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

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

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

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

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

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

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

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

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

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

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

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.

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

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

Background : Circulating cell-derived microparticles (MP) are currently of interest in the fields of hemostasis, thrombosis, and inflammation. Administration of MP has been proposed for use as a hemostatic agent. Efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models [Thromb. & Haemost., 2013;110:751-60]. One of the challenges in developing MP as a hemostatic agent is uncertainty of half-life (T1/2) in circulation, reportedly ranging from several minutes to hours. The purpose of this study was to assess the pharmacokinetics of RMP in rabbits vs. rats, and comparing alternative dosing regimens and assay markers. Although this work was aimed at optimizing the dosage regimen of RMP as a hemostatic agent, it has wider interest because very few studies have addressed the question of MP half-life in circulation. Methods . (i) HumanRMP were produced by high-pressure extrusion [Thromb. & Haemost., 2013; 110:751-60]. The resulting product was washed twice with isotonic saline, lyophilized, and stored at -80°C. (ii) Pharmacokinetics of RMP were measured using either bolus infusion of RMP (3x109 counts/kg) during 1 min., or a combination of bolus (1/3 of total RMP) followed by continuous infusion (2/3 of total RMP) for 30 min to the anesthetized rabbits or rats, via cannulated jugular vein. Blood samples (0.4 mL each) were collected from the cannulated carotid artery at intervals: 5 min pre-injection, and at 1, 2, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 8 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with 2 separated labels: anti-CD235a-PE (anti-glycophorin) and annexin V-FITC (AnV). The former is specific for human RMP and does not label rabbit or rat RMP. The latter is not species-sensitive, and labels both human and host MP. (iii) The procoagulant activity (PCA) of RMP was assayed by thromboelastogram (TEG) ex vivo. Results : (1) BolusInfusion of RMP (3 x109 MP/kg) resulted in a rapid rise of RMP levels peaking at 1.0 min post-infusion, followed by slower decline to baseline by 10 - 15 min. The half-life (T1/2) of clearance determined by anti-CD235a was 6.2 ±0.7 and was 4.2 ±0.5 min for rabbits and rats, respectively. In contrast, the T1/2 measured by AnV was 4.4 ±1.0 and 2.6 ±0.4 min, respectively. These results indicate that these two MP phenotypes were cleared by different mechanisms, i.e., one or more mechanisms predominates in clearing CD235a(+) MP vs. AnV(+) MP. The RMP enriched in phosphatidylserine (AnV+) are cleared faster than those expressing CD235a. (2) Two-step administration was bolus (1/3 total dosage) followed by continuous infusion (2/3 total dosage), and resulted in a smaller initial spike followed by rapid decline to ≈25-30%, then recovering to a nearly steady-state level of 0.3 -0.6 x107 MP/mL after 20 min. The RMP levels returned to baseline within 15 min after cessation of infusion. The area under the curve (AUC) of the PK curve for bolus infusion was about 40 -60% less than that of two-step administration of the same dose. (4) Procoagulant Activity (PCA): For bolus infusion, the ex vivo TEG data revealed some correlation between circulating RMP and PCA at the early phase (1 to 6 min). At later phase (>10 min), although RMP had dropped to almost baseline, the PCA remained active considerably longer than presence of RMP (T1/2= 9.3±1.2 min for PCA). However, bolus followed by continuous infusion resulted in maintaining stable PCA over the course of infusion. Conclusion/Discussion. (i) The clearance rate of RMP in rats is faster than that in rabbits. (ii) When T1/2 is determined by the two different markers, we found significant differences in half-life, indicating differential of mechanisms of clearance. (iii) The TEG data show that the PCA of RMP persists after detectable RMP have entirely cleared. We interpret this to mean that RMP continue to exert PCA by being bound to blood cells, even after free circulating RMP have cleared. (iv) Continuous infusion is expected to perform better than bolus infusion in controlling excessive bleeding in clinical patients. Disclosures No relevant conflicts of interest to declare.

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.

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

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.

