Human blood platelets exposed to complement proteins C5b-9 show increased metabolic conversion of arachidonate through the cyclo-oxygenase pathway and an enhanced secretory response to thrombin. Plasma membrane damage by these pore-forming serum proteins has also been implicated in episodes of vascular thrombosis associated with immunologic disease. The research outlined in this Proposal investigates the molecular events underlying this sublytic interaction of the C5b-9 proteins with the human platelet, with the overall goal of identifying how their insertion into the plasma membrane of this cell alters or bypasses normal transduction processes accompanying platelet activation, and potentially triggers vascular thrombotic reactions. Using gel-filtered platelets exposed to the purified C5b-9 proteins, we aim specifically (i) to measure changes in plasma membrane conductance initiated by C5b-9 assembly on the platelet surface; (ii) to identify the compensatory responses of the platelet that enable this cell to escape lytic plasma membrane rupture after formation of the C5b-9 pore, thereby sustaining its secretory and aggregation responses; (iii) to determine how electrochemical changes in the platelet initiated by the membrane-inserted C5b-9 proteins is related to changes in arachidonate metabolism, lysosomal enzyme release and stimulus-secretion coupling, as well as to selective shedding of plasma membrane components from the platelet surface. In these experiments, time-dependent changes in membrane potential, intracellular Ca++, and internal pH will be monitored with fluorimetric indicators and related to measured transmembrane ion fluxes. Concomitant changes in phospholipid metabolism and granule secretion will be assessed by isotopic, fluorimetric and radioimmunoassay methods. Plasma membrane vesicles released from the platelet surface in response to C5b-9 insertion will be isolated and characterized with respect to composition and """"""""sidedness"""""""" (with respect to the plasma membrane), and the conformational state of the C5b-9 proteins associated with these membrane vesicles defined. The effects of the membrane-inserted C5b-9 proteins on assembly and function of the platelet prothrombinase complex will be examined as a mechanism for increased prothrombin converting activity after immunologic damage. It is proposed that the information derived from these studies will provide new insight into the molecular events that are initiated upon C5b-9 binding to the surface of this key hemostatic blood cell, and contribute to an understanding of the potential role of these serum proteins in the pathogenesis of thrombotic vascular disease.
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