Knowledge of the details of the protein-protein interaction of thrombomodulin (TM) with thrombin may lead to the development of novel therapeutic agents for the treatment of heart disease. The binding of TM to thrombin causes an inhibition of the procoagulant activity of thrombin (cleavage of fibrinogen) and a selective enhancement of the anticoagulant activity of thrombin (activation of protein C). Previous studies have shown that a region of TM containing three domains that resemble epidermal growth factor (EGF domains) are necessary and sufficient to bind to thrombin and to alter thrombin's catalytic activity. Our experiments will determine the molecular details of the interaction of the TM EGF domains with thrombin, and of the concomitant activation of protein C. The fourth, fifth, and sixth EGF domains from TM that have been implicated in the binding and activation of thrombin cleavage of protein C will be expressed in E. coli, and large amounts of the purified domains will be obtained. We will study the function of each of these domains and pairs of domains by assaying their ability to either inhibit the TM-thrombin complexation, or to activate thrombin to catalyze the cleavage of protein C. The solution structures of the functionally important domains will be determined by two-dimensional NMR methods. We will then carry out measurements of transferred nuclear Overhauser effects to protons on thrombin to determine the molecular details of the interaction of the TM EGF domains with thrombin. We will also try to co-crystallize the domains with thrombin to gain structural information about the protein-protein interactions. Information from sequence similarity and analogy to the interaction of epidermal growth factor with its receptor suggests the two beta-turn regions are involved in the interaction. This hypothesis will be probed using site-directed mutagenesis.
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