Aurélie Rousseau

Elevated circulating soluble thrombomodulin activity, tissue factor activity and circulating procoagulant phospholipids: New and useful markers for pre-eclampsia?

OBJECTIVE One of the most frequently proposed mechanisms for pre-eclampsia refers to uteroplacental thrombosis. However, the contribution of classical thrombotic risk factors remains questionable. The aims of this study were to investigate the activities of thrombomodulin, tissue factor and procoagulant phospholipids to assess endothelial cell injury in pregnant women with pre-eclampsia and to compare them with other classical markers of vascular injury and thrombotic risk. STUDY DESIGN Using three new functional assays we studied the plasma levels of these new markers in 35 healthy women, 30 healthy pregnant women, and 35 women with pre-eclampsia. RESULTS We found that plasma levels of thrombomodulin activity, tissue factor activity and procoagulant phospholipids were significantly elevated in women with pre-eclampsia versus normal pregnant and non-pregnant women. CONCLUSION It is thus suggested that elevated levels of these parameters in pre-eclampsia may reflect vascular endothelium damage, and may be a more valuable biomarker than antigen for the assessment of endothelial damage in pre-eclampsia. The high increased levels of procoagulant phospholipids and tissue factor activities in pre-eclampsia could suggest that the procoagulant potential may be implicated in this complication and makes these markers very promising for the understanding, follow-up and therapeutic handling of complicated pregnancy.

Increased levels of tissue factor activity and procoagulant phospholipids during treatment of children with acute lymphoblastic leukaemia

The use of l‐asparaginase (l‐ASP) in paediatric patients with acute lymphoblastic leukaemia (ALL) is associated with thrombotic complications. We evaluated the activities of tissue factor (TFa), thrombomodulin (TMa) and procoagulant phospholipids (PPL) in 26 consecutive children with ALL (25 B‐ALL and one T‐ALL) treated by the French Acute Lymphoblastic Leukemia group (FRALLE)‐2000 protocol. Samples were obtained at diagnosis, after glucocorticoid (GC) therapy, during the induction phase with l‐ASP, vincristine (VCR) and adriamycin (ADR), during the re‐induction and within the week after treatment. Plasma levels of TFa, TMa and PPL increased gradually and significantly during the different phases of the treatment, with higher levels observed during the induction period, and decreased after treatment discontinuation. In vitro studies showed that the different drugs used for ALL treatment could induce a weak expression of TF and procoagulant activity (PCA) on normal and leukaemia blood cells, while a marked effect was observed on endothelial cells. In conclusion, these data indicate that, in addition to the well‐identified increased in coagulation factors and inhibitor deficiencies, the injury of the endothelium could lead to the release of TF and PPL and could contribute to the hypercoagulability of children treated for ALL.

Clinical evaluation of a new functional test for detection of plasma procoagulant phospholipids.

The objective of this study was to validate a simple factor Xa-based clotting test developed to monitor procoagulant phospholipids (PPLs) and platelet-derived microparticles (PMPs). This assay is easily automated, giving it a major advantage over the more laborious and expensive flow cytometry, electron microscopy and ELISA techniques in general usage at present. The intra-assay and inter-assay variation coefficients were less than 5% at both low and high levels of PPLs. The test is not affected by other clotting factors is assured by the use of a phospholipid-free animal plasma, which provides excess factor V, fibrinogen and prothrombin. This test was evaluated in apparently healthy volunteers and in selected patient groups associated with increased levels of PMPs in the circulation (diabetes mellitus, sickle cell disease, thyroid cancer and patients with multiple trauma). The study showed that XaCT has a high discriminating power for PPLs and that the patient groups have significantly highly increased PPLs activities when compared with healthy volunteers. Although of a preliminary nature, the test has shown that it has the sensitivity for discriminating severity of disease, as it could detect patients in sickle cell crisis and differentiate between type 1 and 2 diabetes. In conclusion, the combination of reliability, reproducibility and easy performance makes the XaCT assay a simple test to screen for PPLs in plasma samples.

Plasma thrombomodulin activity, tissue factor activity and high levels of circulating procoagulant phospholipid as prognostic factors for acute myocardial infarction.

Several studies have indicated an association between haemostatic markers and acute myocardial infarction, but few or no studies refer to their activity. We studied plasma levels of 10 coagulation factors (fibrinogen, protein C, protein S, von Willebrand factor, D-dimers, factor VIIa, free tissue factor pathway inhibitor, tissue-type plasminogen activator, plasminogen activator inhibitor-1, thrombomodulin) and using new specific assays analysed the activity of plasma tissue factor (TFa), thrombomodulin (TMa), and procoagulant phospholipid in 46 consecutive patients with acute myocardial infarction at the time of hospital admission, and compared them with 34 healthy normal volunteers. Plasma levels of TFa, TMa, and procoagulant phospholipid were significantly higher in cases than in control patients (P < 0.001). In addition the ratio of TFa/free tissue factor pathway inhibitor was higher in patients than in controls, whereas the tissue-type plasminogen activator (t-PA)/plasminogen activator inhibitor-1 ratio was lower in patients. Interestingly, patients with an unfavourable outcome during a 2-month follow-up had higher levels of TFa, TMa, procoagulant phospholipid, a higher ratio of TFa/free tissue factor pathway inhibitor and a lower ratio of t-PA/plasminogen activator inhibitor-1 than patients who recovered. The combination of these different parameters reveals an increase in procoagulant activity as well as impaired fibrinolytic activity during the acute phase of an acute myocardial infarction. The association of the level of the activity of these three factors may provide a new tool to assess the prognosis of acute myocardial infarction. Further studies are needed to support our findings and to elucidate the clinical interest of measuring these factors.

