MP-associated Procoagulant Activity Research Articles

Procoagulant Activity of Blood and Endothelial Cells via Phosphatidylserine Exposure and Microparticle Delivery in Patients with Diabetic Retinopathy

Background/Aims: The mechanisms for thrombosis in diabetic retinopathy (DR) are complex and need to be further elucidated. The purpose of this study was to test phosphatidylserine (PS) exposure on microparticles (MPs) and MP-origin cells from the circulation and to analyze cell-/MP-associated procoagulant activity (PCA) in DR patients. Methods: PS-positive MPs and cells from healthy controls (n = 20) and diabetic patients (n = 60) were analyzed by flow cytometry and confocal microscopy. Clotting time and purified coagulation complex assays were used to measure PCA. Results: PS exposure on platelets and monocytes was higher in proliferative DR (PDR) patients than in non-PDR patients or controls. The highest levels of MPs (derived from platelets [30%], erythrocytes [13%], leukocytes [28%], and endothelial cells [10%]) were found in patients with PDR. In addition, PS exposure on blood cells and shed MPs in DR patients led to significantly increased FXa and FIIa generation, fibrin formation, and markedly shortened coagulation time. Moreover, lactadherin reduced 70% of PCA by blocking PS, while an anti-tissue factor antibody had a smaller effect. Conclusion: Our results confirmed that PCA in DR patients may be partly ascribed to PS exposure and MP release from blood and endothelial cells. Lactadherin may act as an efficient anticoagulant factor in this process.

Low Molecular Weight Heparin Modulates Thrombin Generation in Cancer Patients Undergoing Antithrombotic Therapy for Overt Venous Thromboembolism

Introduction The incidence and recurrence rate of venous thromboembolism (VTE) are increased in the cancer compared to the non-cancer population. Low molecular weight heparin (LMWH) is recommended for both initial and long-term anticoagulant therapy for cancer-associated thrombosis, being more effective and safer than vitamin K antagonist therapy. However, failure of anticoagulation with LMWH in cancer-associated thrombosis still remains significantly elevated (VTE recurrence = 9-15%). In this study we tested whether thrombin generation (TG), a global hemostatic assay, may represent a modality to evaluate the LMWH anticoagulant level in vivo and help identifying patients at high risk for VTE recurrences. In a prospective cohort of cancer patients with VTE receiving LMWH nadroparin 200 U.I./Kg once a day, we evaluated whether LMWH treatment modulated the plasma TG capacity, together with other hemostatic parameters, i.e. microparticle (MP)-associated procoagulant activity (PCA) and D-dimer levels. Second we wished to explore whether these parameters may predict for VTE recurrence and/or bleeding.Methods Fifty eight consecutive cancer patients with acute VTE were enrolled into the study. Plasma samples were obtained at the following time intervals: at the thrombotic event (T0), at 1 month LMWH therapy (T1), and at discontinuation (T2), i.e. 6 months after VTE. Patients were followed up clinically for a total 9 months to detect overt thrombotic or bleeding events. TG was measured by the CAT assay, MP-associated procoagulant activity (PCA) by the STA Procoag PPL assay, and D-dimer by HemosIL D-dimer test.Results In this study cohort, the most represented malignancies were colon (25.9%), breast (15.5%), lung (15.5%), and gastric cancer (13.8%). Thirty six patients had metastatic disease, 22 had a limited disease. The VTE sites were: subclavian (36.2%), femoro-popliteal (20.7%), pulmonary (13.8%), jugular (8.6%), and brachial (6.9%) veins. Six patients had simultaneous femoro-popliteal venous thrombosis and pulmonary embolism. At T0 patients were characterized by a procoagulant phenotype as reflected by increased TG potential, and elevated MP-associated PCA and D-dimer compared to controls. In particular, cancer patients displayed a higher levels (p<0.01) of endogenous thrombin potential (ETP) and peak of thrombin vs controls (ETP: 1612±538 vs 1210±344 nMol*min and peak: 305±123 vs 182±84 mMol). These data were accompanied by a significant increase in MP-associated PCA (i.e. shorter clotting time 58±18 vs controls 89±12 sec; p<0.01), and D-dimer plasma levels (1430±1615 vs controls 90±96 ng/ml; p<0.01). During LMWH nadroparin therapy, a significant reduction in both TG potential (ETP-T1: 1254±810 nMol*min) and D-dimer levels (397±697 ng/ml) occurred, and after LMWH discontinuation (T2), TG potential as well as D-Dimer levels rose back, becoming similar to the control values. Differently, no reduction in MP-associated PCA was observed during LMWH therapy. Twenty-seven patients (46.6%) died during follow-up because of cancer disease. No patients had VTE recurrence. The analysis according to the stage of disease showed a significant difference with a higher mortality rate among those with a metastatic stage (p < 0.01). No correlations were observed according to chemotherapy.Conclusion Our results show that LMWH therapy modulates the global thrombin generation capacity and affects the hypercoagulable state of cancer patients, whereas MP-PCA is insensitive to it. In this cohort LMWH treatment was effective (0% VTE recurrences) and safe (1 major bleeding episode). As no recurrences were observed, it was not possible to identify a predictive value for the biological parameters. It is worth to define in a large trial the clinical utility of the TG global coagulation assay to monitor LMWH anticoagulation levels in this high risk population. DisclosuresNo relevant conflicts of interest to declare.

