Chemically modified thrombin and anhydrothrombin that differentiate macromolecular substrates of thrombin.

BACKGROUND

Thrombin is a primary inducer of thrombus formation by activations of coagulation cascade and platelet aggregation. Hitherto, several types of thrombin inhibitors have been developed for therapeutic purpose.

OBJECTIVES

We prepared modified thrombin (M-thrombin) and modified anhydrothrombin (M-anhydrothrombin) by chemical modification of carboxyl groups of thrombin and anhydrothrombin, respectively, to present a new strategy for a potent antiplatelet-anticoagulant agent and new tools for investigation of thrombin functions.

RESULTS

M-anhydrothrombin retained high affinity for factor VIII (FVIII), but demonstrated lower affinity than anhydrothrombin for fibrinogen and factor V (FV). Both M-anhydrothrombin and anhydrothrombin prolonged activated partial thromboplastin time (APTT) without affecting prothrombin time, and M-anhydrothrombin prolonged APTT much more than anhydrothrombin. M-anhydrothrombin also retained affinity for the recombinant extracellular domain peptide of protease-activated receptor 1 (PAR1). M-thrombin exhibited marginal clotting activity (4% of thrombin), but induced platelet aggregation in platelet-rich plasma without forming a fibrin clot, which was completely suppressed by anti-PAR1 antibody (ATAP2) and by M-anhydrothrombin, but not by anhydrothrombin. These results indicate that M-thrombin induced platelet aggregation through the activation of PAR1, and M-anhydrothrombin inhibited this process completely. In contrast, neither M-anhydrothrombin nor anhydrothrombin apparently inhibited thrombin-induced platelet aggregation. Only in the presence of the Gly-Pro-Arg-Pro (GPRP) peptide that inhibits polymerization of fibrin, M-anhydrothrombin completely inhibited thrombin-induced platelet aggregation.

CONCLUSION

M-thrombin is PAR1-specific and M-anhydrothrombin is FVIII- and PAR1-specific derivatives, and thereby, are new tools as specific agonist and antagonist, respectively, of PAR1. Furthermore, M-anhydrothrombin may be an attractive model for development of a potent anticoagulant-antiplatelet agent.