François Depasse

Assessment of laboratory assays to measure rivaroxaban – an oral, direct factor Xa inhibitor

Although there is no need for routine coagulation monitoring with rivaroxaban--an oral, direct factor Xa inhibitor--a haemostasis assay might be valuable to measure its pharmacodynamic effects. This study aimed to find assays, among those commercially available, to measure rivaroxaban pharmacodynamics. Several global conventional clotting tests, as well as clotting or chromogenic assays to measure anti-factor Xa activity, were studied. A thrombin generation test using calibrated automated thrombogram was also done. Tests were performed with the indirect factor Xa inhibitor fondaparinux for comparison. A concentration-dependent prolongation of prothrombin time (PT), dilute PT, and activated partial thromboplastin time was observed with rivaroxaban. The results varied depending on the reagents. This variability cannot be standardised with the international normalised ratio system commonly used for vitamin K antagonists. Using a standard calibration curve, PT test results can be expressed in plasma concentrations of rivaroxaban rather than PT seconds or ratio. Standard methods for HepTest and two-step prothrombinase-induced clotting time (PiCT) resulted in a paradoxical response, with low concentrations of rivaroxaban reducing clotting times. This was not observed with shorter incubation times, or when antithrombin-deficient (immunodepleted) plasma was used. The chromogenic tests found a dose-dependent relationship between anti-factor Xa activity and rivaroxaban concentration. Modified specific factor Xa chromogenic assays are being further investigated. One-step PiCT and HepTest with shortened incubation times, as well as the widely available PT assay (using a rivaroxaban calibrator) could be useful to monitor the pharmacodynamic effects of rivaroxaban accurately. Finally, all clotting and chromogenic assays showed a concentration-dependent effect induced by rivaroxaban.

In vitro inhibition of thrombin generation, after tissue factor pathway activation, by the oral, direct factor Xa inhibitor rivaroxaban

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An optimised, rapid chromogenic assay, specific for measuring direct factor Xa inhibitors (rivaroxaban) in plasma

An optimised, rapid chromogenic assay, specific for measuring direct factor Xa inhibitors (rivaroxaban) in plasma -

Towards a standardization of thrombin generation assessment: The influence of tissue factor, platelets and phospholipids concentration on the normal values of Thrombogram-Thrombinoscope assay

BackgroundThrombin generation assay was developed several years ago to mimic physiological coagulation mechanisms but it had important limitations. Thrombogram-Thrombinoscope assay using a fluorogenic substrate, allows obtaining thrombin generation curves in non-defibrinated platelet rich plasma (PRP) in a fully automated manner.MethodsWe standardised the methodology of Thrombogram-Thrombinoscope and we evaluated the precision of thrombin generation parameters (lag-time, maximum concentration of thrombin [Cmax], time required to reach Cmax [Tmax] and endogenous thrombin potential ETP) using different concentrations of recombinant human tissue factor, platelets or phospholipids. Normal values of thrombin generation assay were established in optimal experimental conditions.ResultsIn the presence of low TF concentrations (final dilution of thromboplastin in plasma: 1/1000–1/2000) the Thrombogram assay showed intra-assay and inter-assay coefficients of variation lower than 9%. Thrombin generation parameters showed an important inter-individual variability and the coefficients of variation ranged from 18% to 50%. In PRP the lag-time, Cmax and Tmax but not the ETP, were influenced by TF concentration. Thrombin generation parameters were not influenced by variations of platelet concentration from 50 × 109/l to 400 × 109/l. The addition of synthetic procoagulant phospholipids in PPP strongly influenced all the parameters of thrombogram. For all the parameters of thrombogram a threshold effect was observed in the presence of phspholipid concentrations equal or higher to 4 μM. In frozen-thawed PRP the lag-time and the Tmax were significantly reduced and the Cmax was increased compared to the fresh PRP, but the ETP, the intra assay and the inter-assay coefficients of variation were similar in both test-systems.ConclusionThrombogram-Thrombinoscope assay performed in fresh or in frozen-thawed PRP has an acceptable precision, with low inter-assay and intra-assay coefficient of variations. The concentration of TF is determinant for the normal values of the studied parameters of thrombin generation. When the assay is performed in PPP, thrombin generation parameters are influenced by the concentration of procoagulant synthetic phospholipids. The optimal experimental conditions were obtained in the presence of 1/1000 final dilution of thromboplastin, a platelet count higher than 50 × 109/l and a synthetic phospholipid concentration higher than 4 μM.

