Conversion of thrombin into an anticoagulant by protein engineering

Abstract

AT sites of vascular injury, thrombin interacts with multiple procoagulant substrates1–6 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 observed8,9. 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.

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Gibbs, C., Coutré, S., Tsiang, M. et al. Conversion of thrombin into an anticoagulant by protein engineering. Nature 378, 413–416 (1995). https://doi.org/10.1038/378413a0

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