One of the greatest challenges facing hematologic researchers today is understanding how normal physiologic processes such as blood clotting and wound repair become dysfunctional and contribute to coronary artery disease and atherosclerosis. New therapeutic agents are urgently needed that can specifically target the abnormal accumulation of smooth muscle cells, inflammatory cells, and fibrin deposits that comprise these lesions without disrupting normal blood clotting. Currently used thrombolytic therapies can have effects at non-thrombotic loci which lead to hemorrhagic complications. One potential target is the thrombin receptor. The thrombin receptor links the coagulation events occurring within the lumen of the blood vessel with the cellular signaling pathways that mediate platelet aggregation, cellular proliferation, and wound healing. Thrombin activates the receptor by a unique proteolytic cleavage of the extracellular domain. In an incompletely understood sequence of events, the new N-terminus serves as an intramolecular ligand that binds the body of the receptor. This intramolecular complex then activates an internally located G-protein. The cell must then switch off the irreversibly-cleaved thrombin receptor to prevent overstimulation of downstream signaling pathways. The first goal of these studies is to solve the 3-dimensional structure of the extracellular domain in both resting and activated states by NMR. Another NMR project will study the exodomain in complex with receptor extracellular loops and with thrombin. Capillary electrophoresis and inhibition of small chromogenic substrate hydrolysis will quantitate binding of thrombin to non-cleavable exodomains. A second goal is to create a genetic system to study thrombin receptor activation and G- protein coupling in isolation from all other mammalian proteins. We will capitalize on our recent expression and purification of affinity-tagged thrombin receptor in yeast to produce sufficient quantities of receptor for mapping the ligand binding surface. Functional coupling of the receptor to the yeast G-protein signaling pathway will allow testing of the mechanism of intramolecular ligand-receptor activation and the specificity of G-protein interactions. This genetic system which responds to soluble ligands will be a useful tool in the screening and development of novel anti-thrombin receptor agents. A third goal is to explore the role of anticoagulant serum proteases in truncation of the receptor exodomain. Previous experiments that determined cleavage sites will be correlated with loss of platelet function and cleavage of receptors on platelets and endothelium in a thombolytic animal model.
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