Recombinant factor VIIa partially reverses the inhibitory effect of fondaparinux on thrombin generation after tissue factor activation in platelet rich plasma and whole blood

Fondaparinux (Arixtra), a specific AT-dependent FXa inhibitor, is effective and safe in the prevention and treatment of venous thromboembolism, but some major hemorrhagic events may occur. No specific antidote to fondaparinux has been proposed. Recombinant FVIIa (Novoseven) could be used as an haemostatic treatment, but this option has not been well documented. We studied the effect of rFVIIa (1 micro g/ml) on the inhibition of thrombin generation induced by fondaparinux (0.1 micro g/ml to 1 micro g/ml). Coagulation was triggered in platelet rich plasma (PRP) or in whole blood by recalcification in the presence of diluted thromboplastin. In PRP thrombin generation was assessed using the thrombinoscope assay. In whole blood, prothrombin activation was assessed by measuring the kinetics of F(1+2) formation using an ELISA assay. Fondaparinux at concentrations equal or greater than 0.5 micro g/ml prolonged the initiation phase of thrombin generation, and reduced the velocity of prothrombin activation. It also decreased by 60% the endogenous thrombin potential. In the presence of fondaparinux (0.5 micro g/ml to 1 micro g/ml) rFVIIa accelerated the initiation phase of thrombin generation, but it did not significantly increase the endogenous thrombin potential. However, rFVIIa did not completely reverse the inhibitory effect of fondaparinux on the parameters of thrombin generation and prothrombin activation. This study shows that rFVIIa accelerates thrombin generation, but does not completely reverse the inhibitory effect of fondaparinux on thrombin generation. The potential clinical use of rFVIIa as haemostatic treatment of major bleedings related to fondaparinux has to be evaluated.

Comparison of the effect of fondaparinux and enoxaparin on thrombin generation during in-vitro clotting of whole blood and platelet-rich plasma.

Fondaparinux, a selective antithrombin-dependent inhibitor of activated factor X (FXa), is effective in the prevention and treatment of deep vein thrombosis and seems to be superior to enoxaparin. However, the exact mechanism of fondaparinux antithrombotic action is still unclear. We compared the effect of clinically relevant concentrations of fondaparinux and enoxaparin on the initiation and propagation phase of prothrombin activation and on the endogenous thrombin potential (ETP). Coagulation was triggered either in whole blood or in platelet-rich plasma (PRP) by recalcification in the presence of diluted thromboplastin. Prothrombin activation in whole blood was assessed with an original method by measuring the kinetics of prothrombin F1+2 formation using an enzyme-linked immunosorbent assay. We also assessed the maximum concentration of thrombin (Cmax) and the ETP in PRP using the Thrombogram-Thrombinoscope assay. Concentrations of fondaparinux achieved in prophylaxis (0.11–0.28 anti-FXa IU/ml) prolonged the initiation phase and reduced the velocity of the propagation phase of F1+2 formation. Concentrations of enoxaparin achieved in prophylaxis (0.1–0.25 anti-FXa IU/ml) did not significantly modify these parameters. Concentrations of fondaparinux equal to or higher than 0.57 anti-FXa IU/ml significantly reduced the Cmax of F1+2 or thrombin as well as the ETP. At fondaparinux concentrations equal to or higher than 0.91 anti-FXa IU/ml, a maximum 60% inhibition of thrombin generation was observed. In the presence of enoxaparin concentrations equal to or higher than 0.8 anti-FXa IU/ml, the inhibition of thrombin generation was higher than 80%. Fondaparinux prolonged the initiation phase, decreased the velocity of the propagation phase of thrombin generation and partially reduced the total amount of generated thrombin. The inhibitory effect of fondaparinux on the initiation and propagation phase of thrombin generation seems to be responsible for its antithrombotic action. The more profound inhibition of thrombin generation induced by enoxaparin is due to its supplementary anti-activated factor II activity.

