Search

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: 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.

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.

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.

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.

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

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.

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.

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.

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.

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.

Introduction. Circulating cell-derived microparticlesa (MP) from platelets (PMP), leukocytes (LMP), and endothelia (EMP) have been well-documented for their roles in hemostasis, thrombosis and inflammation but the clinical significance of RMP is less well understood. The purpose of this study is to study the relation of elevated RMP to selected hematologic and thrombotic disorders. Methods. This is a retrospective study on RMP profiles for patients referred to University of Miami Hospital and Clinics for hematologic consultation over the last 8 years. The patient population includes 51 hemolytic anemia (HA), 459 thrombocytopenia (TP), 26 myeloproliferative disorder (MPD), 413 hypercoagulable state (HCS), and 446 thrombotic disorders (TBS). Some patients were analyzed in more than one of the above disorders. Levels of RMP were measured by flow cytometry with PE-conjugated anti-CD235a labeling as previously described [Thromb. & Haemost., 2013;110:751-60]. Levels of RMP above mean +2SD of controls (> 2,000 counts/mL) are designated as "elevated RMP". Results. (I) Prevalence of elevated RMP in patient populartion: Elevated RMP were found in 31 of 51 HA (60.8%), 138 of 459 TP (30.1%), 20 of 26 MPD (78.1%), 175 of 413 HCS (42.4%), and 251 of 446 TBS (56.3%). (II) Association of elevated RMP with thrombosis: Of 31 HA patients with elevated RMP, 11 were positive for TBS, and the remaining 20 were negative. Levels of RMP (mean ±SD) for TBS(+) and TBS(-) were 5,824 ±3713 and 3,265 ±1,048, counts /mL, respectively (p Discussion. (i) The cause of RMP elevation in HA is reasonably attributed to red cell destruction. The cause in TP/ITP is likely due to products of platelet destruction or leukocyte activation. The cause in MPD could result from clearance of the high burden of red cells and platelets. (ii) Patients who were TBS(+) had higher RMP than TBS(-), seen in HCS, HA, TP, raising the question of whether high RMP is a cause or consequence of TBS. To answer this will require further study. (iii) The finding of exceptionally high RMP levels in recurrent TBS vs. non-recurrent TBS (HCS-3 vs. HC-2) indicates that RMP levels reflect severity of TBS. (iv) Finally, these data indicate that RMP may be a useful biomarker of thrombotic risk, particularly because some TBS patients had elevated RMP, yet tested negative by all conventional markers of HCS workups. Disclosures No relevant conflicts of interest to declare.

Background: We have recently observed that increased levels of circulating RMP are associated with several hematological disorders and thrombophilic states, and that levels were significantly higher when thrombosis was present. However, other species of MP, such as from platelets (PMP), endothelia (EMP), leukocytes (LMP) and annixin V-binding (AnV) have also been shown to be associated with thrombophilic states. The purpose of this study was to determine if correlations exist between RMP and other MP. Methods: This is a retrospective analysis of 702 laboratory samples over an 8 year period, limited to elevated values: >2SD above normal controls (>2,000/uL).. About 87% were individuals, the remaining 13% were tested 2-3 times and all tests >3 per patient were excluded. The disorders were hemolytic anemia (HA, n=38), hypercoagulable state (HCS, n=64), immune thrombocytopenia (ITP, n=86), thrombocytopenia of all causes (TP, n=69), myeloprolifereative disorder (MPD, n=29), thrombotic thrombocytopenic purpura (TTP, n=29), antiphospholipid syndrome (APS, n=34), pulmonary embolism (PE, n=21), and all-cause thrombosis (TBS, n=251). Some were classified in more than one way. MP species assayed were RMP by glycophorinA, PMP by CD41 (PMP41), PMP by CD42 (PMP42), EMP by CD31+/42- (EMP31), EMP by CD62E (EMP62E), LMP by CD45, annexin V binding (AnV), and counts by lectin, FITC-Ulex, which efficiently detects total MP including very small. Results: In HCS, the RMP >2,000/uL correlated well with PMP41 (R=0.407, p 2,000/uL and any of the MP markers in APS, TP, or TTP. The MP species markers, EMP31, EMP62E, and AnV, failed to show correlation with RMP in any of the disorders analyzed. In addition, we tested for correlations between elevated RMP and other MP for the entire data set (all disorders combined) and found that only LMP was significant (p 0.05). Discussion: These correlation analyses shows that RMP correlated most frequently with PMP, as seen in HA, ITP, HCS, and TBS, followed by LMP, as seen in HA and TBS. These observations suggest that platelet or/and leukocyte activation may be involved in RMP generation. Of added interest is the finding that the overall data correlated well with LMP only at the higher LMP levels, not at all in the lower quintile of LMP. This suggests that RMP elevations are associated with immunolgic, inflammatory processes. In summary, correlation analysis reveals likely interaction between red cells and platelets or leukocytes during immunologic, inflammatory or in thrombophilic states, resulting in elevated RMP. Disclosures No relevant conflicts of interest to declare.

