Monitoring effects of direct FXa-inhibitors with a new one-step prothrombinase-induced clotting time (PiCT) assay: comparative in vitro investigation with heparin, enoxaparin, fondaparinux and DX 9065a

Abstract

Selective, direct factor Xa-inhibitors are an emerging new class of antithrombotic drugs but their application in therapy may require adequate laboratory monitoring. A recently introduced assay for monitoring anti-FXa-activity using Russell's viper venom is based on the prothrombinase-induced clotting time (PiCT). In this study comparative data on the performance of PiCT using direct and indirect FXa-inhibitors and measurements of FXa-activity and aPTT are reported. Whole citrated blood samples from six healthy volunteers were preincubated with UFH (0-1.0 IU/ml), enoxaparin (0-10 microg/ml), fondaparinux (0-1.0 microg/ ml) and DX 9065a (0-10 microg/ml). PTT, FXa-activity and PiCT in plasma were determined on an ACL coagulation analyzer. PiCT was done with both a 180-sec incubation period before recalcification (2-step), and without (1-step). FXa-activity was based on a chromogenic assay (S2222). FXa-activity was reduced 10-40% by the lowest concentration and by 80-95% by the highest concentration of all agents. At the highest concentration the maximum prolongation in aPTT exceeded 120 sec with UFH, enoxaparin and DX 9065a but was only marginally prolonged (increase 39 +/- 3 sec) by fondaparinux. Prolongation in PiCT was significantly different when the two PiCT-methods were compared e.g. at 1.0 IU/ml UFH, 137 +/- 25 (1-step) vs. 187 +/- 32 sec (2-step) (p < 0.001); at 10 microg/ml enoxaparin 83 +/- 9 sec vs. 130 +/- 15 (p < 0.001); at 1.0 microg/ml fondaparinux 48 +/- 5 sec vs. 73 +/- 9 sec (p < 0.001); at 10 microg/ml DX 9065a 28 +/- 3 vs. 25 +/- 2 (p < 0.01), respectively. The 2-step method was unable to detect a prolongation in the effects of DX 9065a, and at concentrations < 5 microg/ml clotting times were even shorter (e.g. 13 +/- 1 sec at 1.0 microg/ml DX 9065a) than the baseline readings (20 +/- 2 sec). Only the 1-step method (i.e. without pre-incubation) seems suitable for the monitoring of new, direct selective FXa-inhibitors.