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

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

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.

It is now recognized that all circulating blood cells, as well as endothelial cells (EC), continuously shed small membranous vesicles (microparticles [MPs]), which are approximately less than 1 µm, and that levels of circulating MPs are sensitive indicators of disease activity. The first type extensively studied in patients was platelet MP (PMPs) (1). Currently, endothelial-derived MP (EMPs) have risen to the fore as sensitive markers of EC perturbation, recently reviewed (2) and further considered in this article. Although other reviews may differ in viewpoint and emphasis (3,4), it is generally agreed that circulating MPs comprise different subspecies of membrane vesicles released from endothelium and blood cells, such as platelets, leukocytes, and red blood cells (RBCs). MPs containing negatively charged phospatidylserine (PS) and/or tissue factor are highly procoagulant. MPs that express specific adhesion molecules are capable of interacting with leukocytes and endothelia to initiate inflammatory responses.

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

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.

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.

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 / OBJECTIVES. Cell-derived microparticles (MP) are known to promote thrombin generation, and more recently have been shown to contribute substantially to hemostasis. This activity derives mainly from platelet MP (PMP) but recent evidence implicates also red cell MP (RMP). Traditionally, MP-mediated procoagulant activity has been ascribed to presentation of negatively charged surfaces for the assembly of tenase and prothrombinase. However, the mechanism of this activity is becoming more complicated as it is emerging that MP-mediated procoagulant activity includes augmentation of the contact pathway [van der Meijden et al,J Thromb. Haemost., 2012; 10:1355-62, Rubin et al, Transfusion, 2013; 53:1744-54] and of ADP-induced platelet aggregation [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Accordingly, we undertook examination of interactions of RMP with specific elements of the contact factor pathway, and related issues such as whether or not RMP can initiate contact pathway by activating FXII directly. METHODS. Plasmas depleted in each of 8 factors (F’s II, V, VII, VIII, IX, X, XI, XII), obtained from commercial sources, were mixed with pooled normal plasma to produce depletion levels of 75% to 95%, i.e., from 25% to 5% of normal. These plasmas were then assayed by thromboelastography (TEG) with / without addition of RMP. The RMP was prepared by high-pressure extrusion of washed RBC [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Blood was drawn into citrated Vacutainers in presence / absence of 140 µg/mL corn trypsin inhibitor (CTI) and run in TEG. Controls were obtained from healthy staff volunteers (n=8) to establish baseline values, +/- addition of RMP. Tissue factor (TF) at 20 or 200 pg/mL was added to standardize extrinsic pathway activation. Anti-FVIIa (Seksui Diagnostics) was added in certain experiments to abolish the TF pathway. Finally, when contact factor activation was fully blocked (no fibrin generation within 2 hrs), kaolin was added in presence / absence of RMP. RESULTS. (1) In the factor depletion experiments, we found that RMP most strongly potentiated depletion of factors of the contact pathway, namely FVIII, FIX, FXI, and FXII, as judged by shortening of R (lag time), p (2) RMP were capable of enhancing the rate of thrombin generation at low concentration of CTI (40 µg/mL), but at high concentration (140 µg/mL) this effect was completely abolished. When kaolin was added to the high concentration CTI-treated blood, it initiated thrombin generation after long delay (~30 min). Upon addition of RMP, the rate of thrombin generation was potentiated considerably (by ~50%). These data indicate that unlike kaolin, RMP are not capable of activating FXII directly but RMP can potentiate FXIIa-mediated thrombin