline to baseline by 10 - 15 min. The half-life (T1/2) of clearance determined by anti-CD235a was 6.2 ±0.7 and was 4.2 ±0.5 min for rabbits and rats, respectively. In contrast, the T1/2 measured by AnV was 4.4 ±1.0 and 2.6 ±0.4 min, respectively. These results indicate that these two MP phenotypes were cleared by different mechanisms, i.e., one or more mechanisms predominates in clearing CD235a(+) MP vs. AnV(+) MP. The RMP enriched in phosphatidylserine (AnV+) are cleared faster than those expressing CD235a. (2) Two-step administration was bolus (1/3 total dosage) followed by continuous infusion (2/3 total dosage), and resulted in a smaller initial spike followed by rapid decline to ≈25-30%, then recovering to a nearly steady-state level of 0.3 -0.6 x107 MP/mL after 20 min. The RMP levels returned to baseline within 15 min after cessation of infusion. The area under the curve (AUC) of the PK curve for bolus infusion was about 40 -60% less than that of two-step administration of the same dose. (4) Procoagulant Activity (PCA): For bolus infusion, the ex vivo TEG data revealed some correlation between circulating RMP and PCA at the early phase (1 to 6 min). At later phase (>10 min), although RMP had dropped to almost baseline, the PCA remained active considerably longer than presence of RMP (T1/2= 9.3±1.2 min for PCA). However, bolus followed by continuous infusion resulted in maintaining stable PCA over the course of infusion. Conclusion/Discussion. (i) The clearance rate of RMP in rats is faster than that in rabbits. (ii) When T1/2 is determined by the two different markers, we found significant differences in half-life, indicating differential of mechanisms of clearance. (iii) The TEG data show that the PCA of RMP persists after detectable RMP have entirely cleared. We interpret this to mean that RMP continue to exert PCA by being bound to blood cells, even after free circulating RMP have cleared. (iv) Continuous infusion is expected to perform better than bolus infusion in controlling excessive bleeding in clinical patients. Disclosures No relevant conflicts of interest to declare.