Procoagulant Microparticles Research Articles

169 Does atrial differences in endothelium damage, leukocyte and platelet activation contribute to chamber specific thrombogenic status in patients with atrial fibrillation?

In atrial fibrillation (AF), the reasons why most of the thrombi form in the left atrium are mainly unknown. In the vasculature, endothelial damage together with platelet activation and inflammation contribute to initiation of blood coagulation and thrombus growth. The purpose of this study was to investigate whether atrial-specific differences in endothelial damage, leukocyte activation, platelet stimulation occur in patients with AF. Twenty patients (15 men, 5 women; age 55 ± 8 years, 15 paroxystic AF, 5 persistent AF) with AF undergoing ablation were investigated. Blood samples from the left and right atrium were obtained at the start of the procedure. Procoagulant microparticles (MPs), reliable markers of vascular damage were measured by capture assays. Their procoagulant abilities were quantified by functional prothrombinase assay and their cellular origin were determined (endothelium, platelet, leukocyte). In addition, platelet reactivity was evaluated by whole blood flow cytometry for expression of platelet Pselectin (CD62P), active glycoprotein IIbIIIa receptor (PAC-1). Platelet aggregation was evaluated using Arachidonic acid (AA), ADP, TRAP and collageninduced whole blood aggregometry. No atrial-specific differences in the levels of total procoagulant MP, leukocyte-derived-MP and platelet-derived MP could be evidenced. Conversely, endothelial-derived MPs (CD105+) were slightly elevated in the right atrium (RA 0.96 ± 0.53 vs. LA 0.80 ± 0.45 nm PhtdSer Eq.; p = 0.041). Likewise, collagen-induced platelet aggregation was evidenced in the right atrium (Collagen 1 mg/l RA: 48 ± 33% vs LA 37 ± 29%; p 0.035; collagen 2,5 mg/l RA: 76 ± 25% vs LA: 60 ± 29%; p = 0.001). In patients with AF, endothelial damage and collageninduced platelet aggregation appear slightly more pronounced in the right atrium. Our data did not substantiate the view that chamber specific enhanced thrombogenic status could be a reliable explanation for the increased propensity for thrombus formation observed in the left atrium in AF patients.

200 Safety of transvenous pulmonary vein isolation for the treatment of atrial fibrillation: a prospective randomized study comparing radiofrequency energy with cryoenergy

New transvenous devices using cryoenergy have been recently introduced to perform pulmonary vein isolation (PVI) for the treatment of atrial fibrillation (AF). Experimental data suggested that cryoenergy (CRYO) produced less endothelial disruption and platelet activation than radiofrequency energy (RF) offering safety benefits. We aimed to compare both systems with regards to safety in patients by measuring for the first time sensitive laboratory markers of cell damage. platelet activation and inflammation after a PVI using either one of those energies. Sixty patients with symptomatic drug-resistant AF referred for PVI (56 ± 9 years of age, 48 males. 38 with paroxysmal and 22 with persistent AF) were randomly assigned to undergo the ablation procedure using either an open irrigated tip RF catheter (Thermocool®. Biosense Webster) or a cryoballoon catheter (Arctic front®. Medtronic). Systemic markers of cell damage (procoagulant microparticles [MPs of various cellular origin], troponin T, CK and CK-MB). platelet activation (ADP-induced light transmittance aggregation [LTA], expression of the platelet surface proteins P-selectin [pSEL] and activated GPIIb/IIIa [PAC-1]) and inflammatory response (hs-CRP) were determined frequently before and 4, 24 and 48 hours after the procedure. Procedure time was significantly shorter in patients treated with the cryoballoon (177 ± 30 min versus 200 ± 46 min. p = 0.028), but there were no differences in fluoroscopic time, clinical event rate and success rate. Post-procedural increases of MPs Troponin T and hs-CRP were observed but there were no consistent differences in parameters used for comparative laboratory safety assessment of the ablation systems using either cryoenergy or radiofrequency energy. Neither systematic sensitive markers of cell damage, of platelet activation nor of inflammatory response could detect any difference in the safety profile between cryoenergy and RF energy used for transseptal PVI in patients with AF.

