René J. Berckmans

Cell-Derived Microparticles Generated in Patients During Cardiopulmonary Bypass Are Highly Procoagulant

BACKGROUND Microparticles from platelets and other cells have been extensively studied and characterized in vitro. Although the level of platelet-derived microparticles is elevated in a variety of diseases, including cardiac surgery, virtually nothing is known about their functions in vivo. The aim of the present study was to investigate the procoagulant properties of microparticles generated in vivo. METHODS AND RESULTS In 6 patients at the end of cardiopulmonary bypass, 14.8 x 10(9)/L (median; range, 9.7 to 27.4 x 10(9)/L) platelet-derived microparticles were present in pericardial blood, whereas blood obtained from the systemic circulation contained 1.6 x 10(9)/L (median; range, 0.4 to 8.9 x 10(9)/L) of such microparticles, as determined by flow cytometry. Microparticles stained positively for phosphatidylserine as determined with labeled annexin V. In contrast to systemic blood, pericardial blood contained not only microparticles of platelet origin but also microparticles that originated from erythrocytes, monocytes, or granulocytes, and other hitherto unknown cellular sources. Plasma prepared from pericardial blood and to a lesser extent plasma from systemic blood obtained at the same time, stimulated formation of thrombin in vitro. This activity of pericardial plasma was lost after removal of its microparticles by high-speed centrifugation, whereas the corresponding microparticle pellet was strongly procoagulant. The generation of thrombin in vitro involved a tissue factor/factor VII-dependent and factor XII-independent pathway. CONCLUSIONS This study is the first to demonstrate that microparticles generated in vivo can stimulate coagulation.

Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease

It has long been known that patients with sickle cell disease have ongoing activation of their coagulation system, which is exacerbated during painful occlusive crises. In this paper, the authors explore the role of the increased numbers of erythrocyte derived microparticles in this phenomenon and suggest that a surprisingly large proportion of this is dependent on Factor XI. See related perspective article on page 1481. Background Sickle cell disease is characterized by a hypercoagulable state as a result of multiple factors, including chronic hemolysis and circulating cell-derived microparticles. There is still no consensus on the cellular origin of such microparticles and the exact mechanism by which they may enhance coagulation activation in sickle cell disease. Design and Methods In the present study, we analyzed the origin of circulating microparticles and their procoagulant phenotype during painful crises and steady state in 25 consecutive patients with sickle cell disease. Results The majority of microparticles originated from platelets (GPIIIa,CD61) and erythrocytes (glycophorin A,CD235), and their numbers did not differ significantly between crisis and steady state. Erythrocyte-derived microparticles strongly correlated with plasma levels of markers of hemolysis, i.e. hemoglobin (r=−0.58, p<0.001) and lactate dehydrogenase (r=0.59, p<0.001), von Willebrand factor as a marker of platelet/endothelial activation (r=0.44, p<0.001), and D-dimer and prothrombin fragment F1+2 (r=0.52, p<0.001 and r=0.59, p<0.001, respectively) as markers of fibrinolysis and coagulation activation. Thrombin generation depended on the total number of microparticles (r=0.63, p<0.001). Anti-human factor XI inhibited thrombin generation by about 50% (p<0.001), whereas anti-human factor VII was ineffective (p>0.05). The extent of factor XI inhibition was associated with erythrocyte-derived microparticles (r=0.50, p=0.023). Conclusions We conclude that the procoagulant state in sickle cell disease is partially explained by the factor XI-dependent procoagulant properties of circulating erythrocyte-derived microparticles.

Synovial microparticles from arthritic patients modulate chemokine and cytokine release by synoviocytes.

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.

Cell-derived vesicles exposing coagulant tissue factor in saliva

On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds.

Prolactin does not affect human platelet aggregation or secretion

Platelets play an important role in the development of plaque formation and in the events after rupture of the atherosclerotic plaque, leading to atherothrombosis. Multiple hormones, either in excess or when deficient, are involved in the development of atherothrombotic disease, but, to which extent such hormones affect platelet function, is still controversial. It was the objective of this study to assess the ability of the pituitary hormone prolactin to affect platelet functions. Venous blood was collected from six healthy males. Platelet activation was studied by (i) flow cytometry in whole blood (exposure of P-selectin as a measure of platelet secretion, and binding of PAC-1 as a measure of ligand- binding conformation of αIIbβ3), and by (ii) optical aggregation and whole blood aggregation. All studies were performed without and with exposure to several concentrations of ADP (0.1, 0.5 and 1.0 μM) and prolactin (50 and 1,000 μg/l). The presence of the prolactin receptor was investigated by Western blot and flow cytometry. In response to either 50 or 1,000 μg/l prolactin, no evidence of platelet activation or aggregation was found. In addition, ADP-induced platelet activation or aggregation was not enhanced by prolactin. Finally, prolactin receptors could not be detected on the surface of platelets. The present data indicate that prolactin does not directly modulate platelet function.

