Exposure of human platelets or endothelial cells to the terminal complement proteins results in increased plasma membrane permeability to Ca2+ , and the activation of intracellular protein kinases. Consequently, the contents of storage granules are secreted, PADGEM/GMP140 is surface expressed, and membrane sites for assembly of the tenase and prothrombinase enzyme complexes are exposed, accelerating plasma clotting. Expression of this procoagulant activity was shown to be related to a C5b-9-induced vesiculation of plasma membrane, forming small membrane particles that express binding sites for the Va & VIIIa cofactors of these enzyme complexes. Generation of such """"""""microparticles"""""""" of vesiculated plasma membrane was also observed for platelets and endothelial cells stimulated by a variety of other agonists, suggesting a pervasive and causal relationship between induction of plasma membrane vesiculation and the expression of Surface catalytic function for the tenase and prothrombinase reactions. During the next five years we will focus directly on the process of microparticle release from the surface of activated platelets, with the goal of elucidating the cellular and molecular events that underlie both the formation of these vesicles and the exposure and decay of aminophospholipid-dependent sites for the membrane-stabilized coagulation enzymes.
Our specific aims will be to characterize the mechanism by which acidic aminophospholipids are normally translocated and sequestered to the inner leaflet of the platelet plasma membrane, to identify the mechanism by which these phospholipids become exposed on the surface of activated platelets and platelet-derived microparticles, and to determine what cellular processes regulate the formation of these plasma membrane vesicles upon platelet activation. As part of these studies, we aim to identify the molecular defect underlying Scott syndrome, a bleeding disorder that is characterized by defective microparticle formation and deficient expression of membrane procoagulant activity. The adherent properties of the platelet microparticle membrane will be elucidated, with the premise that the interaction of these vesicles with vascular surfaces may influence whether activated platelets contribute to normal hemostasis, or, initiate disseminated intravascular coagulation.
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