Cellular Mechanisms Underlying the Formation of Circulating Microparticles

Microparticles (MPs) derived from platelets, monocytes, endothelial cells, red blood cells, and granulocytes may be detected in low concentrations in normal plasma and at increased levels in atherothrombotic cardiovascular diseases. The elucidation of the cellular mechanisms underlying the generation of circulating MPs is crucial for improving our understanding of their pathophysiological role in health and disease. The flopping of phosphatidylserine (PS) to the outer leaflet of the plasma membrane is the key event that will ultimately lead to the shedding of procoagulant MPs from activated or apoptotic cells. Research over the last few years has revealed important roles for calcium-, mitochondrial-, and caspase-dependent mechanisms leading to PS exposure. The study of Scott cells has unraveled different molecular mechanisms that may contribute to fine-tuning of PS exposure and MP release in response to a variety of specific stimuli. The pharmacological modulation of MP release may have a substantial therapeutic impact in the management of atherothrombotic vascular disorders. Because PS exposure is a key feature in pathological processes different from hemostasis and thrombosis, the most important obstacle in the field of MP-modulating drugs seems to be carefully targeting MP release to relevant cell types at an optimal level, so as to achieve a beneficial action and limit possible adverse effects.

Increased thrombin generation measured in the presence of thrombomodulin in women with early pregnancy loss

Compared with 537 parous controls with no history of pregnancy loss, a lower thrombomodulin-related inhibition of the endogenous thrombin potential was measured in 264 cases with previous unexplained pregnancy loss, especially when losses occurred between 9 and 12 weeks of gestation. Adjusting age, protein S, factor VIII, factor V Leiden, and prothrombin G20210A did not change the results.

An Important Role for Rac1 and RhoA In Regulating Platelet Microparticle Formation and Phosphatidylserine Exposure

AbstractAbstract 2023Platelets activated by physiological agonists such as thrombin and collagen shed procoagulant microparticles (MPs) and externalize the procoagulant phospholipid phosphatidylserine (PS), both of which are critical to hemostasis and play an important role in inflammation. To date, the signaling mechanisms that regulate agonist-induced MP formation and PS exposure in platelets remain unclear. In this study, we demonstrate that the small GTPases Rac1 and RhoA play important roles in regulating the procoagulant activity of platelets. Rac1 null (-/-) mouse platelets or human platelets treated with the Rac1 inhibitor NSC23766 (NSC) displayed a significant defect in MP formation and PS exposure induced by various agonists. Furthermore, Rac1-/- platelets and NSC-treated human platelets displayed a defect in procoagulant activity as demonstrated by a prolonged coagulation time following recalcification of citrated PRP. The stimulatory role of Rac1 in platelet MP formation and PS exposure is distinct from the known function of Rac1 in facilitating platelet granule secretion and secretion-dependent amplification of platelet aggregation, because supplementation of the granule content ADP rescued the defect in platelet aggregation caused by Rac1 inhibition, but failed to rescue the defect in MP formation caused by Rac1 inhibition. In contrast to Rac1, RhoA plays an inhibitory role in regulating platelet procoagulant activity, because treatment of platelets with the Rho inhibitor C3-toxin (C3) significantly enhanced agonist-induced MP formation, PS exposure, and procoagulant activity. The enhancing effect of C3 on platelet procoagulant activity is not caused by an overall enhancement of platelet activation because C3 significantly inhibited platelet secretion and aggregation. Thus, our data demonstrates that while Rac1 and RhoA both play important stimulatory roles in platelet granule secretion and aggregation, they play opposing roles in MP formation and PS exposure in platelets. Rac1 is important for stimulating platelet MP formation, PS exposure, and procoagulant activity, which is antagonized by RhoA. Disclosures:No relevant conflicts of interest to declare.

P2×7 Signaling Triggers Tissue Factor Activation and Protein Disulfide Isomerase-Dependent Release of TF+ Microparticles In Thrombosis

