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.
Summary
Acquired factor V inhibitor (AFVI) is an extremely rare disorder that may cause severe bleeding. To identify factors associated with bleeding risk in AFVI patients, a national, multicentre, ...retrospective study was made including all AFVI patients followed in 21 centres in France between 1988 and 2015. All patients had an isolated factor V (FV) deficiency <50% associated with inhibitor activity. Patients with constitutional FV deficiency and other causes of acquired coagulation FV deficiencies were excluded. The primary outcome was incident bleeding and factors associated with the primary outcome were identified. Thirty‐eight (74 36–100 years, 42·1% females) patients with AFVI were analysed. Bleeding was reported in 18 (47·4%) patients at diagnosis and in three (7·9%) during follow‐up (7 0·2–48.7 months). At diagnosis, FV was <10% in 31 (81·6%) patients. Bleeding at diagnosis was associated with a prolonged prothrombin time that strongly correlated with the AFVI level measured in plasma {r = 0·63, 95% confidence interval (CI) 0·36–0·80, P < 0·05}. Bleeding onset during follow‐up was associated with a slow AFVI clearance (P < 0·001). The corresponding receiver operating characteristics curve showed that AFVI clearance was predictive of bleeding onset with an AFVI clearance of seven months with a sensitivity of 100% (95% CI: 29–100) and a specificity of 86% (95% CI: 57–98, P = 0·02). Kaplan–Meier analysis showed that AFVI clearance >7 months increased the risk of bleeding by 8 (95% CI: 0·67–97, P = 0·075). Prothrombin time at diagnosis and time for clearance of FV inhibitor during follow‐up are both associated with bleeding in patients with AFVI.
Thrombin 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.
We 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.
In 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 x 10(9)/l to 400 x 10(9)/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 phospholipid concentrations equal or higher to 4 microM. 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.
Thrombogram-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 x 10(9)/l and a synthetic phospholipid concentration higher than 4 microM.
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