In this proposal, Dr. Hawiger requests funding for a five year period. The title of the project is Adhesive Proteins Platelet Receptor Regulation. Dr. Hawiger wishes to construct a 3D molecular model of the interaction between fibrinogen and the integrin aIibB3 (GPIIb/IIIa) interaction. This is a basic system contributing to formation of a platelet thrombus. Undesired thrombi are a major problem in clinical medicine. The binding of fibrinogen to its platelet integrin receptor aIibB3 (GBIIb/IIIa) is a basic component of platelet thrombi. They form bridges between platelets and also generate outside-in signaling mediated by the receptor, integrin aIibB3. It has also been recognized that fibrinogen binding by integrin aIibB3 occurs via inside-out signaling which is induced by signal transduction systems inside the platelet. These transducers become activated in the presence of an externally provided agonist or at the site of vascular damage. This interaction is the basic mechanism of response to vascular injury. The interaction between fibrinogen and integrin aIibB3 is under strict regulation, but may go out of control following atherosclerotic plaque rupture in the cardiac and cerebral circulation. Dr. Hawiger will map the gamma chain site on fibrinogen which is responsible for recognition of the platelet integrin aIibB3. He will then develop synthetic peptide inhibitors of this interaction and will subsequently attempt to solve the 3D structure of this segment of the gamma chain. He will determine the 3D structure of the integrin aIibB3 extracellular region which is responsible for the interaction with the carboxy terminal segment of the human fibrinogen gamma chain. The latter will utilize a newly developed technology of carrier protein-driven crystallization. The cytoplasmic tails of integrin aIibB3 will be mapped for their sites of interaction with each other and with the actin cytoskeleton as well as intraplatelet signal transducers. This will be done using a novel technology involving cell permeable peptides for identification of functional domains involved in intracellular protein-protein interaction. This technique does not involve microinjection or permeabilization. The 3D structure of the mapped cytoplasmic domains of integrin aIibB3 will be determined. Thus, the overall thrust of the research will produce new knowledge and concepts concerning the 3D model of the fibrinogen-integrin aIibB3 interaction. This will be coupled with a comprehension of the mechanism of inside-out and outside-in regulation. The long range goal will involve development of better pharmacologic approaches toward developing novel antithrombotic compounds which are selective and specific. The 3D structure of the carboxy terminal segment of human fibrinogen gamma chain has been pinpointed by Dr. Hawiger as an integrin aIibB3 specific ligand. This will be verified further. Known functional domains of this complex which are involved in recognition of the gamma chain segment and of the RGD motif will be obtained. Structure-function analysis of the cytoplasmic domains of aIibB3 involved in interactions with each other and with certain cytoplasmic proteins such as actin binding proteins will be carried out. The cell permeable peptide import procedure will be further used to gain knowledge concerning intra-cellular protein-protein interactions. Further development of the 3D structural model of the cytoplasmic cell adhesion regulatory domain of integrin aIibB3 will be further developed. This will also include information on functionally important cytoplasmic segments of integrin aIibB3 involved in intra-cellular protein-protein interactions. Carrier protein-driven crystallization will be used to define important cytoplasmic segments.
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