AbstractAbstract 344Thrombosis induced by the tissue factor (TF) pathway remains a major cause for morbidity and mortality in cardiovascular diseases. The cellular mechanisms of TF activation in primary cells implicated in pathological thrombosis are poorly understood and a controversy exists about the importance of thiol exchange involving protein disulfide isomerase (PDI) versus cell surface exposure of procoagulant anionic phosphatidylserine (PS). Myeloid cells are a source for TF positive (+) procoagulant microparticles (MP). We found that primed bone marrow-derived macrophages had undetectable TF activity. Upon stimulation of the purinergic P2X7 receptor with ATP, TF was readily decrypted. P2X7 signaling induced both the activation of TF cell surface procoagulant activity and thiol- and PDI-dependent generation of procoagulant MP in a pathway that was independent of cell surface PS exposure. Specifically, thiol blockade with dithio-bis-2-nitrobenzoic-acid (DTNB) prevented ATP-triggered release of procoagulant MP without effects on baseline or agonist-induced PS exposure. Similarly, anti-PDI antibody RL90, previously shown to have antithrombotic activities in vivo, attenuated the ATP-induced release of redox active TF+ MP without appreciable effects on cell surface TF activity or PS exposure. Importantly, P2X7 signaling also regulated TF activation and MP release from smooth muscle cells, implicated in TF thrombogenic pathways. Accordingly, P2X7 signaling-deficient mice displayed an attenuated TF-dependent thrombogenic response to an 8% FeCl3·6H2O carotid artery injury. In a more severe 10% injury, P2X7 deficient (-/-) mice eventually formed occluding thrombi that were inhibited by anti-FXI antibody given at a dose without effect in wild-type controls. In contrast, anti-TF did not prevent thrombosis, implying that P2X7-/- mice under these conditions rely on the intrinsic pathway for vascular occlusion and are generally impaired in TF-dependent thrombosis. Bone marrow transplantation experiments showed that P2X7 inactivation on both hematopoietic and vessel wall cells was required for protection from thrombosis. In further studies on this pathway, we identified another anti-PDI monoclonal antibody with unique activating effects that bypassed defective signaling of P2X7-/- cells, directly induced cell surface TF activity, and restored the thiol pathway leading to release of procoagulant MP from P2X7-/- smooth muscle cells and macrophages. The MP released from P2X7-/- macrophages by this antibody were remarkably similar to ATP-induced MP from wild-type mice and carried the thrombus targeting receptor P-selectin glycoprotein ligand-1 (PSGL-1), integrin b1, and PDI. In vivo, the activating anti-PDI antibody induced stable arterial occlusion in P2X7-/- mice challenged with 8% FeCl3·6H2O, and confirmed to be TF dependent since occlusion under these conditions was prevented by a function blocking anti-TF antibody. In conclusion, this study provides novel evidence that cell signaling is required for the activation of prothrombotic TF in vivo, thus challenging the current thinking that loss of vascular integrity or cell injury and PS exposure are the primary triggers for TF-dependent thrombosis. Our findings position cell surface PDI at a central node that controls P2X7-dependent TF cellular decryption and MP release. This novel activation pathway is critical for the generation of prothrombotic TF by vessel wall and blood cells and may be targeted as an alternative strategy to conventional anticoagulant therapy directed towards coagulation proteases. Disclosures:No relevant conflicts of interest to declare.

Erythrocytapheresis in Sickle Cell Disease Lead to An Elective Reduction of Erythrocytes-Derived Blood Microparticles

AbstractAbstract 2657 Introduction:Erythocytapheresis is a highly effective procedure used in the management of both acute and chronic complications of sickle cell disease (SCD). Indeed, exchange transfusion is recommended as a part of the initial treatment of vaso-occlusive stroke in children. Red cells exchange can also be proposed in primary and secondary stroke prevention in order to maintain a low level of HbS. Microparticles (MPs) are small vesicles released from cells after activation or apoptosis. Microparticles number is increased in SCD both at the steady state and during crisis. The majority of these microparticles are originated from erythrocytes and platelets. In SCD, erythrocytes MPs are strongly correlated with hemolysis and activation of coagulation.In this study, we aimed to measure the level of total, platelets-derived and erythrocytes-derived MPs in patients with SCD undergoing erythrocytapheresis; we compared the level of MPs before and after red cells exchange in order to evaluate if the reduction of stroke observed in patients undergoing erythrocytapheresis could be due to a specific decrease in erythrocytes-derived MPs known to be implicated in coagulation activation in this disease. Materials and Methods:After informed consent, 24 samples were harvested on a tube containing buffered sodium citrate from 22 patients with SCD undergoing erythrocytapheresis. For each patient, a sample was collected before and just after the exchange. 20 samples from donor with neither anemia nor coagulation disorders were used as controls. Each sample was centrifugated at 1500g during 15 mn, the supernatant was collected and centrifugated 2mn at 13000g at 20°C and then frozen at -80°C until analysis. For MPs detection, 38mL of the thawed supernatant was labelled with 5mL of 10X Annexine V buffer, 1mL of Annexine V-FITC, 1 mL of anti-GPA-PE and 5 mL of anti-CD41-APC. 5mL of calibrated beads at known concentration were added to 345 mL of 1X Annexine buffer just before analysis on the MoFlow cytometer (Beckman Coulter). The Megamix beads system was used in order to delimitate the FSC/SSC gate of MPs. Results:Total number of plasma MPs (1755/mL vs 273), erythrocytes-derived MPs (174/mL vs 8) and platelets-derived MPs (1520/mL vs 236) were significantly increased in SCD patients in comparison with controls. For SCD patients undergoing erythrocytapheresis, the mean HbS level was respectively 52% and 24% before and after red cells exchange. No significant difference in the total hemoglobin concentration was noted. The mean number of MPs did not differ before and after the exchange (1755/mL versus 1940/mL respectively, student test p>0.05). The mean number of platelets-derived MPs remained stable before and after the exchange (1520/mL versus 1250mL, student test, p>0.05). In sharp contrast, we observed a significant clearance of the erythrocytes-derived MPs in the plasma of SCD patients after red cells exchange (mean number of GPA+-MPs: 174/mL before exchange versus 77mL after, student test, p<0.05). However, we could also observe a great variation of the number of erythrocytes and platelets-derived MPs between the different patients. Conclusion:The number of erythrocytes-derived MPs decreased specifically after red cells exchange, in contrast with the total and the platelets-derived MPs which remained stable; this selective decrease in the GPA+-MPs of SCD patients could be due to a reduction in the total number of red cells containing a high HbS content in the circulation, because of their replacement by normal erythrocytes that have a far lower capacity to produce MPs. It has been recently shown that in SCD, GPA+-MPs are specifically correlated with coagulation and fibrinolysis activation, underlying the fact that a direct link exists between hemolysis, erythrocytes- derived MPs liberation and procoagulant state. The specific clearance of the GPA+-MPs after red cells exchange could partially explain the positive effects of this procedure in primary and secondary prevention of stroke in SCD. However, that GPA+-MPs remained statistically increased after red cells exchange in comparison with controls; whether a low number of post-exchange MPs could be correlated with a better outcome is actually under investigation. Disclosures:No relevant conflicts of interest to declare.

