Platelet activation begins with conversion of a quiescent, disc-shaped cell to a more-rounded form possessing multiple pseudopodia. Secretion of granule contents active in hemostasis takes place, and the platelets aggregate to form white thrombi. These processes must occur within seconds or fractions of a second to ensure efficient hemostasis in rapidly-flowing blood. Present research is only beginning to consider the initiation and regulation of these important early events. Some evidence exists for activation of nucleotide, lipid and protein metabolism within 5 seconds of platelet activation. However, partly because of methodology, knowledge is limited and sometimes contradictory when correlating chemical and functional events. Our proposed research is therefore directed to understanding the mechanisms by which platelet function is initiated, especially in the critical first 5 seconds. Platelet function will be evaluated by new quenched-flow techniques which provide control of second or sub-second reaction times.
Aims are to assess the significance of the phosphatidyl inositol (PI) cycle in normal platelet activation, and whether fibrinogen interaction with platelets occurs in primary aggregation via the glycoproteins GPIIb/IIIa, or whether this takes place much later during aggregate stabilization. The role of inositol trisphosphate (1P3) as a product of the PI cycle will be evaluated, especially in terms of its ability to increase intracellular calcium levels. Specific inhibitors such as lithium or colchicine will be employed to test effects of regulation of the cycle on efficiency of platelet function. Formation of certain phosphoproteins will also be evaluated. Monoclonal antibodies to GPIIb/IIIa will be prepared and used to test specific association of bound fibrinogen to these glycoproteins. The kinetics and nature of fibrinogen association with platelets during the first few seconds of their aggregation and shape change will be measuerd, and compared to much later when aggregate stabilization occurs.
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