Procoagulant Platelet-Derived Microparticles Are Lower in Calreticulin-Than in-JAK2-Mutated Essential Thrombocythemia

Rationale: Essential Thrombocythemia (ET) is a Philadelphia-negative myeloproliferative neoplasm characterized by an increased risk of thrombosis. Previous reports suggest a role for microparticles (MP) in the pathogenesis of thrombosis in ET. MPs are small plasma membrane vesicles bearing potent procoagulant proteins and phospholipids. They are released into the circulation by various blood and endothelial cells after cellular activation and/or apoptosis. The recently described somatic calreticulin (CALR) exon 9 mutations in almost 20% of ET defines a lower-risk thrombosis ET subtype. We extensively studied phenotype and procoagulant activity of plasma MPs in order to assess MP contribution to the thrombotic risk in CALR versus JAK2 (V617F) mutated ET patients.Patients and methods: We analyzed MP count, phenotype and procoagulant activity in 45 JAK2 V617F+ and 15 CALR+ consecutive and newly diagnosed ET patients in accordance to WHO criteria recruited between november 2010 and april 2013. After given informed consent, blood samples were obtained in all patients before initiating any cytoreductive therapy. Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-Committee and each test was performed in duplicate . Using flow cytometry (FC500 flow-cytometer, Beckman-Coulter), MPs were characterized and measured in platelet-free plasma samples. MPs were characterized by their size and co-expression of bound Annexin V and the following cell-specific monoclonal antibodies: CD41 (Platelet-MP, PMP), CD235a (Red cell-MP, RMP), CD14 (Monocytes-MP, MoMP), CD11b (Granulocytes-MP, GMP), CD144 (Endothelial-MP, EMP), CD62P and CD41 (P-Selectin+ PMP), CD142 and CD41 (Tissue Factor (TF)+ PMP, TF+PMP). MP-associated procoagulant activity was also measured using a-thrombin generation assay (Zymuphen MP-activity). Statistical analysis was performed with SPSS software. Results are expressed as median [interquartile range].Results: Patients characteristics: The platelet count (109/L) was higher in CALR+ than in JAK2+ patients (866 [666 – 918], 659 [571 – 807] respectively, p= 0.049), and all other clinical and hematological characteristics were also distributed in agreement with previous reports. Furthermore, CALR+ patients were preferentially distributed in the lower risk categories of the IPSET-thrombosis and IPSET-survival scores than in JAK2+ patients (p<0.00001 and p= 0.04 respectively).The main results are summarized in table 1. MP count and MP/Platelets ratio were significantly lower in CALR+ than in JAK2+ patients. PMP count, PMP percentage of all MPs and PMP/Platelets ratio were lower in CALR+ than in JAK2+ patients. Furthermore, CALR+ patients had lower PMP surface P-Selectin. MP-associated procoagulant activity/Platelets ratio was significantly lower in CALR+ than in JAK2+ patients. There were no significant differences in TF-carrying PMP or in other cell-derived MP (RMP, MoMP, GMP, EMP).Conclusion: Our results demonstrate a decreased in circulating procoagulant PMP in CALR+ ET-patients compared to JAK2+ patients and lower platelet activation in CALR+ ET patients as measured by P-Selectin expression on PMP. Thus, as CALR+ patients have lower thrombotic-risk according to the literature and the IPSET-survival and IPSET-thrombosis scores observed in our study, the lower level of procoagulant PMP could account, at least in part, for a lower thrombotic risk of CALR+ ET patients.Table 1main resultsCALR +JAK2 V617F +p valueMP (/µL)3289 [1662-4240]4961 [2697-6687]0.04MP/Platelets3.6 [2.8-5.3]6.9 [4.7-9.6]0.01PMP (/µL)3100 [2068-3887]5702 [3423-10257]0.004PMP/Platelets3.38 [2.97-6.33]7.55 [5.12-12.66]0.002% PMP90 [84-92]93 [90-96]0.03MP-activity(nM)/platelet0.015 [0.008-0.028]0.029 [0.019-0.041]0.05P-Selectin+ PMP (/µL)195 [152-219]747 [383-965]0.001RMP (/µL)4 [1.7-8.2]4 [2.5-9.7]nsMoMP (/µL)43 [27-52]74.5 [22-135]nsGMP (/µL)31.5 [24-48]30 [11-55]nsEMP (/µL)99.4 [32-110]95.6 [51-140]nsTF+ PMP (/µL)22.3 [9.8-115]27.1 [20.8-94.4]ns DisclosuresNo relevant conflicts of interest to declare.