Increased Microparticle-Linked Procoagulant Activity In Patients with Primary Immune Thrombocytopenia.

AbstractAbstract 3707Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by isolated thrombocytopenia (platelet count less than 100,000/μL) and the absence of any obvious initiating and/or underlying cause for the thrombocytopenia. In spite of the low platelet number, some thrombocytopenic patients seldom bleed, indicating the existence of other factors that regulate haemostasis in these patients. Elevated levels of plasma microparticles (MPs) had been observed in IPT patients. MPs are vesicles with a size less than 0.5 micrometers, derived from cell membranes after their activation or apoptosis. Most MPs are highly procoagulant, expressing annexin V binding sites and tissue factor. However, relatively little is known of their specific functions in ITP.In the present study we aim to elucidate if a relationship exists between microparticle-linked procoagulant activity and haemostasis in ITP patients.Twenty-two ITP patients, 3 male and 19 female, aged between 25 to 92 years, were included. Sixteen age- and sex-matched healthy individuals were used as control group.Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-micrometer aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time), which is expressed in seconds.MP procoagulant activity was determined with ZYMUPHEN MP-Activity kit (Aniara, Mason, Ohio) and by calibrated automated thrombography (CAT) in plasma samples obtained after 2 centrifugations at room temperature (first: 15 min at 1,500 g, second: 2 min at 13,000 g). These methods measure endogenous thrombin generation. CAT evaluates four parameters of thrombin generation: the endogenous thrombin potential (ETP), lag time, time to peak (TTP) and peak height.PFA-100® determinations with COL-EPI and COL-ADP cartridges in blood samples from ITP patients with less than 50,000/μL showed longer closure times than control group (p<0.05), whereas samples from ITP patients with a platelet count between 50,000/μL and 100,000/μL showed closure times of the same order of magnitude as control ones (platelet count ranging from 162,000 to 368,000)μL).Plasma from these patients had higher MP-mediated procoagulant activity evaluated with ZYMUPHEN kit (control 6.1+3.9 nM, ITP group 10,1±8.2 nM, p<0.05) as well as with CAT (ETP (nM*min): control: 1692.6±341.9, ITP: 2191,8±398.9, p<0.01; lag time (min): control: 19.9±8.2, ITP: 14.3±4.3, p<0.05; TTP (min): control: 22.0±8.3, ITP:16.3±4.4, p<0.05; peak height (nM): control: 389.7±70.6, ITP: 498,8±97.5, p<0.01).Our results indicate that increased MP procoagulant activity in ITP patients may be protective against bleeding events that should be observed in those thrombocytopenic conditions.Three of the ITP patients included in this study had been splenectomyced and we consider of interest to point out that two of them in spite of recovering a normal platelet count still maintain a high MP procoagulant activity. This observation agrees with a recent work that postulates that MPs might contribute to an increased risk of thrombosis, progression of atherosclerosis and cardiovascular disease following splenectomy (Fontana et al, Thromb Research, 2008;122:59). Disclosures:No relevant conflicts of interest to declare.

