Grigoris T. Gerotziafas

Effect of the anti‐factor Xa and anti‐factor IIa activities of low‐molecular‐weight heparins upon the phases of thrombin generation

Summary.u2002 Background:u2002Low‐molecular‐weight heparins (LMWHs), derived from unfractionated heparin (UFH) by different depolymerization procedures, vary in both their relative abilities to enhance the inhibition of FXa (anti‐FXa) and thrombin (anti‐FIIa), and in their physicochemical properties. Objective:u2002We aimed to profile the inhibition of thrombin generation induced by bemiparin, enoxaparin, nadroparin, dalteparin and tinzaparin in platelet‐rich plasma (PRP), and to compare them with UFH and fondaparinux (a synthetic pentasaccharide that specifically enhances FXa inhibition). Methods:u2002Different LMWHs, UFH or fondaparinux were added to normal PRP. Thereafter, tissue factor‐triggered thrombin generation was assessed using the Thrombogram–Thrombinoscope® assay. Results:u2002At equivalent anti‐FIIa activity concentrations, LMWHs and UFH exhibited similar inhibitory effects upon thrombin generation. However, when used at equivalent anti‐FXa activity concentrations, tinzaparin was significantly more active than the other LMWHs at inhibiting thrombin generation, and had similar activity to that of UFH. Enoxaparin, nadroparin and dalteparin all showed similar inhibitory activities. In these experiments, bemiparin exhibited the lowest inhibitory effect on thrombin generation of all the LMWHs. At 0.1u2003μgu2003mL–1 (0.093 anti‐FXa IUu2003mL–1), fondaparinux inhibited the rate of thrombin generation by 50%. A 7‐fold higher concentration of fondaparinux was required to inhibit the endogenous thrombin potential by 50%. Conclusions:u2002LMWHs have a variable inhibitory effect on thrombin generation in vitro when compared by anti‐FXa activity, but are similar when compared by their anti‐FIIa activities. The rate of thrombin generation during the propagation phase, rather than the endogenous thrombin potential, is more sensitive to the anticoagulant activity of fondaparinux and the polysaccharide chains of LMWHs possessing only anti‐FXa activity.

The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles

Sickle cell disease (SCD) is linked to hypercoagulability and is characterised by high concentrations of erythrocyte-derived microparticles (Ed-MPs). However, the impact of procoagulant cell-derived microparticles on the thrombin generation process remains unclear. We analysed the alterations of each phase of thrombin generation (TG) in relation to the concentration of erythrocyte- or platelet-derived microparticles (Ed-MPs and Pd-MPs) in a cohort of patients with steady-state SCD. We studied 92 steady-state SCD patients, 19 of which were under treatment with hydroxyurea, and 30 healthy age- and sex-matched individuals. TG was assessed by calibrated automated thrombogram. Ed-MP and Pd-MP expressing or not phosphatidylserine (PS) were determined by means of flow cytometry. Procoagulant phospholipid-dependent activity in the plasma was evaluated by the Procoag-PPL assay. Levels of thrombomodulin and haemoglobin in the plasma as well as red blood cell and reticulocyte counts were measured. SCD patients, independently of the administration of hydroxyurea, were marked by a significant acceleration in the propagation phase of TG which correlated with the Ed-MP/PS+ concentration. TG was significantly attenuated in hydroxyurea-treated patients. In conclusion, the acceleration of the propagation phase of TG, driven by Ed-MP/PS+, is a major functional alteration in blood coagulation in patients with steady-state SCD. Treatment with hydroxyurea, in addition to the regulation of haemolysis, lowers Ed-MPs and attenuates thrombin generation. The thrombogram could be a useful tool for the diagnosis of hypercoagulability and optimisation of the treatment in patients with SCD.

The Antithrombotic Potential of Tinzaparin and Enoxaparin Upon Thrombin Generation Triggered In Vitro by Human Ovarian Cancer Cells IGROV1.

Background: A documented relationship between ovarian cancer and thrombosis does exist. Low-molecular-weight heparins (LMWHs) are cornerstone drugs in the primary prevention and treatment of venous thromboembolic events in patients with cancer. However, cancer cells may alter the efficiency of these antithrombotic agents. Objective: We aimed to characterize the procoagulant phenotype of human epithelial ovarian adenocarcinoma cells, IGROV1, and to compare the capacity of tinzaparin and enoxaparin to inhibit thrombin generation triggered by these cells. Methods: Thrombin generation induced by different concentrations of IGROV1 cells on platelet poor plasma (PPP) was assessed by the calibrated automated thrombogram assay. Tissue factor (TF) expression was studied using Western blot analysis. Then, the experimental model of thrombin generation was used to compare the inhibitory effect of clinically relevant concentrations of both tinzaparin and enoxaparin. The inhibitory concentration 50 (IC50) of the mean rate index and the endogenous thrombin potential and the 2-fold increase in lag time were analyzed on the basis of the anti-Xa and anti-IIa activities of the LMWHs. Results: IGROV1 cells suspended into PPP resulted in a significant increase in thrombin generation in the absence of any exogenous source of TF and phospholipids. Tissue factor was expressed by IGROV1 cells. Tinzaparin was a more potent inhibitor of thrombin generation than enoxaparin. The inhibition of thrombin generation induced by IGROV1 cancer cells depended mainly on the anti-Xa activity of the LMWHs. Conclusion: This experimental study in ovarian cancer cells demonstrates that the antithrombotic activity of LMWHs is not completely predicted by the anti-Xa or anti-IIa activities measured in PPP.