Features of Microparticle-Associated Procoagulant Activity in Patients with Thrombocytopenias of Immune and Central Origin

Introduction: The risk of bleeding in patients with thrombocytopenia is increased with platelet counts less than 20 or 30 x 109/L. Nevertheless, some patients with thrombocytopenia of peripheral and central origin (immune thrombocytopenia [ITP] and myelodysplastic syndromes [MDS] respectively) have fewer bleeding symptoms than expected. This fact suggests there may be compensatory mechanisms for the thrombocytopenia, such as the presence of microparticles (MPs).Objective: The aim of this study was to evaluate and characterize the microparticle-associated procoagulant activity in ITP and MDS patients with thrombocytopenia.Methods: Thirty-five patients with chronic ITP and twenty-six patients with MDS with a platelet count less than 50 x109/L and twenty-five healthy controls were included. Blood cell counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 1,500 g for 15 min at 23°C. Platelet-poor plasma obtained was additionally centrifuged twice at 23°C (15 min at 1,500 g, and 2 min at 13,000 g, [PFP]) and aliquots were stored at -70ºC until analysis.Phosphatidylserine-MP (Ph-MP) and tissue factor-MP (TF-MP)-dependent procoagulant activities were determined with the ZYMUPHEN kits (Hyphen BioMed, Neuville sur Oise, France) following the manufacturer’s instructions.The identification of MP’s cell origin was determined by flow cytometry labeling MPs with Annexin-V-fluorescein and the following specific monoclonal antibodies (mAb) conjugated with phycoerythrin: anti CD41 mAb for platelets, anti CD14 mAb for monocytes, anti CD144 mAb for endothelial cells, anti CD235 mAb for red cells and anti CD45 mAb for leukocytes.APRIL plasma levels were determined by ELISA (DuoSet ELISA, R&D Systems, Minneapolis,MN, USA).Results: Ph-MP associated procoagulant capacity in MDS and ITP patients was higher than in controls (p<0.01) whereas MP-TF associated procoagulant activity was only increased in MDS patients and practically negligible in ITP and control groups (p<0.01).MPs analysis by flow cytometry of seventeen controls, twenty ITP and six MDS patients showed that ITP patients had an increased percentage of MPs from platelets and red cells, whereas MDS had an increased percentage of monocytes derived MPs. Proportion of MPs derived from other cell types were similar to the control group.Increased percentage of monocyte-derived MPs in MDS patients is in accordance with the higher MP-TF-associated capacity observed in this group since monocytes are tissue factor rich cells. Nevertheless, no differences were found in monocyte count with control and ITP groups and monocyte count did not correlate with MPs associated procoagulant activity and the percentage of monocyte-derived MPs.In the ITP group neither MP-Ph-associated procoagulant activity nor the percentage of red cell-derived MPs correlated with red cell count. On the contrary, MP-Ph-associated procoagulant activity and the percentage of platelet-derived MPs inversely correlated with platelet count.ITP is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction. A proliferation-inducing ligand (APRIL) is a factor that promotes B-cell maturation and survival. All patients with ITP and thrombocytopaenia showed higher APRIL plasma levels than MDS and control groups (p<.01), which inversely correlated with platelet count and correlated to MP-Ph-associated procoagulant activity. These observations support the proposed pathogenic role of APRIL in the development of this disease and that the increase in platelet-derived MPs was due to peripheral platelet destruction.Conclusion: Peripheral and central thrombocytopenias might present a MP-associated procoagulant activity to compensate bleeding risk present in these patients. Cellular origin of these MPs differed according to thrombocytopaenia etiology. DisclosuresNo relevant conflicts of interest to declare.

