The procoagulant signature of cancer cells drives fibrin network formation in tumor microenvironment and impacts its quality. Implications in cancer cell migration and the resistance to anticancer agents.

INTRODUCTION: Cancer cells induce hypercoagulability in the tumoral microenvironment by expressing Tissue Factor (TF). We aimed to study the impact of the procoagulant signature of cancer cells on the quality and structure of fibrin network. We also studied the impact of fibrin clot shield (FCS) on the efficiency of anticancer agents and the migration of cancer cells. MATERIALS AND METHODS: Pancreatic cancer cells BXPC3 and breast cancer cells MDA-MB231 and MCF7, were cultured in the presence of normal Platelet Poor Plasma (PPP), diluted 10 % in conditioning media. Their potential to induce thrombin generation and their fibrinolytic activity were assessed. The structure of fibrin network was analyzed with Scanning Electron Microscopy (SEM). Cancer cells' mobility with fibrin clot and their interactions with fibrin were observed. Cancer cells were treated with paclitaxel (PTX) or 4-hydroxy-tamoxifen (4OHTam) in the presence or absence of FCS. RESULTS: Cancer cells, in presence of PPP, induced fibrin network formation. High TF-expressing cancer cells (BXPC3 and MDA-MB23 cells), led to dense fibrin network with fine fibers. Low TF expressing cells MCF7 led to thick fibers. Exogenous TF enhanced the density of fibrin network formed by MCF7 cells. Cancer cells through their inherent profibrinolytic potential migrated within the fiber scaffold. The BXPC3 and MCF7 cells moved in clusters whereas the MDA-MB231 cells moved individually within the fibrin network. FCS decreased the efficiency of PTX and 4OHTam on the viability of cancer cells. CONCLUSIONS: The procoagulant signature of cancer cells is determinant for the quality and structure of fibrin network in the microenvironment. Original SEM images show the architecture of "bird's nest"-like fibrin network being in touch with the cell membranes and surrounding cancer cells. Fibrin network constructed by triggering thrombin generation by cancer cells, provides a scaffold for cell migration. Fibrin clot shields protect cancer cells against PTX and 4OHTam.

Adherence with brand versus generic bisphosphonates among osteoporosis patients: a new-user cohort study in the French National Healthcare Insurance database.

Several studies documented declines in treatment adherence with generic forms of oral bisphosphonates in osteoporosis compared to branded forms, while others did not support this relation. Our aim was to compare medication adherence with brand versus generic forms of oral bisphosphonates. A new-user cohort study was conducted using routinely collected administrative and healthcare data linked at the individual level extracted from a nationwide representative sample of the French National Healthcare Insurance database. We included all patients aged 50 and older, new users of oral bisphosphonates for primary osteoporosis between 01/01/2009 and 31/12/2015. Two components of adherence were measured: implementation (continuous multiple-interval measure of medication availability version 7; CMA7) and persistence (time to discontinuation). The sample was composed of 1,834 in the "brand bisphosphonate" group and 1,495 patients in the "generic bisphosphonate" group. Initiating oral bisphosphonate treatment with brand was associated with a higher risk of discontinuation within 12 months (Hazard Ratio = 1.08; 95%CI = [1.02;1.14]). The risk of good implementation (CMA7 ≥ 0.90) was significantly lower in "brand bisphosphonate" group (Risk Ratio = 0.90; 95%CI = [0.85; 0.95]). We did not find any evidence to support the hypothesis of a lower adherence to generic bisphosphonates. In fact, prescribing of generic bisphosphonates led to a higher persistence rate and to better implementation at 1 year.

Extracellular vesicles derived from pancreatic cancer cells BXPC3 or breast cancer cells MCF7 induce a permanent procoagulant shift to endothelial cells.

The endothelium could be a potential target of cancer cell derived extracellular vesicles (CaCe-dEV). We investigated in vitro the effect of CaCe-dEV on the hemostatic balance of endothelial cells. Extracellular vesicles released from pancreas adenocarcinoma cells (BXPC3) or human breast cancer cells (MCF7) were isolated by differential centrifugation. Human umbilical vein endothelial cells (HUVEC) were cultured for 72 h in the presence or absence of CaCe-dEV. Subsequently, they were washed and re-cultivated over three cycles to get daughter cell generations (DG) which were not exposed to CaCe-dEV. Thrombin generation of normal platelet poor plasma (PPP) added in wells carrying HUVEC was assessed by the Calibrated Automated Thrombogram®. Tissue factor activity (TFa) and procoagulant phospholipid clotting time were assessed. Some traces of TFa were displayed by non-exposed HUVEC (0.18 ±â€¯0.03 pM) and their EVs (1.2 ±â€¯1.0 pM). Non-exposed HUVEC did not induce any detectable thrombin generation. BXPC3-dEV displayed significantly higher TFa as compared to MCF7-dEV (45 ±â€¯5 pM versus 4.6 ±â€¯2.3pM respectively; p < 0.05). HUVEC exposed to CaCe-dEV enhanced thrombin generation. BXPC3-dEV induced significantly higher thrombin generation as compared to those exposed to MCF7-dEV. The procoagulant properties of HUVEC, acquired upon exposure to CaCe-dEV were transferred to DG. In conclusion, CaCe-dEV lead to a procoagulant shift of endothelial cells which, upon exposure, display TFa and enhance thrombin generation which is transferred to DG of HUVEC. The potency of CaCe-dEV to induce procoagulant shift of HUVEC depends on the histological type of the cancer cells. The procoagulant shift of endothelial cells which is transferable to DG could be an additional mechanism - together with cancer-induced blood hypercoagulability - in the pathogenesis of cancer associated thrombosis.