Circulating procoagulant microparticles in cancer patients

Accumulating evidence indicates that microparticles (MPs) are important mediators of the interaction between cancer and the hemostatic system. We conducted a large prospective cohort study to determine whether the number of circulating procoagulant MPs is elevated in cancer patients and whether the elevated MP levels are predictive of occurrence of venous thrombembolism (VTE). We analyzed plasma samples of 728 cancer patients from the ongoing prospective observational Vienna Cancer and Thrombosis Study. Study endpoint was the occurrence of symptomatic VTE. Sixty-five age- and sex-matched healthy controls were recruited for defining the cut-off point for elevated MPs (4.62 nanomolar phosphatidylserine [nM PS]), which was set at the 95th percentile of MP levels in healthy controls. The measurement of MPs was performed after capture onto immobilized annexin V, and determination of their procoagulant activity was quantified with a prothrombinase assay. During a median observation period of 710 days, 53 patients developed VTE. MP levels (nM PS) were significantly higher in cancer patients than in healthy controls (median [25th-75th percentile], 3.95 [1.74-7.96] vs. 1.19 [0.81-1.67], p<0.001). Multivariate analysis including age, sex, surgery, chemo- and radiotherapy showed no statistically significant association of the hazard ratio of elevated MPs with VTE (0.95 [95% CI, 0.55-1.64], p=0.856). In conclusion, MP levels were elevated in cancer patients compared to healthy individuals in this study. However, elevated MP levels were not predictive of VTE.

Factors influencing the level of circulating procoagulant microparticles in acute pulmonary embolism

Flow cytometry has shown levels of platelet-derived microparticles (PMPs) and endothelial-derived microparticles (EMPs) to be elevated in deep-vein thrombosis. Cardiovascular risk factors can also contribute to hypercoagulability due to circulating procoagulant microparticles (CPMPs). To investigate in a case-control study the respective contribution of pulmonary embolism and cardiovascular risk factors to the level of hypercoagulability due to CPMPs. CPMP, PMP and EMP levels were measured in 45 consecutive patients (age 67.9 +/- 11.6 years; 66.7% men) admitted to an intensive care unit for acute pulmonary embolism (APE), 45 healthy control subjects with no history of venous thromboembolism or vascular risk factors (Controls(noCVRFs)), and 45 patients with cardiovascular risk factors (Controls(CVRFs)). APE was diagnosed by spiral computed tomography or scintigraphy. CPMP levels were assessed using a prothrombinase assay on platelet-depleted plasma (results expressed as nmol/L equivalent). CPMP levels were higher in APE patients than in Controls(noCVRFs) (medians 4.7 vs 3.2 nmol/L, interquartile ranges [IQRs] 2.9-11.1 vs 2.3-4.6 nmol/L; p=0.02). Similar results were reported for PMPs (medians 2.2 vs 1.9 nmol/L, IQRs 1.7-5.8 vs 1.4-2.4 nmol/L; p=0.02), whereas EMP levels were not significantly different. However, CPMP procoagulant activity was not significantly different in APE patients and Controls(CVRFs). CPMPs and PMPs were significantly elevated in APE patients vs Controls(noCVRFs), but this correlation was not significant when APE patients were compared with Controls(CVRFs). Our observations highlight the importance of adjusting for the presence of cardiovascular risk factors in conditions in which microparticle levels are raised.

The effect of prion reduction in solvent/detergent-treated plasma on haemostatic variables

Octapharma PPGmbH has recently modified its manufacturing process for solvent/detergent-treated plasma to incorporate a prion reduction step, in which a 3 log reduction has been demonstrated. The current study was undertaken to assess the impact of this procedure on haemostatic variables in the new product OctaplasLG in comparison with standard Octaplas. Production batches of standard Octaplas (n=4) and OctaplasLG (n=16) were assessed for levels of coagulation factors, physiological protease inhibitors, markers of activation and procoagulant microparticles. Global haemostasis was assessed by a thrombin generation test (TGT) and rotational thromboelastometry (ROTEM). Mean levels of factors: II, V, VII, IX, X, XI, XII and XIII, VWF:Ag, antithrombin, protein C and free protein S were all >75 u/dl. ADAMTS-13 activity levels were normal. Factor VIII and VWF:RCo were >55 u/dl. TGT and ROTEM were similar in both preparations, and microparticles were present at negligible levels. Two units of OctaplasLG had slightly elevated levels of Prothrombin Fragments 1+2, but D-Dimer and thrombin-antithrombin complexes were normal in all batches. These studies indicate that the affinity chromatography procedure used in OctaplasLG does not appear to adversely affect the proven haemostatic quality of Octaplas, while offering a selective reduction in the concentration of pathological prion proteins.

The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression

Introduction High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs). Material and Methods DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI. Results In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p = 0.02) and to granulocytes (p = 0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p = 0.02-0.002). In the MI population, the DTI reduced the TF activity (p < 0.001). Conclusion DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production.

