Effect of different types of thrombin inhibitors on thrombin/thrombomodulin modulated activation of protein C in vitro.

The objectives of this study were to investigate whether the affinity of thrombin for small-molecule, active site-directed thrombin inhibitors and substrates is affected by the presence of thrombomodulin (TM), and to what extent thrombin inhibitors inhibit TM-bound thrombin. Inhibition of human alpha-thrombin was studied in the presence and absence of solubilised rabbit lung TM in a buffer containing CaCl(2). TM inhibited thrombin-induced proteolysis of human fibrinogen with a dissociation constant (K(D)) of 4 nmol/l. With at least 16-fold molar excess of TM over thrombin the affinity of thrombin both for the small thrombin substrates (S-2366 and S-2238) and the reversible, active site-directed thrombin inhibitors (inogatran and melagatran) increased twofold. In contrast, the ability of hirudin to inhibit thrombin was reduced by TM, since hirudin competes with TM in binding to thrombin. The effect of thrombin inhibitors on protein C activation by thrombin bound to human kidney cells transfected with cDNA for human TM was also studied. The mean binding capacity of the transfected cells was approximately 320,000 quantified by flow cytometry with antibodies against TM. Hirudin, inogatran and melagatran inhibited the activation of protein C by thrombin complexed with cell-bound TM in a dose-dependent manner, with mean IC(50) values+/-S.D. of 4.4+/-0.8, 20.0+/-1.1 and 6.4+/-0.2 nmol/l, respectively. Antithrombin inhibited protein C activation with an IC(50) value of 290+/-10 nmol/l, which was enhanced fourfold (IC(50) 60 nmol/l) by the addition of heparin 0.5 U/ml. Heparin alone, up to a concentration of 1 U/ml, had no effect on the activation of protein C. Small direct thrombin inhibitors thus inhibited both free and TM-bound thrombin and therefore also inhibited the activation of protein C. Whether this will influence their clinical efficacy or safety versus heparin and warfarin, which also inhibit protein activation, respectively, lowers the concentration of protein C, remains to be studied in clinical trials.