generation. (3) In whole blood treated with low concentration TF (20 pg/mL) and high concentration CTI, RMP increased rate of thrombin generation by 25% compared to controls (p< 0.05). When blood was treated with anti-FVIIa, RMP retained the ability to enhance thrombin generation by roughly 30% (p CONCLUSIONS. First, these results demonstrate highly selective stimulation of elements of the contact pathway by RMP, as opposed to non-specific augmentation of coagulation. However, we do not have evidence of direct activation of FXII to FXIIa by RMP. Instead, reslults can be explained by marked potentiation of small amounts of activated enzyme. Second, it is shown that this activity is entirely independent of the TF pathway. Third, results support the potential value of RMP for use as an infusible hemostatic agent [Jy et al, Thrombosis & Haemostais, 110:751-60 (2013)], for treating a wide variety of clinical conditions involving excessive bleeding. Finally, in view of new evidence showing that MP-mediated procoagulant activity is diverse in action, new avenues of MP-mediated hemostasis are open to exploration. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION: Spontaneous intracerebral hemorrhage (SICH) is associated with significant morbidity and mortality, yet no effective treatment for it exists. It has been shown that the size of hematoma plays a major role in determining the outcomes of SICH. Therefore, the paradigm that reduction of hematoma growth, and/or prevention of continued bleeding in SICH has been an attractive therapeutic target. Various hemostatic agents including NovoSeven® have previously been investigated for SICH, without benefit. Red blood cell-derived microparticles (RMP) have diverse hemostatic activity. It has been shown in vitro that RMP enhance platelet functions as well as accelerating coagulation, augmenting both primary and secondary hemostasis. Efficacy of RMP as hemostatic agent has been documented in animal bleeding models [Jy et al, Thrombosis & Haemostais, 2013; 110:751-60]. The present report provides preliminary data on efficacy of RMP infusion for reducing hematoma expansion in a rat model of SICH. METHODS: RMP were produced by high pressure extrusion of washed RBC as described [Jy et al, Thrombosis & Haemostais, 2013; 110:751-60]. SICH was induced in male Sprague Dawley rats by injection of 0.17U of collagenase (2 µl at the rate of 0.4 µl / min) into the left striatum sterotexically. It has been shown that after injection of collagenase, intracerebral bleeding continued for several hours [Del Bigio et al. Stroke, 1996; 27:2312-9]. Rats were randomly divided into two groups (1) Placebo (vehicle-treated) group, n=9; and (2) RMP-treated group, n=10. RMP were injected via femoral vein at 1 hour and again at 2 hours post-SICH, at concentration 3x109particles / kg, per injection. Rats were sacrificed at ~24 hours post-collagenase injection, and brains were sectioned. Sectioned brains were scanned and hematoma area on brain section images was evaluated by Image J software. The neurological score was evaluated at ~24 hr as described earlier [Bederson et al. Stroke. 1986; 17:472-476; De Ryck et al. Stroke. 1989; 20:1383-1390]. Induction of SICH and evaluation of hematoma area and neurological score was carried out by an investigator blinded to the experimental conditions. RESULTS: (i)Effect of RMP treatment on hematoma area: We observed that total hematoma area for the placebo group was 105 ± 13 mm2 (mean ±SEM) at ~24 hr post-collagenase injection. In contrast, in the RMP-treated group, total hematoma area was 72 ± 9 mm2, which was 31% lower than in the placebo group; p CONCLUSIONS: These preliminary results are promising, but indicate a need for longer-term observation and larger sample size. Modified dosage regimen might also improve efficacy since we have observed in other studies that continuous infusion of RMPs is more effective than bolus. In summary, results demonstrate that RMPs have the potential to arrest or limit hematoma growth in SICH. If further work confirms and improves this benefit, RMP treatment could save many lives. Disclosures No relevant conflicts of interest to declare.

