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.

Background Rivaroxaban is widely used in clinical practice. Although routine coagulation monitoring is not required, quantitative determination of rivaroxaban might be valuable in certain clinical circumstances. Variation in response sensitivity of prothrombin time (PT) reagents to rivaroxaban is well described in the literature, and the conventional international normalised ratio cannot be used for rivaroxaban. Purpose This multicentre study assessed the intra and interlaboratory precision of measurements of rivaroxaban plasma concentrations using the PT assay together with rivaroxaban calibrators and controls. Materials and methods Participating laboratories (Europe and North America) were provided with rivaroxaban calibrators (0, 41, 219 and 430 ng/ml), rivaroxaban pooled human plasma controls (19, 160 and 643 ng/ml) and PT reagent. Evaluation was performed over 10 consecutive days by each laboratory using local PT reagents as well as the centrally provided PT reagent (STA Neoplastine CI Plus; Diagnostica Stago). A calibration curve was produced each day, and day-to-day precision was evaluated by testing three control plasma samples. The control was diluted and re-tested if the level was above the highest concentration of the calibration curve. Results Intralaboratory variations in PT were dependent on the sensitivity of the local PT reagents, regardless of the type of instrument used. A large inter-laboratory variation (in seconds) was observed with local PT reagents; the coefficient of variation (CV) was 13.6–29.7%. When the results were expressed as rivaroxaban concentration (ng/ml), the inter-reagent variations were reduced; less variation was found with both local reagents (CV: 5.1–15.5%) and the central reagent (CV: 2.2–7.5%). However, over-estimation was observed with both local and central reagents. The CV for the calibrator containing 41 ng/ml rivaroxaban was 5.8% when the central reagent was used. Conclusions The PT assay may be useful for measuring rivaroxaban peak plasma concentrations (2–3 h after drug intake) using rivaroxaban calibrators and controls.

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Rivaroxaban is an oral, direct Factor Xa (FXa) inhibitor that has been recommended for approval by the Committee for Medicinal Products for Human Use for the prevention of venous thromboembolism after elective hip and knee replacement, and is in advanced clinical development for the prevention and treatment of thromboembolic disorders. There is no need for routine laboratory monitoring with rivaroxaban. However, a hemostasis assay might be valuable to measure the pharmacodynamic (PD) effects of rivaroxaban in special situations. The aim of this study was to identify a widely available assay (clotting assay, or colorimetric or fluorometric assay) that could be used in clinical practice. Increasing concentrations of rivaroxaban were spiked into citrated pooled human platelet-poor plasma. The global clotting assays prothrombin time (PT), diluted PT (dPT), and activated partial thromboplastin time (aPTT) were compared, as well as the specific clotting assays HepTest® (heparin test), prothrombinase-induced clotting time (PiCT®), and diluted Russell’s viper venom time (dRVVT). In addition, the anti-FXa activity-based colorimetric assays STA Rotachrom® and Stachrom® and thrombin generation test (TGT) were measured. A concentration-dependent prolongation of PT, dPT, and aPTT was observed with rivaroxaban, although the results varied depending on the reagents used. When testing PT over time using frozen plasma spiked with defined concentrations of rivaroxaban, PT values did not change during storage. Using a standard calibration curve, the results of the PT test can be expressed in rivaroxaban (ng/mL) rather than as PT ratio or international normalized ratio (INR). A concentration-dependent prolongation of the clotting time was also observed with dPT, a test that may be clinically more significant than conventional PT because it is closer to in vivo conditions (i.e. it uses lower tissue factor concentrations). Conventional methods for the HepTest and two-step PiCT (incubation times of 120 seconds and 180 seconds, respectively) resulted in a paradoxical response, with low concentrations of rivaroxaban reducing clotting times. This paradoxical response was not observed with shorter incubation times (0 or 30 seconds for one-step PiCT and 30 seconds for HepTest), or when antithrombin-immunodepleted plasma was used. Rivaroxaban also increased the dRVVT concentration dependently. Rivaroxaban influenced the various parameters of the TGT concentration dependently; peak effect was the most informative parameter. Rivaroxaban concentration-dependently inhibited FXa activity in both the Rotachrom and Stachrom assays– the results were expressed as anti-FXa international unit/mL. PT assays, which are widely used, appear to be valuable assays for monitoring the PD effects of rivaroxaban. One-step PiCT and HepTest with shortened incubation times or anti-FXa assays could also be useful to monitor rivaroxaban, if standardized methods were used.