I propose to study the factors that regulate the formation of the microfilament network of the human platelet. Microfilaments exist in stimulated platelets in two distinct morphologies: a loose, filamentous network and bundles of actin filaments which are found in the microspikes. I will study the factors that maintain actin in an unpolymerized form in the resting platelet and examine the hypothesis that profilin, gelsolin, and a newly recognized actin polymerization inhibitor (Mr 65,000 daltons) are capable of maintaining an unpolymerized pool of cytoplasmic actin, after complete characterization of the 65,000 dalton protein and gelsolin. The emphasis will be on a quantitative analysis of the ability of these proteins to control the vast amount of actin in the platelet. I will then study the interaction of these proteins in regulating the polymerization of actin filaments, with particular attention being paid to calcium concentration and pH. These studies will hopefully allow an elucidation of the regulation of actin filament formation in a quantitative manner. I will then study the formation of the actin bundles found in platelet microspikes by applying the principles of polymer chemistry and physics. formation of actin bundles will be studied in detail by examining the interactions of actin, tropomyosin and Alpha-actinin. Gelsolin will be used to alter actin filament length so the predictions of phase transition theory can be tested in this biologic system. Localization of these newly recognized platelet contractile proteins will be undertaken by raising antibodies to each and labeling the antibodies with ferritin an colloidalgold. Protein localization will be performed using both flourescent light microscopy and the electron microscope.
