Search

Background: Spontaneous intracerebral hemorrhage (sICH) is the deadliest stroke subtype with no effective therapies. Limiting hematoma expansion is a promising therapeutic approach. Red blood cell-derived microparticles (RMPs) are novel hemostatic agents. Therefore, we studied the potential of RMPs in limiting hematoma growth and improving outcomes post-sICH. Methods: sICH was induced in rats by intrastriatal injection of collagenase. RMPs were prepared from human RBCs by high-pressure extrusion. Behavioral and hematoma/lesion volume assessment were done post-sICH. The optimal dose, dosing regimen, and therapeutic time window of RMP therapy required to limit hematoma growth post-sICH were determined. We also evaluated the effect of RMPs on long-term behavioral and histopathologic outcomes post-sICH. Results: RMP treatment limited hematoma growth following sICH. Hematoma volume (mm 3 ) for vehicle- and RMP- (2.66×10 10 particles/kg) treated group was 143±8 and 86±4, respectively. The optimal RMP dosing regimen that limits hematoma expansion was identified. RMPs limit hematoma volume when administered up to 4.5-hour post-sICH. Hematoma volume in the 4.5-hour post-sICH RMP treatment group was lower by 24% when compared with the control group. RMP treatment also improved long-term histopathologic and behavioral outcomes post-sICH. Conclusions: Our results demonstrate that RMP therapy limits hematoma growth and improves outcomes post-sICH in a rodent model. Therefore, RMPs have the potential to limit hematoma growth in sICH patients.

Spontaneous intracerebral hemorrhage (sICH) is a subtype of hemorrhagic stroke with enormous mortality and morbidity, and tobacco use is one of the major risk factors for sICH. We have shown that red cell microparticle (RMP) treatment limits hematoma growth following sICH in nicotine-exposed rats. Presently, we determined the therapeutic time window of RMP efficacy. RMPs were generated from RBCs using high pressure extrusion. Rats were treated for a period of 2-3 weeks with nicotine (4.5 mg / kg b.w./ day, s.c.) using osmotic pumps. sICH was induced using intra-striatal collagenase administration. Rats of either sex were randomized between saline (vehicle) or RMP treatment groups (n=10 each for 4.5 h and 6 h RMP treatment time points, n=15 each for pooled vehicle time points). Males treated at 4.5 h and females treated at both timepoints received ~1.34х10 11 particles/ kg b.w., i.v. over 2 h period. While males treated at 6 h received ~ 1.58х10 11 particles/ kg b.w., i.v. over 2 h duration. After ~24 hours, functional deficit was assessed using neurological scores, and hematoma volume was measured in a blinded manner. The hematoma volume of male rats treated with RMPs 4.5 h post-sICH (94±3 mm 3 ) was significantly lower (p3 ). Moreover, the neurological score of male rats treated with RMPs 4.5 h post-sICH (8.9±0.5) was significantly lower (p3 ) and neurological score (10.1±0.6) of the 6 h post-sICH RMP treatment male group were not different from the controls. The hematoma volume observed in female rats treated with RMPs 4.5 h post-sICH (60±6 mm 3 ) was significantly lower (p3 ). Further, neurological score of the rats treated with RMPs 4.5 h post-sICH (7.7±0.6) was significantly lower (p3 ) and neurological score (10.4±0.3) in female rats treated with RMPs 6 h post-sICH were not different from the controls. Our results show that RMPs limit hematoma growth in nicotine-exposed rats when administrated 4.5 h post-sICH induction. Thus, RMPs may lower hematoma growth in sICH patients following tobacco use. Support: James Esther King Biomedical Research Grant 9JK08.

Spontaneous intracerebral hemorrhage (sICH) is a disabling stroke sub-type, and tobacco use is a prominent risk factor for sICH. We showed that chronic nicotine exposure enhances bleeding post-sICH. Reduction of hematoma growth is a promising effective therapy for sICH in smoking subjects. Red-blood-cell-derived microparticles (RMPs) are hemostatic agents that limit hematoma expansion following sICH in naïve rats. Considering the importance of testing the efficacy of experimental drugs in animal models with a risk factor for a disease, we tested RMP efficacy and the therapeutic time window in limiting hematoma growth post-sICH in rats exposed to nicotine. Young rats were chronically treated with nicotine using osmotic pumps. sICH was induced in rats using an injection of collagenase in the right striatum. Vehicle/RMPs were administered intravenously. Hematoma volume and neurological impairment were quantified ≈24 h after sICH. Hematoma volumes in male and female nicotine-exposed rats that were treated with RMPs at 2 h post-sICH were significantly lower by 26 and 31% when compared to their respective control groups. RMP therapy was able to limit hematoma volume when administered up to 4.5 h post-sICH in animals of both sexes. Therefore, RMPs may limit hematoma growth in sICH patients exposed to tobacco use.

