Abstract
Introduction: Thromboplastin reagents used for prothrombin time (PT) clotting assays vary in their sensitivity to anticoagulant drugs that directly inhibit Factor Xa (FXa). The International Sensitivity Index (ISI)/International Normalized Ratio (INR) system was introduced for monitoring warfarin, and corrects for differences in PT assay sensitivity. However, it does not adequately correct for differences in assay sensitivity to direct FXa inhibitors. The objective of this study was to determine how the composition of thromboplastin reagents affects PT sensitivity to the novel oral, direct FXa inhibitor rivaroxaban and how this correlates with the INR.
Methods: Several recombinant thromboplastin reagents were prepared using different concentrations of NaCl, tissue factor and phospholipids (PL). They also contained different % of phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). These locally prepared thromboplastin reagents and five commercial thromboplastin assays were evaluated. PT ratios (PTR = PT with drug/PT without drug) were measured using normal human plasma to which rivaroxaban 1 μg/mL was added in vitro. Some PTRs were converted to INRs using locally determined ISI.
Results: PT obtained with commercial thromboplastins was prolonged by rivaroxaban (Table), but the magnitude varied more than 3-fold, depending on the thromboplastin. Converting PTR to INR failed to normalize these results and made discrepancies more pronounced. Using locally prepared thromboplastin reagents, the PT sensitivity toward rivaroxaban was found to increase by decreasing the concentration of tissue factor or by increasing the concentration of PL or NaCl. Increasing the % PS generally decreased rivaroxaban sensitivity, while including PE generally increased rivaroxaban sensitivity. There was also a trend toward higher rivaroxaban sensitivity as the baseline PT increased. As with commercial thromboplastins, converting these PTR values to INR frequently made the discrepancies more pronounced.
Conclusions: Changing the composition of thromboplastin reagents had disparate effects on the sensitivity of PT clotting tests to rivaroxaban. Furthermore, converting PTR values to INR failed to eliminate, and in some cases even exacerbated, the apparent differences in assay sensitivity of these PT clotting tests to rivaroxaban. This study sheds new light on the shortfall of the ISI/INR system to adequately correct for variation in the sensitivity of PT tests to direct FXa inhibitors. However, data show that other global clotting tests, such as PT, can be used to assess the efficacy of direct FXa inhibitors.
Table 1: PT results for normal plasma spiked with 1 μg/ml rivaroxaban using commercial thromboplastins Commercial thromboplastins ISI PTR INR INR, International Normalized Ratio; ISI, International Sensitivity Index; PTR, prothrombin time ratio Recombiplastin 0.94 5.07 4.60 Innovin 0.98 2.25 2.22 Thromborel S 1.07 2.37 2.52 Neoplastine CL+ 1.09 7.32 8.76 Thromboplastin C+ 1.50 3.77 7.31
Effect of daptomycin on prothrombin time and the requirement for outlier exclusion in International Sensitivity Index calibration of thromboplastin