Impact of blood hypercoagulability on in vitro fertilization outcomes: a prospective longitudinal observational study

BackgroundBlood coagulation plays a crucial role in the blastocyst implantation process and its alteration may be related to in vitro fertilization (IVF) failure. We conducted a prospective observational longitudinal study in women eligible for IVF to explore the association between alterations of coagulation with the IVF outcome and to identify the biomarkers of hypercoagulability which are related with this outcome.MethodsThirty-eight women eligible for IVF (IVF-group) and 30 healthy, age-matched women (control group) were included. In the IVF-group, blood was collected at baseline, 5–8 days after administration of gonadotropin-releasing hormone agonist (GnRH), before and two weeks after administration of human follicular stimulating hormone (FSH). Pregnancy was monitored by measurement of βHCG performed 15xa0days after embryo transfer. Thrombin generation (TG), minimal tissue factor-triggered whole blood thromboelastometry (ROTEM®), procoagulant phospholipid clotting time (Procoag-PPL®), thrombomodulin (TMa), tissue factor activity (TFa), factor VIII (FVIII), factor von Willebrand (FvW), D-Dimers and fibrinogen were assessed at each time point.ResultsPositive IVF occurred in 15 women (40%). At baseline, the IVF-group showed significantly increased TG, TFa and TMa and significantly shorter Procoag-PPL versus the control group. After initiation of hormone treatment TG was significantly higher in the IVF-positive as compared to the IVF-negative group. At all studied points, the Procoag-PPL was significantly shorter and the levels of TFa were significantly higher in the IVF-negative group compared to the IVF-positive one. The D-Dimers were higher in the IVF negative as compared to IVF positive group. Multivariate analysis retained the Procoag-PPL and TG as predictors for the IVF outcome.ConclusionsDiagnosis of women with hypercoagulability and their stratification to risk of IVF failure using a model based on the Procoag-PPL and TG is a feasible strategy for the optimization of IVF efficiency that needs to be validated in prospective trials.

Comparison of antithrombin-dependent and direct inhibitors of factor Xa or thrombin on the kinetics and qualitative characteristics of blood clots

Venous thromboembolism (VTE) is associated with significant morbidity and mortality.

Disseminated Intravascular Coagulation: An Update on Pathogenesis, Diagnosis, and Therapeutic Strategies

Disseminated intravascular coagulation (DIC) is an acquired clinicobiological syndrome characterized by widespread activation of coagulation leading to fibrin deposition in the vasculature, organ dysfunction, consumption of clotting factors and platelets, and life-threatening hemorrhage. Disseminated intravascular coagulation is provoked by several underlying disorders (sepsis, cancer, trauma, and pregnancy complicated with eclampsia or other calamities). Treatment of the underlying disease and elimination of the trigger mechanism are the cornerstone therapeutic approaches. Therapeutic strategies specific for DIC aim to control activation of blood coagulation and bleeding risk. The clinical trials using DIC as entry criterion are limited. Large randomized, phase III clinical trials have investigated the efficacy of antithrombin (AT), activated protein C (APC), tissue factor pathway inhibitor (TFPI), and thrombomodulin (TM) in patients with sepsis, but the diagnosis of DIC was not part of the inclusion criteria. Treatment with APC reduced 28-day mortality of patients with severe sepsis, including patients retrospectively assigned to a subgroup with sepsis-associated DIC. Treatment with APC did not have any positive effects in other patient groups. The APC treatment increased the bleeding risk in patients with sepsis, which led to the withdrawal of this drug from the market. Treatment with AT failed to reduce 28-day mortality in patients with severe sepsis, but a retrospective subgroup analysis suggested possible efficacy in patients with DIC. Clinical studies with recombinant TFPI or TM have been carried out showing promising results. The efficacy and safety of other anticoagulants (ie, unfractionated heparin, low-molecular-weight heparin) or transfusion of platelet concentrates or clotting factor concentrates have not been objectively assessed.