Extracellular vesicles exposing tissue factor for the prediction of venous thromboembolism in patients with cancer: A prospective cohort study

INTRODUCTION The procoagulant activity of extracellular vesicles (EV) exposing tissue factor (TF) is a promising biomarker for venous thromboembolism (VTE) in cancer patients. We evaluated an in-house EV-TF activity assay (the fibrin generation test) for the prediction of cancer-associated VTE. We also compared the results with the fibrin generation tests to an EV-TF-dependent factor Xa generation assay in samples from pancreatic cancer patients. MATERIALS AND METHODS Data collected in a multinational, prospective cohort study were used. Patients with various types of advanced cancer were enrolled if chemotherapy was scheduled or started in the previous 3 months. Patients were followed for 6 months for the occurrence of VTE. The fibrin generation test was performed at baseline to measure EV-TF procoagulant activity. RESULTS The fibrin generation test was performed in 648 patients with advanced cancer. The mean age was 62 years; 58% had distant metastasis. Forty patients (6.1%) developed VTE. Overall, a high fibrin generation test result was associated with a two-fold increased risk for VTE (HR 2.0; 95%-CI, 1.1-3.6). The association was stronger in patients with pancreatic cancer (HR 4.1; 95%-CI, 0.91-19) than in those with other tumor types (HR 1.5; 95%-CI, 0.72-3.1). Correlation between the FGT and the TF-dependent factor Xa generation assay in patients with pancreatic cancer was poor (Spearmans R = 0.35). CONCLUSION This study shows that a high EV-TF procoagulant activity as measured by the fibrin generation test is associated with an increased risk of VTE in cancer patients, in particular in those with pancreatic cancer. Future studies should aim to further improve the feasibility and accuracy of EV-TF activity assays.

Cellular origin and procoagulant activity of tissue factor-exposing microparticles in cancer patients

Background: In patients with cancer, tissue factor-exposing microparticles (TF-exposing MP) have been associated with disease progression and thrombosis. The cellular origin and coagulant activity of TF-exposing MP, however, remain disputed. Therefore, we investigated the cellular origin of the TF-exposing MP and the procoagulant activity in cancer patients. Methods: The cellular origin of TF-exposing MP was investigated by flow cytometry in a cohort of 209 cancer patients (59 pancreatic, 97 gastrointestinal, 23 breast, 15 lung, 5 prostate cancer and 10 other types), and 22 healthy controls. We first determined the numbers of TF-exposing MP in plasma from all patients and measured TF-exposing MP coagulant activity in a fibrin generation test. Based on previous results, a prolongation of the clotting time in the presence of an inhibitory antibody against factor VIIa above 13% was considered abnormal. Second, we selected those patients with numbers of TF-exposing MP above the 95th percentile, and determined the cellular origin of TF-exposing MP in these patients. Results: The numbers of TF-exposing MP were increased in the cancer patients compared to the healthy subjects (median: 2.0 vs 0.40×105/mL; p=0.01). 30% of the cancer patients had an abnormal FGT test, indicating TF-exposing MP coagulant activity. There was no correlation between the number and coagulant activity of TF-exposing MP (r=0.029, p=0.685). 13 patients had TF-exposing MP above the 95th percentile. Of these TF-exposing MP, 5.9% (median; interquartile range (IQR) 0.69-54) double stained with CD227 (MUC-1) and 19% (0.62-41) with CD24, both markers of cancer cells (Fig. 1). Furthermore, 28% (11-63) of the TF-exposing MP stained for CD61, platelet glycoprotein IIIa, 3.9% (0.39-55) for the monocyte LPS-receptor (CD14), while 3.0% (1.4-3.5) of TF-MP stained for the erythrocyte marker CD235, and 0.97% (0.53-4.5) for the granulocyte marker CD66b. (Figure presented) Discussion: Levels of TF-exposing MP in cancer patients are higher than in healthy subjects. Strikingly, but not unexpectedly, we could not demonstrate a relationship between levels of TF-exposing MP and coagulant activity. Therefore, at least part of the TF exposed on circulating MP may be involved in other TF-dependent functions, such as angiogenesis or signal transduction. Most TF-exposing MP seem to originate from cancer cells, as they stained double positive with typical tumour cell markers such as CD227 and CD24. However, most TF-exposing MP also labelled positive for typical blood cell CD antigens, therefore these microparticles might be of mixed cellular origin, being shed after a tumour cell has incorporated blood cell characteristics. This phenomenon is specific rather than an artefact, since no double-labelling was observed with some markers such as CD235.

