Thrombin Receptor-G Protein Signaling Mechanisms

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DESCRIPTION (provided by applicant): Proteolytic cleavage of the G protein-coupled protease-activated receptors activates an extraordinarily diverse array of physiologic responses. These include platelet activation and aggregation, cellular proliferation/apoptosis, cell migration and protease-homing, angiogenesis, and the hemostatic and inflammatory responses to vascular injury. Four protease-activated receptors have been identified: PAR1, PAR2, PARS, and PAR4. Matrix metalloproteases have also emerged as important proteases in vascular biology. Initially described as extracellular matrix remodeling enzymes involved in tissue repair and cancer invasion, a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in atherothrombotic disease and platelet hemostasis. Quite recently, we found that a matrix metalloprotease, MMP-1, can also function as a protease agonist of PAR1 cleaving the receptor to generate PAR1-dependent Ca2+ signals, migration, platelet activation and cell shape changes. MMP-1 is expressed in high levels in platelets and endothelium. Circulating levels of MMP-1 have been shown to be significantly elevated in plasma from patients following acute myocardial infarction pointing to activated platelets and endothelium as potential sources for MMP-1. However, it is not clear what role(s) MMP1-PAR1 signaling plays in hemostasis, thrombosis and the control of vascular integrity or whether MMP1 activates different G protein pathways as compared to thrombin. PAR1, has been shown to couple to Gq, Gi, Gi2/i3, and (3y under a variety of in vitro conditions. The specifics of how PAR1 interacts with the G proteins and the relative importance and temporal ordering of differential G protein activation under in vivo conditions is still unknown. Far less is known about the identity of non-G protein effectors that might also interact with the intracellular loops of the PARs. The major goals of these studies are: 1) to investigate the molecular basis of G protein activation by PAR1 under in vivo conditions, 2) to understand the mechanism and physiologic relevance of MMP1-PAR1 signaling in platelets during arterial thrombus formation 3) to investigate the mechanism of endothelial MMP1 activation of PAR1 and the functional consequences of MMP1-PAR1 signaling on endothelial function during sepsis, and 4) to determine how the newly identified PAR1 effector, BicD1, modulates PAR1 signaling, signal termination, and cell motility. Platelet activation has been shown to be heightened in the setting of angioplasty and stenting which may cause clinical complications including acute heart attacks and death. The PAR1 receptor has long been recognized as an obvious candidate for therapeutic intervention of patients with heart conditions, the work proposed here may lead to the development of novel treatments for patients with arterial blood clots and those suffering from life- threatening sepsis.