Effects of thrombopoietin receptor agonists on procoagulant state in patients with immune thrombocytopenia

Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of immune thrombocytopenia (ITP), an anti-platelet-antibodies autoimmune disease. The observation of a low frequency of bleeding episodes despite their thrombocytopenia suggests the existence of a compensatory mechanism. This study aimed to evaluate the effect of TPO-RA treatment on platelet function and on the procoagulant state in ITP patients before (ITP-bR) and after responding (ITP-aR) to treatment. Plasma- and microparticle (MP)-associated procoagulant capacity from ITP patients was similar before and after responding to the TPO-RA regimen but higher than the healthy control values. High MP-associated procoagulant activity did not seem to be due to increased platelet activation, since platelet stimulation by agonists was reduced in ITP-bR and ITP-aR patients. It could be related to increased platelet apoptosis, evaluated in terms of surface phosphatidylserine (PS), observed in both ITP groups. In summary, TPO-RA treatment increased platelet count but did not ameliorate their function and did not change plasma- and MP-associated procoagulant state of ITP patient responders to this therapy.

Microparticle-Associated Thrombogenic Mechanism Might Compensate Bleeding Tendency in Patients with Myelodysplastic Syndromes.

Abstract 2821 Introduction:Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders with clonal bone marrow anomalies characterized by ineffective hematopoiesis, morphologic and functional hematopoietic cells abnormalities, and an increased risk of transformation to acute myeloid leukemia. Previous work from our lab has shown that MDS patients had an impairment in platelet activation, more externalization of phosphatidylserine (PS) indicating an increased platelet apoptosis and, in some cases, thrombocytopenia. However, these patients do not bleed at the frequency that would be expected taking into account these facts. Objective:The aim of this work was to identify the existence of some compensatory mechanisms that could be counteracting the expected bleeding tendency in these patients.Since apoptosis is accompanied by membrane blebbing and production of PS-exposing procoagulant microparticles (MP) shed from the plasma membrane, we assessed the thrombogenic capacity of MP and evaluate their correlation with exposure of PS. We also studied the ability of plasma to generate thrombin after stimulus. Methods:Seventy-five patients with MDS, 36% female, mean age 77 years (range: 41 to 93 years) and sixty-eight healthy controls, 29% female, mean age 52 years (range 20 to 76 years) were included. Whole blood was collected in citrated tubes and centrifuged at 1,500 g for 15 min at 23°C to obtain platelet poor plasma (PPP). PPP was subjected to 2 additional centrifugations at room temperature (first: 15 min at 1,500 g and second: 2 min at 13,000 g) following SSC-ISTH recommendations to obtain platelet free plasma (PFP). MP procoagulant activity was determined in PFP with the ZYMUPHEN MP-Activity kit (HYPHEN BioMed, Neuville sur Oise, France).Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) as described by Hemker at final concentration of 1pM tissue factor and 4 microM phospholipids. Four CAT parameters were recorded: endogenous thrombin potential (ETP, total amount of thrombin generated over time), lag time (time to the beginning of the explosive burst of thrombin generation), time to peak (TTP, time to reach the maximum thrombin concentration) and peak height (maximum thrombin concentration obtained). Apoptosis was determined by assessing the level of PS externalization by the binding of FITC-Annexin V to platelets by flow cytometry. Thrombocytopenia was defined as platelet count less than 50,000 platelets/microliter. Results:Platelet count in MDS patients varied from 4,000 to 478,000 platelets/microliter. We observed an increased MP-associated procoagulant activity in MDS patients with thrombocytopenia compared to controls (p<0.05). This would be related to the increased apoptosis observed in platelets from MDS patients with thrombocytopenia (Spearman test r = 0.655, p <0.01). CAT evaluation of plasma procoagulant activity did not show differences between patients and controls in any of the measured parameters. Conclusions:Our results showed an increased procoagulant activity associated to plasma MPs in MDS with thrombocytopenia that might contribute to the minor risk of bleeding observed in these patients. However, further studies are necessary to assess the clinical significance of this finding. Disclosures:No relevant conflicts of interest to declare.