PO-61 Measurement of procoagulant activity of microparticles by the fibrin generation test as a predictor for venous thrombosis in cancer patients

Background: Although in patients with cancer the risk of venous thromboembolism (VTE) is increased, the incidence is too low to routinely give prophylactic treatment. Procoagulant microparticles (MPs), especially tissue factor (TF)-bearing MPs, contribute to the risk of VTE in cancer patients. In the present study, we assessed the MP-associated procoagulant activity using a functional assay, the fibrin generation test (FGT), to identify cancer patients who will develop VTE. Methods: As an ongoing study, plasma was collected from cancer patients, mainly with stage III or IV pancreatic, gastro-intestinal, breast or lung cancer. The MP-associated procoagulant activity was determined via the FGT with the addition of an inhibitory antibody to factor VII. The prolongation of the clotting time in the presence of anti-factor VII is a measure for the contribution of TF-bearing MPs to the clotting time. Patients were followed up for 6 months. Preliminary results: 100 patients were included, of which 77 had complete follow-up. The first 43 patients were used to establish a cut-off value of the FGT. Receiver operating characteristics showed that a prolongation of the clotting time of 13% after addition of anti-factor VII, was the optimal cut-off value. In the entire group, 8 of 77 patients (10%) developed VTE, of which 7 could have been predicted by the FGT. Using this cut-off value, 23 patients (30%) had a FGT-result above the cut-off (positive test) and 54 patients had a FGT-result below the cut-off (negative test). The prevalence of VTE was 30% in the FGT-positive patients and 2% in the FGT-negative patients (sensitivity 88%, specificity 77%). Conclusion: The FGT seems an excellent predictor for VTE in cancer patients. The next step will be to test the efficacy of prophylactic anticoagulants in patients with cancer and a high thrombosis risk based on the FGT.

PO-51 Elevated numbers and altered subsets of procoagulant microparticles in breast cancer patients using endocrine therapy

PO-51 Elevated numbers and altered subsets of procoagulant microparticles in breast cancer patients using endocrine therapy

Strenuous exercise promotes shear-induced thrombin generation by increasing the shedding of procoagulant microparticles from platelets

Vigorous exercise increases the risk of vascular thrombotic events. Shear stress enhances the shedding of procoagulant microparticles from platelets, and triggers thrombin generation (TG) in blood. This study explicates the manner in which strenuous exercise affects platelet-derived microparticles (PDMP) release and PDMP-mediated TG under various physio-pathological shear flows. Twenty-four sedentary healthy men performed a graded exercise test (up to VO2max) on a bicycle ergometer. At rest, immediately after and 2 hours after exercise, platelet-rich plasma (PRP) was exposed in a cone-and-plate viscometer to imitate static (0 dyne/cm2), physiological low (LS, 10 dyne/cm2) and pathological high (HS, 100 dyne/cm2) shear stresses ex vivo. The PDMP characteristics and dynamic TG were measured by two-colour flow cytometry and calibrated, automatic thrombinography, respectively. The results demonstrated that there is an increased level of total PDMP together with elevated peak height and rate of TG in PRP in response to both LS and HS conditions. Furthermore, HS, but not LS, promoted the binding of FV/Va or FVIII and the exposure of phosphatidylserine (PS) on platelets. Application of HS after strenuous exercise increased the factor (F)V/Va-/FVIII-/tissue factor-rich and PS-exposing PDMP counts, enhanced the PDMP-promoted peak height and rate of TG, as well as increased the ability of FV/Va or FVIII to bind to PDMP or platelets. However, the enhancement of HS-induced procoagulant activity reversed to the pre-exercise status 2 hours following this exercise. Therefore, we conclude that strenuous exercise modestly contributes to the acceleration of shear-induced TG by increasing the release of procoagulant microparticles from platelets.

Circulating procoagulant microparticles in acute pulmonary embolism: A case–control study

We investigated whether circulating procoagulant microparticles (CPMPs) contributed to hypercoagulability in 45 patients with acute pulmonary embolism (APE) and in 45 controls with and 45 controls without cardiovascular risk factors. Concentrations of CPMPs and platelet-derived microparticles (PMPs) were statistically significantly higher in patients with APE than in controls without cardiovascular risk factors. PMPs appeared to be the main source of procoagulant microparticle release in APE, but this correlation disappeared when APE patients were compared to controls with cardiovascular risk factors. CPMPs may have a role in venous thrombosis as mediators of cardiovascular risk factors.

Comparison of Thrombin Generation of Sickle Cell Patients in Microparticle Rich and Microparticle Poor Plasma Using Thrombin Generation Assay (TGA).