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

Spontaneous intracerebral hemorrhage (sICH) is a serious form of stroke without any effective therapy. Limiting hematoma growth is a promising therapeutic strategy. Published data showed that red blood cell-derived microparticles (RMPs) enhance both primary and secondary hemostasis. Previously, we observed that RMPs lower hematoma growth in a rat model of sICH. The goal of our present study was to determine the optimal therapeutic time window for RMP administration. Optimal therapeutic dose and treatment paradigm were used based on our previous studies. RMPs were prepared from human RBCs using high-pressure extrusion. sICH was induced in young male rats by injecting collagenase into the right striatum. Rats were treated with either saline (vehicle) or RMP (2.55х10 10 particles/kg, b.w. i.v.) starting at 2, 3, 4.5, or 6 h post-sICH induction. Rats were randomly assigned to each treatment group (n=10 for each RMP treatment time window, n=5 for each vehicle time window). Neurological scores (12 indicates maximum impairment) were evaluated ~24 h post-collagenase injection, and then, the rats were perfused using saline. Brains were collected and sectioned for hematoma volume assessment using ImageJ by a blinded investigator. The hematoma volumes (mm 3 ) of 2 (53±4, p

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.

Currently there is no effective treatment for spontaneous intracerebral hemorrhage (sICH). Limiting hematoma extension in sICH is an attractive therapeutic target. Earlier studies demonstrated that red blood cell-derived microparticles (RMP) enhance platelet function and accelerate coagulation, augmenting both primary and secondary hemostasis. In the present study, we determined the efficacy of RMP in reducing hematoma expansion in a rat model of collagenase-induced sICH. RMPs were prepared from human RBCs using a high-pressure extrusion method. At ~24 hours post-collagenase injection, neurological scores were evaluated, rats were sacrificed, and the hematoma areas on images of brain sections were measured. In an earlier study (presented in an abstract form) we determined optimal RMP dose required to lower hematoma growth following sICH. The hematoma volume was lower by 17, 40 (p9 particles / kg b.w.), medium dose (2.55 x 10 10 particles / kg b.w.), and high dose (9.48 x 10 10 particles / kg b.w.) treated groups when compared to placebo group, respectively. For those experiments, total RMP dose was delivered over 2 hours using continuous infusion starting 1 h post-collagenase injection. In the present study we evaluated optimal treatment paradigm using optimal protective (medium) dose as identified above. We evaluated the efficacy of RMP when delivered starting at 1 h post-collagenase injection either via four bolus injections (6.375 x 10 9 particles / kg b.w. per injection) 30 min apart (paradigm B) and when delivered using continuous infusion (2.55 x 10 10 particles / kg b.w.) over 1 hour (paradigm C), and compared with when RMP dose was delivered (2.55 x 10 10 particles / kg b.w.) over 2 hours using continuous infusion (paradigm A). We observed that hematoma volume (mm 3 ) for vehicle, and paradigm A, B and C was 71 ± 3 (n = 35), 43 ± 2 (n = 10, p

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.

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

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.

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

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: Uncontrollable bleeding is a major cause of mortality and morbidity worldwide. Effective hemostatic agents are urgently needed. Red cell microparticles (RMP) have been introduced as a promising new hemostatic agent [Thromb Haemost 2013, 110:751-60]. It acts to promote both primary (platelet function) and secondary (coagulation, thrombin generation) hemostasis, thus meeting the criteria for a universal hemostatic agent. However, although no overt adverse effects were observed in limited animal studies, more rigorous evaluation of safety as well as biodistribution of RMP were addressed in this report. METHODS: RMP were prepared from Type O+ human RBC by high-pressure extrusion. Male rats were treated with either a 1× dose (=6x1010 RMP/kg BW), or 4×, or 20× dose, then monitored for at least 60 min for physiological responses of blood pressure, body and head temperature, hematocrit, and blood gases. A group of RMP-treated rats (n =6) were perfused with saline followed by formaldehyde - acetic acid - methanol mixture (1:1:8) at 4 hr post-RMP. Heart, brain and lung were harvested, sectioned and evaluated in blinded fashion by independent animal pathologist for signs of thrombosis or other pathology. For biodistribution study, RMP were labeled with Alexa-488 and unbound dye was removed by dialysis. At 15 min and 24 hours after infusion of labeled RMP, animals were perfused with saline for 2 min to remove labeled RMP in blood vessels, then organs were harvested, homogenized and sonicated. Fluorescence of the homogenates was measured by a fluorescence plate reader. RESULTS: (i) Toxicity. No adverse effects of RMP were detected by monitoring the above physiological parameters at any of the 3 dose regimens. In addition, histological examination of brain, heart and lung sections by an animal pathologist revealed no microthrombi or other abnormality. (ii) Biodistribution. After injection of fluorescence-labeled RMP, levels of RMP in blood rose rapidly (1 min), then declined to baseline at 15 min, demonstrating rapid clearance. Fluorescence of tissue homogenates at 15 min revealed the highest fluorescence (FAU/g tissue) in liver, followed by spleen, lymph nodes, and lung. At 24 hr, fluorescence of the above-mentioned organs had decreased by 20 - 60%. Surprisingly, the organ of greatest fluorescent intensity at 24 hr had shifted to the kidneys, followed by liver, spleen, and lymph nodes. DISCUSSION. Results further support safety of RMP as hemostatic agent. This is unexpected in view of reports finding proinflammatory properties of RMP obtained from stored blood or by calcium ionophore. This apparent discrepancy may be explained by the high-pressure extrusion method of production, since preliminary experiments show absence of lipid rafts on these RMP compared to RMP prepared by other ways. Lipid rafts are implicated as inflammatory mediators. Our biodistribution data demonstrated that RMP were sequestered mainly by liver, spleen, lymph nodes, and lung at 15 min post-RMP infusion. Our results also suggest that at 24 hr post-RMP infusion, many sequestered RMP have been degraded, leading to redistribution of fluorescent label to the kidney. 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.