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

Introduction Microparticles (MP) can be elevated in cancer and thromboembolic disease. We hypothesized a role for MP in the hypercoagulable state in breast cancer patients using endocrine therapy, in whom both cancer and the use of endocrine therapy are independent risk factors for the development of thrombosis. Design and methods Plasma samples were collected from 40 breast cancer patients using endocrine therapy (20 patients without metastases receiving adjuvant therapy and 20 patients with metastatic disease treated in a palliative setting) and from 20 female healthy controls. The endocrine therapy used was either an anti-estrogen or an aromatase inhibitor. Numbers and cellular origin of MP subsets were analyzed by flowcytometry. MP-associated procoagulant activity was measured using a thrombin generation assay using conditions that allow analysis of MP induced thrombin generation. Results Breast cancer patients using endocrine therapy had higher levels of MP positive for Annexin V (median 10000 vs 6500 × 10E6/l), P-selectin (330 vs 200 × 10E6/l), tissue factor (33 vs 15 × 10E6/l), and of MP derived from platelets (CD41) and leukocytes (CD45). Thrombin generation in plasma was dependent on the presence of MP and thrombin generation performed after addition of isolated MP to normal plasma showed a higher endogenous thrombin potential (1105 vs 1029 nM.min) in breast cancer patients. No differences were observed in MP levels and thrombin generation parameters between the metastatic and adjuvant group. Conclusion Breast cancer patients using endocrine therapy have an increased MP number and a higher MP-dependent thrombin generation, irrespective of the presence of metastatic disease. Altered MP subset characteristics in these patients, especially the higher number of (activated) platelet derived MP and leukocyte derived MP, may in part explain a heightened procoagulant state in breast cancer patients using endocrine therapy.

Prothrombotic state in glioblastoma multiforme: an evaluation of the procoagulant activity of circulating microparticles

The relationship between venous thromboembolism (VTE) and cancer is supported by several pathogenetic factors, including circulating microparticles (MP) originating from different cells and often bearing tissue factor. Since VTE often complicates the clinical course of patients with glioblastoma multiforme (GBM; WHO grade IV astrocytoma) and the role of MPs in these patients population is still not clear, this prospective study was conducted to evaluate the procoagulant activity of circulating MP (MP activity) in GBM patients. We enrolled 61 GBM patients undergoing gross-total or subtotal surgical resection followed by combined radio-chemotherapy; 20 healthy volunteers were tested as controls. Blood samples for MP activity and hemostatic profiles were obtained before and then 1 week and 1, 4, and 7 months after surgery. GBM patients had significantly higher mean MP activity levels than healthy controls before and 7 days after surgery. During the follow-up, MP activity levels became significantly lower 1 and 4 months after surgery (P = 0.007 and P = 0.018, respectively) than prior to surgery, but this decrease was only seen in the subgroup achieving complete tumor resection. MP activity levels increased in 7 (63.6%) of 11 patients who developed VTE. The different incidence of the increase in MP activity levels between patients with and without VTE was statistically significant (χ (2) = 4.93, P = 0.026; relative risk 1.38, 95% CI 1.03-1.86). GBM patients may have an increase in MP-associated procoagulant activity that could contribute to any prothrombotic states and increases the likelihood of VTE complications; this procoagulant activity drops during control of disease.

Microparticle-Associated Thrombin Generation and Procoagulant Activity Is Increased In Patients with Essential Thrombocythemia

