Recent reports that analogues of erythrocyte membrane skeleton proteins are widely distributed in other cells, including the platelet, suggest the functional importance of these components of the cytoskeleton. Platelet proteins immunologically similar to human erythrocyte and brain membrane skeleton proteins have been identified by immunoblotting. This study will focus on protein 4.1, characterizing the molecular basis of its role in coupling the platelet membrane skeleton to the membrane, and defining its function during platelet activition. The specific aims are: a) the development of a protocol for isolating platelet protein 4.1, b) comparative structural analysis of platelet and erythrocyte protein 4.1, c) identification of components of platelet membrane and cytoskeleton with which protein 4.1 associates, and d) analysis of factors (e.g., membrane phospholipids, phosphorylation of protein 4.1) which influence the interaction of protein 4.1 with the membrane and cytoskeleton. Platelet protein 4.1 will be isolated by selective salt extraction and purified by gel filtration, ion exchange, and affinity chromatography. Its structure will be investigated using limited chemical and enzymatic cleavage followed by two-dimentional peptide mapping of the resulting fragments. Results will be compared with those obtained with erythrocyte protein 4.1. The interaction of protein 4.1 with platelet membrane proteins and cytoskeleton components will be analyzed using affinity chromatography and sucrose gradient centrifugation. Platelet glycoproteins will be isolated, fractionated by chromatography on lectin affinity columns, incorporated into phospholipid vesicles, and the binding to protein 4.1 determined. In addition, the composition of cytoskeleton complexes isolated from detergent-solubilized platelets by affinity techniques directed against specific membrane and cytoskeleton proteins will be determined. The effects of a) the presence of specific phospholipids (i.e., polyphosphoinositides) on the interaction of protein 4.1 with membrane proteins, and b) phosphorylation state of protein 4.1 on its interactions with membrane and cytoskeleton proteins will be studied. By investigating the structure and function of a hitherto unstudied component of the platelet cytoskeleton, this project will extend the understanding of the role of the cytoskeleton and its interaction with membrane proteins in the response of platelets to stimuli during the processes of hemostasis and thrombosis.