Thromb Haemost 2008; 99: 657–658 Aortic valve stenosis (AVS) is quite common, affecting some 30% of those over age 65 (1). It frequently progresses to severe obstruction necessitating surgical repair or replacement of the stenotic valve. Annually more than 50,000 aortic valves are replaced surgically in the USA, posing common and serious medical problems (2), yet our knowledge of the pathogenesis and optimal treatment of this condition is surprisingly limited. It may represent a manifestation of atherosclerosis affecting the endothelium lining the heart valve but this is still debated. There is mounting evidence that AVS is associated with systemic inflammation (3, 4). Some but not all studies found an association of AVS with elevated C-reactive protein (CRP) (3) and active atheroinflammatory processes (4), implying a role of inflammation in the pathogenesis of AVS. Even if true, however, there is little insight on exactly how or why mechanical perturbation of blood flow due to AVS leads to systemic inflammation, or what inflammatory processes promote the initiation and progression of AVS. Elucidation of this key issue could provide a rational basis for prevention and therapeutic strategies. The intriguing report by Diehl et al. in this issue of Thrombosis and Haemostasis (5) may offer an insight into this problem, and may be of potential clinical importance. They hypothesize that AVS induces generation of circulating cell-derived microparticles (MP) by shear stress, and that the resulting MP then promote systemic inflammation. Briefly, their scenario goes like this: high shear stress generates platelet microparticles (PMP), which in turn interact with leukocytes to activate them and elicit release of leukocyte MP (LMP) and monocyte MP (MoMP). These procoagulant and inflammatory MP, along with activated monocytes, can induce endothelial injury at the lining of the heart valve, thereby setting up a “vicious cycle,” as the authors put it, leading to further calcific stenosis of aortic valves as seen inAVS. Endothelial cell injury is reflected by release of endothelial MP (EMP) (6). This hypothesis is novel and intuitively appealing.To support it, they measured MP species including PMP, LMP, and EMP in 22 AVS patients, and report a significant increase in all of them relative to controls. In parallel, elevation of markers of systemic inflammation was observed, such as soluble P-selectin, interleukin-6, and activation of monocytes.These data convincingly support the hypothesis, although falling short of conclusively establishing a cause-effect relationship between MP-cell interaction and progression of AVS. In addition, since this is a cross-sectional study, more definitive support for an active role of MP in the pathogenesis of AVS will require a prospective longitudinal study correlating MP-cell interaction with progression ofAVS, as the authors acknowledge in their discussion. Thus, a central theme of this paper is the physiological relevance of MP-cell interactions. They demonstrate copious production of PMP in AVS, and correlation of PMP levels with valvular shear stress in vivo. Others have previously shown in vitro that PMP are readily generated by abnormally high shearing forces (7–9). It has also been shown that PMP can induce leukocyte activation (10, 11), and can act directly on endothelial cells (8, 12). High shear can also induce endothelial activation and release of EMP (13); and in turn, EMP can activate leukocytes (14–17). Indeed, EMP were reported to act on the endothelial cells that produce them (18). Mixed MP isolated from patients were shown to be deleterious on cells in vitro or ex vivo (19, 20). Finally, the LMP elicited by PMP or other MP are known to be injurious to endothelial cells (21, 22). In view of these and other reports cited by Diehl et al. (5), there is ample evidence in vitro to support their hypothesis, that interactions of shear-generated MP with blood cells and endothelium could be pivotal in the progression of AVS. Their report struck a chord with us because of our anecdotal observation some years back of a patient with severe AVS, who refused surgical intervention. Her PMP counts were among the highest we had ever seen. She suffered from numerous recurrent transient ischemic attacks, progressing to advanced dementia. Thus, it is gratifying to see our observation confirmed in a significant number of patients by the work by Diehl et al. What implications might this report have for therapeutic strategies? If the hypothesis of Diehl et al. reported in this issue is correct, then it may be time to begin thinking about ways to modulate MP-mediated injury. Two approaches come to mind

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.