Differential contribution of tissue factor and Factor XII to thrombin generation triggered by breast and pancreatic cancer cells.

Most cancer cells trigger thrombin generation (TG) to various extent. In the present study, we dissected the mechanisms responsible for the procoagulant activity of pancreatic adenocarcinoma cells (BXPC3), a highly thrombogenic cancer type, and breast cancer cells (MCF7), a less thombogenic tumor type. TG of normal plasma was assessed by the Thrombinoscope (CAT®) in the presence or absence of cancer cells. TG was also assessed in plasma depleted of clotting factors, in plasma spiked with tissue factor (TF) and/or procoagulant phospholipids, in plasma spiked with an anti-TF monoclonal antibody or with corn trypsin inhibitor (CTI). The presence of alternatively spliced TF (asTF), TF activity (TFa) and cancer procoagulant (CP) levels were determined. TFa and asTF were highly expressed by BXPC3 cells, compared to MCF7 cells, while CP levels were higher in MCF7 cells. BXPC3 cells had a stronger effect on TG than MCF7 cells. Accordingly, anti-TF had more inhibitory activity on TG triggered by BXPC3 cells while CTI had more pronounced inhibitory effect on TG triggered by MCF7 cells. TG enhancement by both BXPC3 and MCF7 cells was mediated by FVII and intrinsic tenase while FXII and FXI were also important for MCF7 cells. The induction of TG by BXPC3 cells was mainly driven by the TF pathway while TG generation triggered by MCF7 cells was also driven by FXII activation. Therefore, hypercoagulability results from a combination of the inherent procoagulant properties of cancer cell-associated TF as well as of procoagulant phospholipids in the plasma microenvironment.

Procoagulant microparticles derived from cancer cells have determinant role in the hypercoagulable state associated with cancer.

Hypercoagulablity is a common alteration of blood coagulation in cancer patients. However, the procoagulant activity of cancer cells is not sufficient to induce hypercoagulability. The present study was aimed to identify the mechanism with which hypercoagulabilty is produced in the presence of cancer cells. We focused on the analysis of the procoagulant elements carried by cancer cell-derived microparticles (CaCe-dMP) and we evaluated the impact of microparticles associated with the cancer cells from which they stem on thrombin generation. CaCe-dMP from the cancer cells were isolated from the conditioned medium and analyzed for tissue factor (TF) and procoagulant phospholipid expression. Thrombin generation of normal plasma was assessed by the Thrombinoscope (CAT®) in the presence or absence of pancreas adeno-carcinoma cells (BXPC3) or breast cancer MCF7 cells supplemented with the respective CaCe-dMP. Both BXPC3 and MCF7 cells express abundant amounts of active TF. Phosphatidylserine was identified on the surface of CaCe-dMP, unlike the cancer cells themselves. The expression of TFa by the microparticles was significantly higher to that observed on the cancer cells. Culture of the cancer cells with their microparticles resulted in thrombin generation significantly higher as compared to the upper normal limit. In conclusion, cancer cells 'enrich' the microenvironment with procoagulant elements, especially procoagulant micro-particles which express TF and procoagulant phospholipids. The association of cancer cells with procoagulant microparticles is necessary for a state of hypercoagulability, at the level of the tumoral microenvironment. The intensity of the hypercoagulability depends on the histological type of the cancer cells.

Cancer cells BXPC3 and MCF7 differentially reverse the inhibition of thrombin generation by apixaban, fondaparinux and enoxaparin.