AbstractAbstract 1985Essential Thrombocythemia (ET) is a chronic myeloproliferative neoplasm characterized by an increased thrombotic risk. Numerous quantitative and qualitative abnormalities of platelets and leukocytes, arising from the clonal proliferation of hematopoietic progenitor cells, have been described as responsible of the thrombophilic state in this disease. Recently, a high number of plasma microparticles (MP) of different cellular origin has been described in ET patients. MP are a key component of the hemostatic response and have been found increased in diseases at high thrombotic risk. To explore the contribution of plasma MP to the hypercoagulable state of patients with ET, in this study we aimed to characterize the MP functional procoagulant features. We used two different methods: the calibrated automated thrombogram (CAT), to determine the MP-associated thrombin generation (TG), and the P-PPL/1 assay (Stago R&D) to measure the MP-associated procoagulant activity (PCA). Both assays were performed in platelet free plasma (P-FP) obtained from 69 ET patients (24M/45F; 33 carriers of JAK2V617F mutation) and 67 control subjects (32M/35F). In a subgroup of 23 ET patients and 23 controls MP-associated TG and PCA were also determined in MP-free plasma (MP-FP). PFP was obtained by two serial centrifugations (4,000 rpm for 15 min, then 11,000 rpm for 10 min) and MP-FP by re-centrifuging P-FP at 14,000 rpm for 30 min. MP were isolated from the pellet. For the TG assay, 80 ul of P-FP or MP-FP and 20 ul of buffer were mixed, and TG started by adding CaCl2. The results were expressed as lag-time, peak, area under the curve (ETP), and time-to-peak (ttPeak). For P-PPL/1 assay, 100 ul of P-FP or MP-FP were mixed with 50 ul of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds.The results show that P-FP from ET patients generated significantly higher quantity of thrombin compared to controls, as demonstrated by the shorter lag-time (22.1±8.7 vs 29.5±14.7 min; p=0.004) and time to peak (26.6±8.4 vs 33.3±13.2 min; p=0.004) and the significantly greater peak (52.9±24.9 vs 38.24±20.9 nM; p=0.002) and ETP (765.6±206.4 vs 537.5±295 nM*min; p=0.001). Similarly, the MP-associated PCA was significantly increased in ET patients (79±11 sec) compared to controls (89±11 sec; p<0.05). This increase was due to the presence of MP, as no TG and little PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples to the original values of P-FP for both assays. TG was significantly (p<0.05) increased in the JAK2V617F mutation carriers (lag-time: 19.5±7.4 min; peak: 59.8±26.9 nM; ttpeak: 23.9± 4.9 min) compared to wild-type subjects (lag-time: 24.5±9.1 min, peak: 45.4±20.6 nM, ttpeak: 29.3±10.3 min), while no significant differences were found for PCA. Significant correlations were found between the PCA by PPL-assay and the different parameters of TG assay [lag-time (R2= 0.414), peak (R2= -0.542), ETP (R2= -0.514)].In conclusion, our results show that MP-associated TG capacity, as well as PCA, are increased in plasma from ET patients. The highest MP-associated TG was found in JAK-2 mutation carriers, who also are at higher risk for thrombosis compared to wild-type subjects. Our data provide evidence for a contribution of MP to the thrombophilic state of these patients and suggest to test MP associated TG and PCA in prospective studies to predict thrombosis in ET patients. Disclosures:No relevant conflicts of interest to declare.

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.

Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis

Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles (MP) plays an important role in etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells and platelets, exhibits high affinity for PS and has been shown to inhibit several of these PS-mediated pathophysiological processes. We determined plasma annexin A5 levels and MP-associated procoagulant activity, a measure of MP–PS exposure, in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty-five HbAA blood donors served as controls. Both annexin A5 and MP–PS were highest in HbSS patients (5.7 ng/mL, IQR 3.7–7.6 and 37.9 nM, IQR 31.9–69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7–7.6 and 20.9 nM, IQR 10.9–29.6) and healthy controls (2.5 ng/mL, IQR 1.4–4.4 and 13.1 nM, IQR 9.5–18.5) ( p = 0.01 and p < 0.001, respectively). At presentation with a painful crisis, annexin A5 and MP–PS had increased in 16 of 17 patients ( p = 0.001 and p < 0.001, respectively). Most interestingly, in 7 HbSS patients the proportional increase in MP–PS exposure was higher than the proportional increase in plasma annexin A5 concentration, leading to lower annexin A5/MP–PS ratio of HbSS patients during crisis than HbAA controls (0.0027 (0.0017–0.0049) vs 0.0048 (0.0027–0.0085), p = 0.05). In conclusion, patients with SCD have elevated plasma levels of annexin A5- and PS-exposing MP. During crisis both levels increase, but in most HbSS patients MP–PS exposure increases more than annexin A5. Future studies must address a potential role of annexin A5 in modulating PS-related pathophysiological processes in SCD.

Elevated Procoagulant Microparticles Expressing Endothelial and Platelet Markers in Essential Thrombocythemia

Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET).Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay.Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p<0.001).Conclusions: ET patients showed higher number of circulating MPs with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This is confirmed by an increased mature vWF level and an abnormal mature vWF/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for MPs in thrombosis in ET.