Background Red cell microparticles (RMP) have come to recent attention as putative mediators of hemostasis. We reported that RMP improve hemostatic defect of blood samples of thrombocytopenia and thrombocytopathy and augment platelet function. To investigate possible mechanisms of this activity, we measured the effect of RMP on shear-dependent platelet adhesion and aggregation in whole blood. Methods RMP were produced by high-pressure extrusion of washed, packed RBC. The RMP produced in this way are similar to natural circulating RMP in phenotype and most functional assays. Blood was collected in citrate Vacutainers from normal healthy staff volunteers, and first 3 mL discarded to minimize artifact of platelet activation due to tissue factor. It was tested within 2 hours of drawing. Variable shearing rates were applied by a cone-and-plate device, the DiaMed Impact-R, which yields photomicrographs of objects adhering to the plate, and data including percent surface coverage (SC), number objects adhering (OBJ) per mm2, and mean aggregate size (AS) in μm2. Initially, 4μL of either RMP (1.0 x 108/μL) or saline were mixed with 126μL of whole blood obtained as above, and incubated in a microcentrifuge tube for 60s. The mixtures were pipetted to a well of the device and run at a selected shear rate (range 900s-1 to 2700s-1) for 2 minutes. Blood was then carefully drawn off and the wells were washed gently with deionized water. Wells were stained with May-Grünwald stain for 60s, and excess removed. When dry, micrographs of the well surfaces were taken. The most promising shear rates (1125 s-1, 1575s-1, 1800s-1, 2025s-1) were retested with higher numbers of RMP in the 130μL volume. Results Addition of RMP vs. saline (control) induced increased AS over a range of shear rates. For example, 4μL RMP (1x108/μL) running at 2250s-1 increased AS by 34.8%, from 59.2 ±25.1μm2 to 79.8 ±24.7μm2 (n=15, p=0.01). To investigate effect of RMP concentration at fixed shear rate of 1575s-1, we found that 8μL RMP induced increase of 12.0 μm2, 12μL RMP by 24.8 μm2, 16μL by 26.7 μm2, and 20μL by 26.3 μm2; p Conclusion/Discussion RMP increased the size of platelet aggregates under shear, indicating enhanced platelet adhesion by RMP. This effect varied with shear rate: larger amounts of RMP had maximum effect at lower shear rates; smaller amounts of RMP required higher shear to exert maximum effect. These effects are seen at physiologically relevant shear rates (200 - 2000s-1 or higher in pathology such as hypertension or valve defects). Due to the absence of endothelia from this model, high shear was required to induce maximum adhesion. Previous study has indicated the important role of GPIIb/IIIa in platelet adhesion and aggregate size under high shear [Varon, et al; Am Heart J, 1998]. We postulate that RMP may be acting in a GPIIb/IIIa dependent manner to enhance aggregate size. This was consistent with the findings that RMP-induced augmentation of platelet aggregation at relevant levels of ADP or AA concentrations in heparinized blood [Jy, et al; Thromb Haemost, in press]. We are continuing to investigate conditions affecting the interactions between RMP and platelets. Disclosures: No relevant conflicts of interest to declare.

Introduction Blood transfusion is associated with serious adverse events (SAE) such as poor surgical outcome, higher surgical mortality. The cost of transfusion is escalating due to increasing demand and limited blood supply. There is a worldwide growing interest in improving blood utilization methods and storage. Identification of reliable predictors of surgical bleeding is of upmost importance in order to reduce the number of unnecessary transfusions, save limited blood resources, and lower the related costs. Cell derived microparticles (MP) are small membrane vesicles of 0.1 µm in size, released during cell activation and apoptosis. They play an important role in hemostasis and thrombosis. Methods Data presented herein are from an NIH funded prospective randomized study to investigate role of cell derived MP in surgical complications in CABG. Of a total of 122 patients, 81 received transfusion during and/or after surgery (Tx group) whereas the remaining 41 did not (NoTx group). In addition to routine laboratory tests, special assays were performed pre-surgery including concentrations of MP from platelets (PMP), red cells (RMP), endothelia (EMP), and leukocytes (LMP). The two groups were compared with respect to pre-surgical variables in order to assess potential predictors of the need for surgery. Results There were no significant differences between the two groups with respect to most of the pre-surgical demographic, clinical, and lab variables, except that Tx group was associated with higher incidence of female gender, type O blood group, and heparin use. On the other hand, the NoTx group was associated with higher pre-surgical levels of RMP and PMP. Using stepwise logistic regression analyses, four factors were identified as the most significant predictors of the need for transfusion. As depicted by an the areas under the curve (AUC) of the ROC curve, the 95% Confidence Interval of the AUC, and their corresponding p-value for each factor, were as follows. For the model combining all four variables, the AUC and its 95% CI were 0.86 [0.78 - 0.94]. In addition, analysis of post-surgery data revealed better outcomes for the NoTx group in terms of complications such as hours on mechanical ventilation (mean ± SD: 9.2 ± 7.0 vs 15.5 ± 17.1, p = 0.005), number of SAE other than death (0 vs 9 (11.1%), p =0.028), and number of in-hospital deaths (0 vs 5 (6.1%), p= 0.167). Discussion This is the first report to show that pre-surgical levels of circulating MP are highly significant predictors of need for transfusion in CABG. This finding is consistent with other evidence that MP play a major role in hemostasis. Implementation of pre-surgery MP assay will greatly imporove assessment of risk of bleeding in surgery, and requirements for transfusion. This could reduce the number of transfusions, with corresponding improvement in patient outcomes, as well as reduced costs and strains on limited blood supplies. Furthermore, it may be feasible to manipulate pre-surgical MP levels in patients to minimize bleeding and Tx requirements. This could be done by increasing rate of MP generation or retarding rate of clearance, or both. Alternatively, MP could be produced in vitro (autologous or not) for infusion during surgery to minimize blood loss. Such approach is in progress [Jy, et al Thromb Haemost, in press]. Disclosures: No relevant conflicts of interest to declare.

