M-AAA-Thrombin: Potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1

Abstract

Background Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus, several antithrombotic agents targeting thrombin have been developed. We previously reported on a thrombin derivative prepared by dual chemical modifications designated as 'M-anhydrothrombin', which possessed both anticoagulant and antiplatelet properties. In order to obtain a more potent antithrombotic thrombin derivative, we prepared a recombinant thrombin mutant and its chemically-modified derivative, and examined their antithrombotic efficacies. Methods and Results We prepared a thrombin mutant, 65A43A205A-Th (designated as “AAA-Th”) in which Lys65(70) 1 1 Numbers in bracket are those of chymotrypsinogen numbering system. , and His43(57) and Ser205(195) were replaced by Ala, and its chemically-modified derivative at the carboxyl groups (designated as "M-AAA-Th"). M-AAA-Th possessed no enzymatic activity, but retained high affinity and specificity for factor VIII, and prolonged the APTT with a slight effect on PT and no effect on TT. Platelet aggregation induced by PAR1 activation was also suppressed by M-AAA-Th. In contrast, conventional thrombin inhibitors, argatroban and hirulog, substantially prolonged the TT rather than the APTT and the PT. In thromboelastgraph assays, M-AAA-Th suppressed whole blood clotting in a dose-dependent manner, and its effect was synergistically enhanced in the presence of soluble thrombomodulin (s-TM). M-AAA-Th also demonstrated a potent antithrombotic property in the FeCl 3-induced carotid arterial thrombosis model in guinea pigs with minimum effects on the APTT and PT and no prolongation of the TT. Conclusion M-AAA-Th may be a potent anticoagulant and antiplatelet thrombin derivative with differential affinity for factor VIII and PAR1.