Three different patterns of calibrated automated thrombogram obtained with six different anticoagulants

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Comparison of a direct Factor Xa inhibitor, edoxaban, with dalteparin and ximelagatran: A randomised controlled trial in healthy elderly adults☆

INTRODUCTION Edoxaban (the free base of DU-176b) is a new, oral direct Factor Xa inhibitor. This is the first study to compare the hemostatic response to edoxaban, ximelagatran, and dalteparin in healthy, elderly adults. MATERIALS AND METHODS In this open-label, active-controlled clinical trial, 40 adults (65-75 years), were randomised to: oral edoxaban (60 mg, twice-daily, 7 doses), subcutaneous dalteparin (5000 IU, once-daily, 4 doses), oral ximelagatran (24 mg, twice-daily, 7 doses) or no drug. Blood samples were taken before, and 1.5, 4, 12, 24, 72, 84, 96, 108, 120, and 144 hours after, the first dose. The primary outcomes were changes in thrombin-antithrombin complex, prothrombin fragment 1+2 and D-dimer, and adverse events. Additional biomarkers of coagulation, and endothelial cell and platelet activation were compared (ANOVA). RESULTS All subjects completed the study. Inhibition of thrombin generation lag time, peak, and constant velocity index were significantly greater, and extended for a longer period of time, following edoxaban administration, compared with dalteparin. We found that the traditional assay for anti-FXa activity was not appropriate for the new anticoagulants. Biomarker changes following edoxaban administration (including prolongation of prothrombin time) reflected inhibition of Factor Xa; there was no effect on platelet, tissue factor or endothelial activation. There were no clinically significant changes in primary outcomes. No serious adverse events were reported. CONCLUSION Oral administration of edoxaban resulted in effective Factor Xa and TG inhibition, and was well-tolerated. Studies are needed to confirm edoxaban (60 mg daily) use in clinical practice. SPONSORSHIP Daiichi Sankyo Pharma Development.

Deux nouveaux anticoagulants disponibles en 2010 – Dabigatran Etexilate et Rivaroxaban: progrès attendus – problèmes posés

After having been used for decades, heparins (unfractionated heparin [UFH] or low molecular weight heparins [LMWH]) and vitamin K antagonists (VKA), which are only parenterally active or which are responsible for frequent iatrogenicity respectively, have to face the competition of new anticoagulant drugs targeting either factor Xa or factor IIa (thrombin). Rivaroxaban (Xarelto(®)) and Dabigatran Etexilate (Pradaxa(®)) are the two leading components. They are more convenient to use and do not require routine coagulation monitoring. They are already marketed for venous thromboembolism prevention in major orthopaedic surgery. Although manufacturers claim that no biological monitoring is required, these two compounds may interfere in routine coagulation tests such as PT or aPTT, and in esoteric assays such as anti-Xa activity (the results of which are usually expressed in international anti-Xa units either UFH or LMWH unit) for Rivaroxaban or anti-IIa activity for Dabigatran Etexilate. Noteworthy is the fact that, in the case of these new anticoagulant drugs, results should be expressed in active product units (nanogram per millilitre of Rivaroxaban or Dabigatran). The new anticoagulants are associated with a bleeding risk comparable to that of VKA and heparins.

On the mechanism of inhibition of tissue factor pathway by the synthetic pentasaccharide during coagulation of human plasma.

The tissue factor (TF) pathway is preponderant in the initiation of blood coagulation in normal haemostasis and in thrombotic states. In the present study we investigated the mechanisms by which the synthetic pentasaccharide may influence the regulation of the TF pathway during clotting of human platelet poor plasma (PPP). Clotting of normal PPP or plasmas immuno-depleted of a single clotting factor (factor VII, factor XII, factor XI, factor IX, factor VIII, factor X, factor V, factor II) was initiated by triggering the TF pathway in the presence of fondaparinux (0.5 μg/ml). Activated factor VII (FVIIa) levels were measured in serum obtained at several time intervals after re-calcification of PPP. A clotting assay highly specific for FVIIa was used. The synthetic pentasaccharide inhibited the generation of FVIIa by 66%. The inhibitory effect of fondaparinux on FVIIa was completely abolished when antithrombin activity of plasma was inhibited by a specific antibody. Following the activation of TF pathway in plasmas depleted of factor X or factor IX, the inhibitory effect of fondaparinux on FVIIa generation was completely abolished, whereas it was not significantly modified when the other clotting factor-depleted plasmas were clotted. When fondaparinux was added in the serum, after the maximal generation of FVIIa, it inhibited by 20–30% the activity of the FVIIa–TF complex. These data suggest that fondaparinux enhances the antithrombin-dependent downregulation of the TF pathway by decreasing the generation of FVIIa via the inhibition of the generation and the activity of activated factor IX and activated factor X, and by inhibiting the activity of the FVIIa–TF complex.