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

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

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 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.

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.

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

Endothelial microparticles (EMP) are small vesicles released from disturbed endothelial cells (EC). Owing to the central importance of EC injury in thrombotic and inflammatory conditions, assay of EMP as a marker of EC disturbance has come under intensive development by several laboratories. The review begins with established markers of EC injury, commonly soluble markers such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, von Willebrand factor (vWF), etc., pointing out that many of these are in fact mixtures of true soluble molecules with membrane-bound forms, for example, EMP. Assays of EMP from different labs are reviewed and standardization of assay is recommended. EMP are heterogeneous: those released in activation vs. apoptosis are distinctive in phenotypic markers and procoagulant properties. Application of EMP phenotype analysis can distinguish EC state of activation from apoptosis. Some EMP carry functional vWF with properties different from soluble vWF. Certain EMP bind to and activate monocytes; EMP-monocyte conjugates were found to be a marker of inflammatory disease such as multiple sclerosis (MS), and to enhance transendothelial migration of leukocytes in vitro. Clinical studies have revealed elevated plasma levels of EMP in lupus anticoagulant (LA), multiple sclerosis (MS), thrombotic thrombocytopenic purpura (TTP), coronary artery disease (CAD), hypertension, preeclampsia, and diabetes. Further refinement of EMP assay could open new windows for evaluating and monitoring endothelial injury in thrombotic and inflammatory disorders.

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

Endothelial Microparticles (EMP) are small fragments of endothelial cell membrane shed during apoptosis or activation. Our group has previously reported elevations of EMP in patients with coronary artery disease (CAD), thrombotic thrombocytopenic purpura (TTP), pre-eclampsia, multiple sclerosis (MS), and severe hypertension (HTN). In the present study, we evaluate the possible relationship between EMP levels and the angiographic severity and characteristics of coronary obstructive lesions.We studied a total of 43 patients undergoing coronary angiography. Fifteen had presented with acute myocardial infarction (MI), 20 with unstable anginas (UA), 5 with stable angina (SA) and 3 with congestive heart failure. Coronary angiography was reviewed and coronary lesions were classified using the Ambrose classification. Coronary stenoses were classified as high and low risk. High-risk included lesions with eccentric appearance (type II), presence of thrombi, or multiple irregularities. Low-risk lesions were defined as concentric or type I. Lesions were also analyzed by degree of stenosis and history of acute coronary syndrome (ACS). EMP in plasma was assayed by flow cytometry.EMP in eccentric type II or multiple irregular lesions (high-risk) were 2.5-fold higher than in type I or concentric (low-risk) lesions, p0.05. Lesions with thrombi had three-fold higher EMP than those without (p=0.05). Mild stenosis (20%-45%) had three-fold higher EMP than more severe (45%), and five-fold higher than those without stenosis (p0.01). Among patients with type II lesions, those with first ACS episode had four-fold higher EMP levels than those with recurrent ACS (p0.01).High EMP was associated with high-risk angiographic lesions including eccentric type II, multiple irregular, and lesions with thrombi. Mild to moderate stenosis was associated with higher EMP levels than severe stenosis. EMP may be a useful marker in detecting endothelial injury and risk of ACS as defined by angiography.

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

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

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

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

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