The efficiency of anti-activated factor X and anti-activated factor II anticoagulants

Six thrombin-generation inhibitors or thrombin inhibitors were compared in the extrinsic coagulation activity assay (EXCA), where the normal thrombin generation is about 1 IU/ml within 1 min (37 degrees C). Unfrozen pooled normal citrated plasma was supplemented on flat-bottom wells (23 degrees C) with increasing concentrations of dalteparin, danaparoid, heparin, fondaparinux, hirudin, or argatroban. To 50 microl plasma, 5 microl of 1.5 ng/ml tissue factor, 6% bovine serum albumin, and 250 mmol/l CaCl2 were added. After 1 and 2 min coagulation reaction time at 37 degrees C (EXCA-1 and EXCA-2), 100 microl of 2.5 mol/l arginine and 0.16% Triton X 100, pH 8.6, were added. After 3 min (23 degrees C), 25 microl of 1 mmol/l CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, were added, and the linear increase in absorbance with time was determined at 405 nm. The 50% inhibitory concentrations of plasmatic anticoagulants tested in the EXCA-1 (37 degrees C) were 0.025 IU/ml dalteparin, 0.13 U/ml danaparoid, 0.12 IU/ml heparin, 1.3 microg/ml fondaparinux, 2.4 ng/ml hirudin, and 1 microg/ml argatroban. From the 50% inhibitory concentration of hirudin it can be concluded that inhibition of about 30 mIU/ml thrombin halves the normal EXCA-1 value (i.e. if about 0.1 IU/ml thrombin are inactivated, then thrombin cannot self-amplify its generation 10-fold). The efficiency of any clinically used plasmatic anticoagulant can be monitored in the EXCA.