Abstract 2557Poster Board II-534 Introduction:The pathophysiology of sickle cell disease (SCD) is complex, with increasing evidence of abnormalities in nearly every component of hemostasis and a pronounced prothrombotic state. Many factors contribute to this prothrombotic state, including: 1) an increase in thrombin production and 2) an increase in circulating procoagulant microparticles. Currently little is known about procoagulant microparticles and their role in thrombin generation in SCD. We have therefore attempted to define the role of thrombin and procoagulant microparticles in SCD utilizing a thrombin generation assay (TGA) and to correlate the results with D-dimer levels, which are known to be abnormal in SCD. The use of TGA allows examination of lag phase, acceleration phase, peak thrombin, and endogenous thrombin potential (ETP). Patients and Methods:The study included a total of 20 HbSS adult and pediatric patients (mean age=22 years, range 3–35) who were admitted for vaso-occlusive crisis. Patients were excluded if they had been admitted within the last 2 weeks, were on chronic transfusion, or were on antiplatelet or anticoagulant medications. Blood was drawn within 36 hours of admission (crisis) and again following discharge, at least 2 weeks later (steady state). Nine of the enrolled patients were followed from inpatient to an outpatient follow up visit. All plasma was promptly separated and frozen, stored at −80C, thawed once and subsequently tested within 4 hours. For each patient, D-dimer and TGA were performed on platelet poor plasma (PPP). In addition, following initial thaw of PPP, samples were spun for 30 minutes at 30,000g to remove microparticles and isolate particle free plasma (PFP). TGA was also performed on PFP from both inpatient and outpatient samples. Results:The mean D-dimer during inpatient crisis (5358 ± 2052 ng/ml) was significantly higher than during outpatient steady state (1256 ± 298, p=0.042). In comparing crisis and steady state by TGA, there was a significant increase in mean ETP (1267 ± 56 nM vs 923 ± 231, p=0.032) and mean acceleration phase (5.14 ± 0.34 min vs 14.21 ± 6.75, p=0.038). Both lag phase (p=0.066) and peak thrombin (p=0.057) were not statistically different between crisis and steady states. Analysis of PPP and PFP by TGA revealed that all phases including ETP (p=0.070) and peak thrombin (p=0.080) were not statistically different between microparticle rich and poor plasma. Linear regression models for inpatient D-dimer versus age were also performed and a significant decrease in D-dimer was seen with increasing age (r2=0.20, p=0.044). This decreasing trend with age was also seen with outpatient D-dimer, although it did not reach significance (r2=0.21, p=0.076). There was no significant trend seen in linear regression models performed for TGA phases versus age. Linear regression models were performed for D-dimer with each phase of TGA and no significant correlation or trend was seen. Conclusions:Our initial results using TGA to evaluate the increase in hypercoagulability seen in patients with SCD during an acute crisis indicate that the expected increase is also seen with certain phases of the TGA, when compared to outpatient non-crisis state. ETP and acceleration phase were significantly elevated during crisis, indicating that the rate of thrombin production and total thrombin are abnormally increased during this time. Peak thrombin and lag phase also approached significance. In evaluating the significance of microparticles, we were unable to show a significant decrease in thrombin production in microparticle free plasma by use of the TGA. With inclusion of both pediatric and adult patients, it was also found that D-dimer significantly decreased with age, which is not consistent with other chronic disease states which exhibit a gradual increase with age. It is possible that a broader age range of patients may be needed to better determine this trend. Finally, comparison of D-dimer with each phase of TGA did not reveal a significant correlation. This may be due to other influencing factors on D-dimer which do not affect TGA. Thus, TGA appears to be a promising technique with which to assess thrombin generation and the hypercoagulable state in SCD; further studies of increased numbers of patients are needed to validate the use of TGA, and define the contribution of microparticles, in SCD. Disclosures:Shah:Thrasher Foundation: Research Funding.

Elevated Levels of Tissue Factor Bearing Microparticles in Patients with Antiphospholipid Antibodies: Analysis of Fresh, Unfrozen Samples.

