Dissociation of thrombin's substrate interactions using site-directed mutagenesis

Abstract

Thrombin is an allosteric enzyme that interacts with multiple procoagulant substrates such as specific clotting factors and cell surface thrombin receptors, as well as the anticoagulant substrate protein C. Functional mapping of thrombin's interactions with its various substrates has been carried out using a collection of thrombin mutants generated by systematic alanine scanning mutagenesis. A thrombin mutant, E229K, has been identified that has essentially lost all of its procoagulant properties while retaining its ability to activate protein C, thus functioning as an anticoagulant in vitro and in vivo. It is also found that specific and distinct domains are involved in thrombin's interaction with thrombomodulin (TM) and the subsequent activation by the thrombin/TM complex of protein C and the thrombin-activatable fibrinolysis inhibitor (TAFI).