Thrombin, a serine protease central to blood coagulation and hemostasis, interacts with G protein-coupled proteinase activated receptors (PARs) on a variety of cell types. On endothelial cells, thrombin causes expression of endothelial adhesion molecules P-selectin and ICAM-1 and increases endothelial permeability with resultant increases in leukocyte infiltration. In addition, thrombin stimulates endothelial cell proliferation. This suggests that thrombin effects on endothelial cells may play a role in inflammation angiogenesis, thrombosis and atherosclerosis. Three members of the proteinase activated receptor family (PAR-1, PAR-3, and PAR-4) can be cleaved by thrombin. In contrast to intensely studied roles of G proteins activated by PARs on fibroblasts, platelets and smooth muscle cells, their roles in endothelial cell thrombin-mediated processes are poorly understood. Since thrombin effects on endothelial cells are potential therapeutic targets for a variety of pathological processes, we propose to elucidate the roles of G proteins in mediating various thrombin-activated endothelial responses. We will determine the G proteins responsible for mediating specific downstream events in endothelial cells by blocking specific G protein-PAR interactions in cells. To do this, minigene vector constructs encoding sequences from C- termini of particular Galphasubunits will be transfected into endothelial cells to block specific PAR-G protein interaction. To understand the molecular basis of receptor-G protein interaction, a combinatorial library approach will be used to identify peptide sequence derived from the carboxyl terminus of the Galpha subunit(s) that bind with high affinity to PARs. High affinity peptide minigenes will be constructed and used to potently inhibit downstream events including signaling and functional responses. High affinity peptides will also be tested for their severity in blocking the other members in the PAR family as well as other G protein coupled receptors that are important in endothelial activation. The proposed studies will provide a novel understanding of PAR-G protein interactions and PAR activation mechanisms in endothelial cells. By targeting the PAR-G protein interface our studies will lay the framework for design of therapeutic agents that block vascular endothelial activation induced by thrombin.
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