Venous thromboembolism in patients with pancreatic cancer: implications of circulating tissue factor

Among cancers, pancreatic cancer is known to be associated with a higher incidence of venous thromboembolism (VTE). The aim of the study was to determine the implication of circulating tissue factor (TF) in VTE related to active pancreatic cancer. One hundred and sixty-four consecutive patients who participated to the Etude des Determinants et Interactions de la Thrombose veineuse (EDITH) study between January 2005 and August 2007 for symptomatic VTE related to active pancreatic cancer (n = 8), active cancer of other location (n = 42) or classified as unprovoked (n = 114) were included. TF activity (TFa) was measured in a one-stage kinetic chromogenic method. There were no differences of median TFa levels between patients with VTE related to cancer of other type than pancreas [2.01 pmol/l range (0.05–43.92)] and patients with unprovoked VTE [1.78 pmol/l (range 0.05–63.72), P = 0.21]. Median TFa levels were higher in patients with VTE related to pancreatic cancer [12.67 pmol/l (range 0.05–112.04)] than in patients with VTE related to cancer of other type [2.01 pmol/l (range 0.05–43.92), P = 0.02]. Higher levels of circulating TFa during the course of pancreatic cancer may explain the higher incidence of VTE associated with this type of cancer.

Prevalence of antiphospholipid antibodies in psychiatric patients users and non-users of antipsychotics

Past reports have suggested that antiphospholipid (aPL) antibodies may emerge as a response to antipsychotics treatment, as a high prevalence of aPL antibodies in antipsychotics users has been observed. However, no control group of non‐medicated psychiatric patients was included in these reports. In a cross sectional study we determined the prevalence of aPL antibodies in 333 psychiatric inpatients. We compared the proportions of positive aPL antibodytests between users and non‐users of antipsychotics with adjustments for potential confounders. The proportion of antipsychotics users carrying at least one aPL antibody ranged from 10·8% to 27·0% compared with 6·8% to 27·2% in non‐users (P = 0·24, P = 0·24) depending on the method of detection of lupus anticoagulant (LA). The prevalence of LA detected by dilute Russell viper venom time or partial thromboplastin time‐LA was not different between antipsychotics users and non‐users (8·1% vs. 5·4%, P = 0·53 and 18·4% vs. 18·2%, P = 0·22), as well as the prevalence of IgM and IgG anti‐β2‐glycoprotein‐I antibodies, IgM and IgG anti‐cardiolipin antibodies(3·8% vs. 2·0%, P = 0·75, 0·0% vs. 0·0%, P = not applicable, 1·1 vs. 1·4%, P = 0·91, 2·7% vs. 3·4%, P = 0·71). In conclusion, aPL antibodies were frequently found in patients with psychiatric diseases and no significant increase in the prevalence of aPL antibodies was observed in antipsychotics users.

Contribution of procoagulant phospholipids, thrombomodulin activity and thrombin generation assays as prognostic factors in intensive care patients with septic and non-septic organ failure.

Abstract Background: Multiple organ dysfunction syndrome (MODS) observed in patients with sepsis and in non-septic patients organ failure (OF) is associated with a high mortality rate. We investigated whether new coagulation assays [quantification of procoagulant phospholipids (PPL) activity, functional assays measuring the activity of thrombomodulin (TMa) or tissue factor (TFa) and thrombin generation using calibrated automated thrombography (CAT)] could constitute new tools to better understand the physiopathology of MODS and have any prognostic value. Methods: We measured TMa, TFa, PPL and CAT in 32 healthy controls, 24 patients with sepsis and 26 patients with non-septic OF. We compared these parameters with usual coagulation assays [prothrombin time, activated partial thromboplastin time, protein C (PC), protein S, D-Dimers (D-Di), soluble thrombomodulin (sTM)] and markers of inflammation (IL-6, CRP). Samples were collected within 24 h of the diagnosis. Results: TMa, TFa, PPL, the lag time and time to thrombin peak levels were increased in both groups of patients. For both groups D-Di, IL-6, CRP and endogenous thrombin potential (ETP) were higher in non-survivors than in survivors, while PC and PPL were lower in non-survivors than in survivors. TMa increase was more marked in non-survivors patients with OF, while the ratio TMa/sTM was low in non-survivors with sepsis. Received operating characteristic (ROC) curve analysis indicated that thrombin peak and ETP were the more powerful discriminating factors in patients with sepsis or non-septic OF, respectively. Conclusions: PPL, TMa and CAT assays could represent promising tools to identify patients with increased risk of mortality in MODS and could procure insights into pathogenesis of MODS.