INTRODUCTION: Cancer cells may alter the efficiency of the antithrombotic agents. To explore this possibility, the present study compared the capacity of the LMWH enoxaparin and the specific inhibitors of Xa (apixaban and fondaparinux) to inhibit thrombin generation triggered by pancreas adenocarcinoma cells (BXPC3) and human breast carcinoma cells (MCF7). MATERIALS AND METHODS: Samples of platelet poor (PPP) or platelet rich plasma (PRP) spiked with apixaban, fondaparinux or enoxaparin were added in micro wells carrying cancer cells and assessed for thrombin generation. In the control experiment thrombin generation was triggered with tissue factor reagent. RESULTS: The three antithrombotics inhibited thrombin generation in a concentration dependent manner. The BXPC3 and MCF7 cells reversed in a different intensity the effect of the studied agents. According to the histological type of the cancer the antithrombotic efficiency of apixaban was preserved or partially reversed. Fondaparinux, was more vulnerable to the presence of cancer cells as compared to apixaban. The effect of BXCP3 or MCF7 cells on the antithrombotic potency of enoxaparin was of similar magnitude as that on apixaban. CONCLUSIONS: The type of cancer cells is determinant for the antithrombotic efficiency of the specific factor Xa inhibitors. In contrast it does not significantly influence the potency of enoxaparin. The present study shows that the impact of the type of cancer cells on the antithrombotic activity of the specific Xa inhibitors should not be neglected. This has to be taken into consideration for the design of dose-finding studies of the direct orally active FXa inhibitors in patients with different histological types of cancer.

Impact of breast cancer stage, time from diagnosis and chemotherapy on plasma and cellular biomarkers of hypercoagulability.

BACKGROUND: In breast cancer patients routine thromboprophylaxis is not recommended but individualized risk assessment is encouraged. The incorporation of hypercoagulability biomarkers could increase the sensitivity of risk assessment models (RAM) to identify patients at VTE risk. To this aim we investigated the impact of cancer-related characteristics on hypercoagulability biomarkers. METHODS: Thrombin generation (TG) assessed with the Thrombogramme-Thrombinoscope®, levels of platelet derived microparticles (Pd-MP) assessed with flow cytometry, procoagulant phospholid dependent clotting time (PPL-ct) measured with a clotting assay and D-Dimers (were assessed in a cohort of 62 women with breast cancer and in 30 age matched healthy women. RESULTS: Patients showed significantly higher TG, Pd-MP, D-Dimers levels and shortened PPL-ct compared to the controls. The PPL-ct was inversely correlated with the levels of Pd-MP, which were increased in 97% of patients. TG and D-Dimers were increased in 76% and 59% of patients respectively. In any stage of the disease TG was significantly increased as compared to the controls. There was no significant difference of TG in patients with local, regional of metastatic stage. There was no significant difference in Pd-MP or Pd-MP/PS+ between the subgroups of patients with local or regional stage of cancer. Patients with metastatic disease had significantly higher levels of Pd-MP and Pd-MP/PS+ compared to those with regional stage. The D-Dimers increased in patients with metastatic stage. In patients on chemotherapy with less than 6 months since diagnosis TG was significantly higher compared to those on chemotherapy who diagnosed in interval > 6 months. Patients with metastatic disease had significantly higher levels of Pd-MP and D-Dimers compared to those with non-metastatic disease. CONCLUSION: In breast cancer patients the stage, the time elapsed since the diagnosis and the administration of chemotherapy are determinants of cellular and plasma hypercoagulability. The levels and the procoagulant activity of Pd-MP are interconnected with the biological activity and the overall burden of cancer. TG reflects the procoagulant properties of both breast cancer and chemotherapy in the initial period of cancer diagnosis. Thus the weighted incorporation of the biomarkers of cellular and plasma hypercoagulabilty in RAM for VTE might improve their predictive value.

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-ß2-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.

BACKGROUND: Multiple organ dysfunction syndrome (MODS) observed in patients with sepsis and in nonseptic 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.

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.

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.

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.

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.

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.

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.

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.

Human seminal plasma fibrinolytic activity.

The distribution of total fibrinolytic activity in seminal plasma, as well as specific tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor (PAI), has been studied using antigen and activity techniques in 170 ejaculates of men attending for assessment because of infertility without genital urinary pathology. Among these 170 patients, 18 showed oligoasthenoteratospermia, 28 showed azoospermia, and 124 showed normozoospermia. The seminal values were 50 times higher (262 to 289 ng/mL in antigen and 179 to 199 x 10 (3) IU/L for activity) than values in blood for t-PA and 15 times higher than values in blood for u-PA (18.4 to 26 ng/mL and 1.5 to 2.4 IU/mL, respectively). There was no correlation between the two levels in antigen or activity, but a higher concentration was observed in all first fractions from split ejaculates measurements. Moreover, t-PA was significantly lower in semen with abnormal liquefaction compared with semen exhibiting normal liquefaction. Zymography confirms the active forms. PAI was absent or at the detection limit for normozoospermia, whereas patients with oligoasthenoteratospermia or azoospermia showed high PAI antigen and activity levels. These data demonstrate that seminal PA activity may be related to sperm fertilizing capacity.