Introduction In thrombotic thrombocytopenic purpura (TTP), autoantibodies bind and deplete ADAMTS13, a vWF cleaving protease, leading to accumulation of large molecular weight vWF multimers, excessive platelet adhesion and aggregation in microvasculature, formation of platelet rich thrombi and disruption of microcirculation. This results in microangiopathic hemolytic anemia, severe thrombocytopenia and multi-organ dysfunction. Under higher shear stress, red cells undergo shape changes, fragmentation and release of red cell microparticles (RMP). Similarly, MP are released from activated platelets (PMP) and endothelium (EMP). It is now well established that cell derived MP play an important role in hemostasis and thrombosis. Roles of circulating MP in TTP are not well elucidated. Exchange plasmapheresis and infusion of FFP is now a standard therapy for TTP. It is assumed that plasmapheresis removes autoantibodies against ADAMTS 13 and plasma infusion supplies the deficient protease to resume normal hemostasis. It is possible that plasmapheresis also removes procoagulant MP, contributing to remission. We studied MP profiles in patients with acute TTP and investigated efficacy of exchange plasmapheresis to remove procoagulant MP. Methods Three patients presenting acute TTP were admitted for exchange plasmapheresis. In addition to routine CBC, platelet counts, reticulocyte counts, blood chemistry, LDH, special assays of MP derived from platelets (PMP), red cells (RMP), leukocytes (LMP) and endothelium (EMP) were performed daily and before and after each exchange plasmapheresis. Samples were drawn immediately before and immediately after plasmapheresis into citrated vacutainer tubes. Microparticles were assayed by flow cytometry using fluorophore conjugated mAb’s specific to endothelial, platelet, leukocyte, or erythrocyte lineage. Thromboelastography (TEG) was employed to study thrombus formation in vitroand correlate microparticles and clinical parameters. Results 1) Effects of plasmapheresis on MP levels: Circulating RMP and PMP levels fell rapidly following ExPh. Each round of plasmapheresis reduced RMP by 45% (p=0.001) from starting baselines. Repeated plasmapheresis over one week removed 74% of RMP from the peak level (p 2) Effects of plasmapheresis on LDH, HGB, and platelets: LDH was reduced by 30% on average over a week period, from 845 ±211U/L to 545 ±33U/L, but this represented only 45% normalization. Platelet count normalized completely, increasing almost fivefold from an average of 46 ±22 to 219 ±68 after seven days (p 3) Effect of plasmapheresis on TEG parameters: Exchange plasmaphresis tended to prolong the R time (8.4 ±2.2 minutes pre vs. 10.9 ±6.1 minutes post). It also increased K time by 38% and reduced CI by 42%. Although these changes did not reach statistical significance due to high degree of variation in the post-plasmapheresis group, the trend indicated plasmapheresis reduced procoagulance and hypercoagulability. Conclusion Exchange plasmapheresis is very effective in reducing procoagulant and proinflammatory RMP and PMP. Clearance of microparticles resulted in improved TEG parameters and reduced hypercoagulability in TTP. Benefits were pronounced even in the first round of plasmapheresis. Exchange plasmaphresis has additional therapeutic benefit in the treatment of TTP by removing procoagulant microparticles and reducing hypercoagulability in TTP. Disclosures: No relevant conflicts of interest to declare.

