CONVERSION OF THROMBIN INTO AN ANTICOAGULANT BY PROTEIN ENGINEERING

AT sites of vascular injury, thrombin interacts with multiple procoagu lant substrates(1-6) to mediate both fibrin clotting and platelet aggr egation. But upon binding to thrombomodulin on the vascular endotheliu m, thrombin instead activates protein C, thereby functioning as an ant icoagulant and attenuating clot formation(7). Upon infusion in vivo, b oth the procoagulant and anticoagulant effects of thrombin were observ ed(8,9). Preliminary studies indicating that thrombin's protein C acti vating and fibrinogen clotting activities could be dissociated by muta genesis(10) suggested to us that a thrombin variant that lacked procoa gulant activity while retaining anticoagulant function might be an att ractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's s pecificity in favour of the anticoagulant substrate, protein C. In mon keys, this modified thrombin functioned as an endogenous protein C act ivator demonstrating dose-dependent, reversible anticoagulation withou t any indication of procoagulant activity. Notably, template bleeding times were not prolonged, suggesting a reduced potential for bleeding complications.