Comparative evaluation of Tissue factor and Thrombomodulin activity changes during normal and idiopathic early and late foetal loss: The cause of hypercoagulability?

Various components of the coagulation and fibrinolytic pathways are involved in normal embryonic implantation, trophoblast invasion, placentation, and recurrent miscarriages are characterized by defective placentation and microthrombi in the placental vasculature. Although recurrent miscarriage is a heterogeneous condition the relationship between abnormalities in the haemostatic pathways and pregnancy outcome is increasingly recognized. The challenge we face is how to discriminate between women who are destined to miscarry from those whose pregnancy will be successful. Considering the crucial role of thrombomodulin and tissue factor in coagulation and in embryonic development, we have performed a study using specific assays for thrombomodulin, tissue factor activity and procoagulant phospholipids in association with other parameters in 30 early (under 12weeks) and 32 late (over 22weeks) pregnancy loss women and compared them with 62 normal pregnancy women and 35 non-pregnant women. Plasma levels of tissue factor activity, thrombomodulin activity, and procoagulant phospholipids were significantly higher in patients than in control subjects. In addition the tissue factor activity/free tissue factor pathway inhibitor ratio was higher in patients than in controls. Interestingly, patients with late pregnancy loss had higher tissue factor activity/free tissue factor pathway inhibitor ratios than patients with early pregnancy loss. The combinations of these different parameters reveal an increase in procoagulant activity which could be secondary to endothelial damage or coagulation activation and then are involved in the pathogenesis of pregnancy loss. Their simultaneous measurement of these activities might provide a new tool to assess the prognosis of pregnancy loss.

Decreased activity of soluble thrombomodulin and plasma procoagulant phospholipids in childhood bone marrow transplantation with severe complications

BACKGROUND Complications of bone marrow transplantation (BMT) are usually considered to be related to the secretion of inflammatory cytokines, which generate membrane microparticles rich in procoagulant phospholipids (PPL) from different cellular origins and release of endothelial proteins such as thrombomodulin (TM). The use of soluble TM quantified by ELISA (TM:Ag) as a marker of endothelial injury is complex in children since it is age-dependent. MATERIALS AND METHODS Using a functional assay which quantifies the activity of sTM activity (TMa), we performed a pilot study to analyze the ratio TMa/TM:Ag in a control group of 25 healthy children, 8 children with autologous and 16 children with allogeneic BMT. In this last group, 8 experienced BMT complications. In addition, we used a functional assay which quantifies PPL. RESULTS In healthy children the ratio TMa/TM:Ag was independent of age and stable in children with a favorable outcome but significantly (p<0.05) reduced by the use of antithymocyte globulin during the conditioning regimen, and regularly decreased in children with BMT complications. Surprisingly, low plasma PPL levels were associated with a poor outcome. CONCLUSION The ratio TMa/TM:Ag could constitute a marker of endothelium injury, and its follow-up could be of interest for an early discrimination of children with high risk of complications during allogeneic BMT. The decrease of PPL could be also another marker of a poor evolution and deserves further investigations.

Decreased procoagulant phospholipids in patients treated by vitamin K antagonists

INTRODUCTION The stimulation of cells by thrombin is associated with the release of microparticles (MPs) from cell membranes. These MPs can express procoagulant activity. As vitamin K antagonists (VKA) decrease the generation of thrombin, we compared plasma procoagulant phospholipids (PPL) levels in patients with a previous history of venous thrombosis who were being treated with VKA and compared them with an untreated group. MATERIALS AND METHODS Plasma PPL were measured using a factor Xa-based coagulation assay. sGPV, a marker of platelet activation by thrombin, was measured by ELISA. Platelet MPs were also evaluated using standard flow cytometric techniques. Ninety-six VKA-treated patients and 80 patients not undergoing VKA therapy were tested and the results compared. RESULTS PPL activity was significantly reduced (p<0.0001) in VKA-treated patients compared with the untreated group. PPL were correlated with platelet and white blood cell count and with sGPV levels in the untreated group, but not in VKA-treated patients. PPL were correlated with fibrinogen levels in both groups, but not with C-reactive protein. Polymorphonuclear neutrophils (PMN) were significantly lower (p=0.01) in VKA-treated patients than in untreated patients. CONCLUSION The difference between PPL levels in VKA-treated patients and patients without treatment could be related to the decrease in PMN count. It remains to be established if this decrease of PPL is directly related to the capacity of activated PMN to generate MPs, or indirectly by reducing the amount of pro-inflammatory cytokines or reactive oxygen species produced by PMN.