Background Increasing evidence indicates that natural circulating red cell microparticles (RMP) contribute significantly to hemostasis. Accordingly, we produced RMP in vitro by high pressure extrusion of washed RBCs. We previously reported the hemostatic activity of our RMP product in vitro as well as its efficacy in arresting ear bleeding in rabbits, suggesting the potential of RMP as a hemostatic agent (Jy et al Thromb. Haemost., in press). Here we studied its hemostatic efficacy in a rabbit model of acute liver injury, as well as its short-term toxicity. Methods Male New Zealand White rabbits (mean weight 3.8kg) were randomly selected and assigned, one at a time, to an experimental (n = 9) or a control group (n = 10). All animals were sedated with 35mg/kg ketamine, 5mg/kg xylazine, and 0.01mg/kg glycopyrrolate 15 minutes before surgery. They were intubated and anesthetized with 2% isoflurane, and mechanically ventilated at approximately 20 breaths/min. The carotid artery and the jugular vein were canulated, and maintenance saline was administered at 20mL/hr. The abdomen was cleaned and shaved, and an incision was made from lower tip of the sternum to the bladder. A standardized injury of 9 incisions 30mm long by 4mm deep was inflicted to the liver, and two infusions of RMP (1.25x1011/kg) or saline were delivered via jugular catheter, first immediately post-incision, and again 10 minutes post-incision. Blood was collected in gauze pads at 10 min. intervals and weighed to calculate volume lost. Heart rate, blood pressure (BP), and temperature were continually monitored. The blood pressure (BP) of some of the animals fell very low (MAP Results At early time points (30 – 60 min post-injury) when bleeding was most rapid, administration of RMP had no significant effect: mean (± SD) blood loss at 30 min was 46.1 ± 20.6 mL in the control vs. 26.6 ± 9.0 mL (p=0.07) in the RMP group. At 60 min it was 67.2 ± 24.3 mL vs. 53.4 ± 6.9 mL (p=0.23), respectively. Although at these early time points there were not significant differences, a trend of reduced blood loss was observed. At the 90 min time point, blood loss was 78.6 ± 25.9 mL in the control group compared to 55.7 ± 5.6 mL in the RMP group (p Vitals were not significantly different between the control and RMP groups at any time point. Drop in BP tended to be greater in controls than in RMP rabbits. The difference in BP was marginally significant at 120 min. post-injury: systolic BP fell by 39.6 ±17.2 mmHg in controls vs. 18.6 ±15.0 mmHg in RMP-treated (p=0.07). In a short-term toxicity test, quadruple dose (5.0x1011/kg of RMP) was administered and effects observed for three hours: no obvious adverse effects on heart rate, BP, or temperature were observed. Conclusion/Discussion We previously reported dose-dependent reduction by RMP infusion in ear bleeding time of thrombocytopenic rabbits. Efficacy of infusions of RMP is evident in this severe hemorrhagic liver injury model after 30 - 60 min of acute injury when rapid bleeding was substantially slowing down. At 90 and 120 min post-injury, significantly less blood loss was observed in the RMP group compared to the controls, with relative differences of 30% and 36%. Studies are in progress to improve the model and to optimize dosages and scheduling of RMP administration for severe bleeding. Disclosures: No relevant conflicts of interest to declare.

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