Background: Immune thrombocytopenic purpura (ITP) is frequently associated with chronic hepatitis C (HpC-ITP). Methods: Recombinant interleukin-11 (rIL-11), which has both thrombopoietic and anti-inflammatory properties, was evaluated in 12 patients with HpC-ITP in this pilot study. Group 1 (7 patients) was treated at high dose (50 µg/kg daily) while group 2 (5 patients) at low dose (15–35 µg/kg three/week). Results: In group 1, mean platelet counts rose from initial 54 × 109/l to 103 × 109/l (p = 0.02) and in group 2, from an initial 51 × 109/l to 74 × 109/l (p = 0.04). Antiplatelet antibody (aPlt-Ab) decreased in group 1. LFT improved in both groups. The mean HCV-RNA decreased in group 1 (p = 0.04), not in group 2. Side effects were common and troublesome, but were minimized with individualized dosing. One patient achieved good remission of both ITP and HpC lasting >2 years with low-dose maintenance. Conclusion: When used based on individual tolerance, rIL-11 appears useful in HpC-ITP.

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.

Introduction: It has been suggested that Helicobacter pylori eradication often increases platelet counts in patients with chronic idiopathic thrombocytopenic purpura (ITP). In addition, H. pylori has been shown to induce platelet activation (CD62p or P-selectin expression) in previous studies. We assessed the response of platelet count and CD62p expression after eradication therapy in patients with ITP and H. pylori infection. Methods and Results: We prospectively studied 15 ITP patients diagnosed with H. pylori infection by serology and breath test. A follow-up breath test was used to document eradication. Two out of 15 patients showed improvement in platelet counts after 6 months, 1 of which may have had drug-induced thrombocytopenia. Overall, certain platelet response rate in our series was 6.7% (1/15). We found that platelet CD62p expression by flow cytometry was elevated in 10/15 (66.7%) H. pylori-infected patients, which is a statistically significant difference when compared with 3/33 (9.1%) control ITP patients seronegative for H. pylori (p = 0.002). In addition, eradication therapy decreased CD62p expression (p = 0.04). However, reduction in platelet activation was not associated with an increase in platelet counts (mean 72.4 × 109/l before and 68.7 after therapy; p = 0.4). Conclusion: In our series, platelet activation was common in ITP patients with H. pylori, and eradication therapy decreased platelet activation but seldom increased platelet counts. Increased platelet CD62p expression is a putative link between chronic infections and atherosclerosis, but further study is needed to clarify the implications of our observation.

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.

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

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

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.

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

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.

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

To determine the effects of dietary fats on surface antigen expression, we tested the effects of amount and type of dietary fat on murine lymphocytes. Mice were fed diets with 12 en%, 23 en%, or 47 en% fat containing coconut, olive, safflower, or linseed oil. After 2 wk of ad libitum feeding, the mice were killed and splenic lymphocytes were harvested. Lymphocytes were incubated with fluorescent-tagged monoclonal antibodies and assayed for mean and total surface expression using flow cytometry. Our results show that high-fat (47 en%) diets suppress expression of CD3 and CD25 antigens. We also found that linseed-oil diets suppress expression of CD11a but enhance expression of CD25 antigens. Both CD3 and CD25 are critical for lymphocyte activation, and we conclude that immunosuppression associated with high-fat diets may be associated with suppression of these surface antigens.

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

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

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.

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

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

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

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