Abstract 148 Introduction:The antiphospholipid syndrome (APS) is characterized by venous or arterial thrombosis, and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies (APL). The pathogenesis of this disorder may involve, in part, the activation of vascular endothelial cells, platelets, and monocytes. Upon activation, these cells shed components of their plasma membranes as microparticles (MP), and procoagulant MPs are associated with an increased risk for thromboemboli. The goal of these studies was to characterize, for the first time, the frequency by which increased levels of endothelial cell, platelet and monocyte-derived microparticles are observed in freshly-obtained, unfrozen plasma from patients with APL. Patients and Methods:Whole citrated (3.2%) blood samples from patients/healthy donors were centrifuged at 1500xg for 15 min, and the supernatant further centrifuged at 13000xg for 2 min to obtain platelet free plasma (PFP). To detect microparticles, and their origin, PFP was labeled with the following conjugated monoclonal antibodies and analyzed by flow cytometry using an LSR II flow cytometer (BD): Annexin V-APC; CD105-PE and CD144-PE (endothelial cells); CD41-PECy5 (platelets); CD14-PE (monocytes); and anti-tissue factor mAb-Alexa Fluor 647 (a kind gift from Dr. Kenneth Mann, Univ of VT). The binding of control antibodies labeled with identical fluorophores as the monoclonal antibodies was subtracted to adjust for nonspecific binding. We assessed microparticles in 47 patients with APL and 144 healthy controls. ResultsLevels of annexin-V, CD105, CD144, CD41 and tissue factor positive microparticles were significantly higher in patients (47,000±7900, 10,140±2164, 46,690±8783, 127,200±28,050 and 7270±2810 microparticles/ml, respectively) versus controls (17,000±1800, 3,824±374, 17,320±2636, 54,910±7132 and 2,537±747 microparticles/ml, respectively). These differences were statistically significant. In contrast, there was no significant difference in CD14 positive microparticle levels (1035±167 in APL patients versus 1409±465 in controls; p=0.655). Conclusions:Our results demonstrate elevation of annexin V+, CD105+, CD144+, CD41+ and tissue factor-bearing microparticles in freshly-obtained, unfrozen plasma obtained from patients with APL. These results suggest that ongoing activation of platelets and endothelial cells occurs in many patients with APL even in the absence of active thrombosis, and may contribute to the prothrombotic phenotype of these patients. Data is currently being analyzed for associations of microparticle levels with APL serologies, as well as the expression of functional microparticle-associated tissue factor activity. Disclosures:No relevant conflicts of interest to declare.

Plasma Cholinesterase Activity in ITP Patients with/without Thrombosis.

Abstract 3997Poster Board III-933 IntroductionIn the process of immune mediated destruction, platelets may become activated and release procoagulant microparticles in ITP (Immune Thrombocytopenic Purpura). Although bleeding is common manifestation, some patients with ITP may suffer thrombotic complications, often presenting with TIA like syndromes. Brain MRI revealed findings consistent with ischemic small vessel disease in this subgroup (J Lab Clin Med 119: 334, 1992, Thromb Res 107:337, 2002). Recently acetylcoholinesterase (AChE) of RBC and/or non-specific plasma cholinesterase (ChE) was reported to be associated with the inflammation of blood vessels and may be a marker of some inflammatory states. In the present study we investigated plasma ChE activity in patients with ITP with or without thrombotic complication. Methods(i) Patients. We measured ChE activity in 49 patients with ITP. They were sub-grouped as having thrombosis (TBS), F/M (11/7), and non-thrombosis (Non-TBS), F/M (23/8). The TBS group included 9 with TIA like syndrome, 6 with CAD and 3 with venous thrombosis. (ii) Assay of ChE was essentially by Ellman's method. In our system, milli-absorbance units/min (mA/min) x 0.065 = umols substrate cleaved/min. Values reported here are in units of mA/min per mL plasma. Normal controls (NC, n=14) had a cutoff of 3000 mA/min per mL plasma (=mean +2SD). (iii) Sample handling. Platelet-poor plasma (PPP) was prepared by centrifuging 10 min at 1800 xg, then frozen in aliquots. For assay, it was diluted 1:20 with saline, then 5 uL and 10 uL were used in 96-well microtiter plate. Results(i) We observed higher level of ChE in the TBS group compared to Non-TBS in ITP. The TBS group (+/- SD) had mean value 2859 ±866, while the Non-TBS group had 2267 ±777 (units as above). This difference was significant, p<0.02. (ii) In TBS groups, we compared TIA subgroup with other TBS subgroup and observed that the n=9 patients with TIA had higher activity compared to the other TBS: 3250 vs 2466 mA/min per mL plasma. However, this difference did not reach significance in this small patient population. Further, 5/9 TIA patients (66%) had ChE activities >3000 (normal cutoff values) compared to only 2/9 (22%) of those with other TBS >3000. Conclusion(i) The ChE activity of ITP patients with TBS is significantly increased compared to Non-TBS in ITP patients. It has been suggested that Blood ChE/AChE may reflect some inflammatory, prothrombotic states. (ii) ITP patients with TIA exhibited higher ChE activity among TBS subgroups in ITP, suggesting that ChE activity may serve as a useful biomarker in TIA like syndrome associated with ITP. However, further study in a larger number of patients is needed to confirm these preliminary findings. Disclosures:No relevant conflicts of interest to declare.

Thinking small, but with big league consequences: procoagulant microparticles in the alveolar space

pulmonary edema fluid is not just water but is instead a complex biological medium. The daedal nature and variability of its makeup are particularly apparent when samples of alveolar edema or bronchoalveolar lavage fluid from patients with acute lung injury (ALI)/acute respiratory distress syndrome