Cell activation by thrombin is critical for hemostasis and thrombosis, and may be important in inflammatory and proliferative responses in blood vessels. The goal of these studies is to determine the molecular mechanisms by which thrombin activates platelets and other cells. Thrombin activates cells by interacting with a specific cell surface molecule, the thrombin """"""""receptor"""""""". The thrombin receptor has not been identified. Moreover, whether thrombin activates its receptor by a novel proteolytic mechanism or by classical ligand binding is unknown. This basic information is critical for an understanding of thrombin signalling and for the rational design of thrombin inhibitors.
The specific aims of this proposal are: 1) to develop novel recombinant thrombins for use as reagents to define the role of thrombin's protease activity in thrombin-induced cell and platelet activation, and as ligands for affinity purification of the thrombin receptor, The role of thrombin's protease activity will be examined by using recombinant thrombins containing amino acid substitutions designed to abolish catalytic activity without perturbing substrate binding. The ability of these mutants to activate cells and platelets or to block activation by wild type thrombin will be determined. The goal of these studies is to develop not only thrombin receptor agonists or 'antagonists, but also novel affinity reagents for purifying the thrombin receptor. 2) to characterize and obtain a cDNA clone for the thrombin Two complementary approaches will be pursued to clone the thrombin receptor. (a) An assay for the mRNA encoding the thrombin receptor has been established using mRNA expression in xenopus oocytes. Both established and novel screening strategies will utilize this assay to obtain the thrombin receptor cDNA. (b) Recombinant thrombins which are proteolytically inactive yet retain receptor binding properties will be used as affinity agents to purify the thrombin receptor. With the thrombin receptor cDNA in hand we will address the following questions: Is the thrombin receptor cleaved by thrombin, and how does cleavage relate to activation? Do other thrombin receptor-like molecules exist and define a family of protease receptors? Which structural domains in thrombin and its receptor mediate binding? and What are the signal transduction molecules to which the receptor is coupled?
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