Microparticle tissue factor activity is increased in cancer patients prior to the development of venous thromboembolism

Primary immune thrombocytopenia (ITP) is characterized by isolated thrombocytopenia. The mechanisms leading to a low platelet count include antibody-mediated destruction of platelets and suppression of megakaryocyte and platelet development. The causes of loss of tolerance and autoantibody production are unknown and it is likely that both genetic and environmental factors are involved. Platelet-specific autoantibodies are directed against a restricted number of ‘dominant’ epitopes of GPIIbIIIa, less frequently of GPIbIX or other platelet glycoproteins. There is evidence to suggest that these autoantibodies are produced from a limited number of clonal B cells by antigen-driven selection. Abnormalities of T cells certainly play a role in causing or perpetuating ITP. Chronic ITP is characterized by a Th1 profile and an impaired T-regulatory cell function, both of which are reversed upon successful treatment. Furthermore, cytotoxic T cells have been shown to cause platelet destruction in vitro and can probably suppress megakaryopoiesis. Chronic infections such as HIV, HCV and Helicobacter pylorican present with isolated thrombocytopenia and therefore mimic ITP. Antibodies cross-reacting with platelet antigens have been identified in thrombocytopenia associated with these infections, supporting a role for molecular mimicry in the development of this disorder. Disclosure of Interest: Honararia for participation on advisory boards and as a speaker at medical education events supported by GlaxoSmithKline, Amgen and RocheThe antiphospholipid syndrome (APS) is an autoimmune disease associated with the presence of antiphospholipid antibodies (APL) and the occurrence of thrombosis and pregnancy complications. One of the assays to detect APL is based on the prolongation of phospho-lipid dependent coagulation assays caused by these antibodies; lupus anticoagulant (LAC). To prevent thrombotic complications, APS patients use long-term anticoagulant treatment that could interfere with LAC determination. Rivaroxaban is a new direct factor Xa inhibitor. In this study we assessed whether rivaroxaban interferes with the detection of LAC. We tested normal pooled plasma (NPP), LAC-positive plasma of SLE patients and LAC-negative plasma from SLE patients. These plasmas were spiked with rivaroxaban. LAC ratios were determined by measuring the LAC-dependent and -independent coagulation times (dRVVT screen/dRVVT confirm, aPTT low and high phospho-lipids, taipan venom and ecarin times). Taipan venom and ecarin are snake venoms that directly activate prothrombin. The taipan venom time is LAC sensitive, whereas the ecarin time is not. Rivaroxaban added to plasma of healthy individuals prolonged the dRVVT LAC ratio, leading to a false positive lupus anticoagulant signal. Rivaroxaban had no influence on the aPTT LAC ratio of normal plasma, but slightly increased the ratio in plasma of SLE patients. For some plasmas negative for LAC, the presence of rivar-oxaban lead to a false positive LAC signal in the aPTT. We observed that the ratio of the taipan venom time over ecarin time remained uninfluenced by the presence of rivaroxaban, both in the absence or presence of antiphospholipid antibodies. This study shows that rivaroxaban can interfere with conventional LAC assays. The taipan venom time/ecarin time ratio may be a good alternative for the detection of LAC in patients using rivaroxaban.Introduction: Cancer greatly increases the risk of venous thromboem-bolism (VTE). Here, we investigated the contribution of microparti-cle-dependent procoagulant activity to the prothrombotic state in these patients. Methods: In 43 cancer patients without VTE at entry and 22 healthy volunteers, markers of in vivo and microparticle-dependent coagulation were measured and patients were prospectively followed for 6 months for the development of VTE. Procoagulant activity of microparticles (MPs) was measured using a phospholipid dependent test (STA Procoag PLL), a factor Xa-generation assay (Xa-assay) with and without anti-tissue factor (TF), and a fibrin generation test (FGT) with and without anti-factor VII(a). Results: Markers of in vivo coagulation activation and total number of circulating MPs were elevated in cancer patients compared to controls (F1+2 246 vs. 156 pM, thrombin-antithrombin complexes 4.1 vs. 3.0 mg/L, D-dimer 0.76 vs. 0.22 mg/L and 5.53 x 106 vs. 3.37 x 106 microparticles per mL; all P <0.001). Five cancer patients (12%) developed VTE during follow-up. Patients with VTE had comparable levels of coagulation activation markers and phos-pholipid dependent MP procoagulant activity. However, TF-mediated Xa-generation (0.82 vs. 0.21 pg/mL, P = 0.016) and the VIIa-dependent FGT (13% vs. 0%, P = 0.036) were higher in the VTE group compared with the non-VTE group.

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.

Prediction of venous thrombosis in cancer patients using a microparticle based clotting test

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 prone to 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 MPassociated 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. Results: 100 patients were included, of which 77 have completed follow-up until now. 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 FGTnegative patients (sensitivity 88%, specificity 77%). Conclusions: 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.