Conversion of thrombin into an anticoagulant by protein engineering

Abstract

AT sites of vascular injury, thrombin interacts with multiple procoagulant substrates^1–6 to mediate both fibrin clotting and platelet aggregation. But upon binding to thrombomodulin on the vascular endothelium, thrombin instead activates protein C, thereby functioning as an anticoagulant and attenuating clot formation⁷. Upon infusion in vivo, both the procoagulant and anticoagulant effects of thrombin were observed^8,9. Preliminary studies indicating that thrombin's protein C activating and fibrinogen clotting activities could be dissociated by mutagenesis¹⁰ suggested to us that a thrombin variant that lacked procoagulant activity while retaining anti-coagulant function might be an attractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's specificity in favour of the anticoagulant substrate, protein C. In monkeys, this modified thrombin functioned as an endogenous protein C activator demonstrating dose-dependent, reversible anticoagulation without any indication of procoagulant activity. Notably, template bleeding times were not prolonged, suggesting a reduced potential for bleeding complications.