The long range objective of this research is to study the relationship between the interactions of the cytoskeletal proteins of human platelets and the physiological phenomena observed when platelets are stimulated, namely shape change, filopodia extensions, secretion, aggegation and clot retraction. To this end we have succeeded in isolating and purifying the five major protein components of the cytoskeleton, actin binding protein (ABP), alpha-actinin, actin, myosin and tropomyosin and we have begun a systematic investigation into how these proteins reassemble, in vitro, to reform a structure resembling the native cytoskeleton. What factors govern this reassembly and how the reformed cytoskeleton interacts with the platelet membrane is a logical extension of our interests in these porteins. Because the membrane-cytoskeleton complex no doubt acts as a means of transmembrane communication whereby the binding of an external molecule to surface receptors results in the movement and/or restructuring of the cytoskeleton on the cytoplasmic side of the membrane, our long range goal of relating cytoskeletal interactions with physiological events appears closer to realization. To achieve these goals our studies during the next grant period will focus on 1) the phosphorylation - dephosphorylation of ABP and the resulting consequences to cytoskeletal formation, membrane interactions and susceptibility to proteolysis by CAP; 2) the interaction of individual and combinations of cytoskeletal proteins with the isolated platelet membrane and the glycoproteins present in the membrane; 3) role fo Ca++, CAP and actin capping or severing proteins on cytoskeletal assembly- disassembly and the resulting effects that these factors have on cytoskeletal membrane interactions; 4) the role of a newly isolated metalloprotease from platelets which is externalized upon thrombin activation of platelets and which proteolyzes surface membrane glycoproteins, in vitro; 5) the isolation of active proteolytic fragments of ABP which still bind to actin and the localization of the essential phosphate residues and 6) a continuation of our studies on two forms of platelet actin one of which associates preferentially with the cytoskeleton and the other predominantly with myosin.
