MECHANISMS ON VON WILLEBRAND FACTOR-INDUCED PLATELET ACT

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The broad, long-term objective of this proposal is to gain a better understanding of the interactions between platelets and blood vessels which result in normal and pathologic hemostasis. The specific aim of this research plan is to determine the nature of the biochemical responses of platelets to von Willebrand factor (vWF) and, in so doing, elucidate the physiologic and pathophysiologic consequences of the vWF/platelet interaction. Previous investigations of platelet-vWF interactions have focused predominantly on the structure-function relationships of vWF and the determinants of its binding to platelet membrane receptors; this proposed research, in contrast, will primarily examine the mechanisms of signal-response coupling and pathways of activation of platelets following exposure of vWF. The principal investigator has spent two years of full-time research training towards his development in the field of platelet signal transduction; his sponsor is an established investigator in this field; and his collaborators have extensive experience in the protein chemistry and molecular biology of human von Willebrand factor. The hypothesis to be tested is that vWF binding to platelets results in specific intracellular signals that initiate or promote platelet activation and thrombus formation. The experimental design is based on the systematic performance of quantitative and temporal measurements of intracellular processes that result in platelet activation. The studies will focus on the role of the following biochemical events in mediating platelet activation by vWF: (1) phosphoinositide turnover and the generation of inositol phosphates, diacylglycerol, phosphatidic acid, and activated protein kinase C; (2) changes in cytosolic calcium; and (3) eicosanoid production from phospholipase A2- or phospholipase C-mediated phospholipid hydrolysis. These measurements will be made under conditions in which the following are varied: (1) the von Willebrand factor (native, desialylated, and recombinant molecules); (2) plasma constituents other than vWF (e.g. fibrinogen, ADP, and thrombin); and (3) the intact human platelet (normal and with altered surface glycoprotein structure or function). Through the results of these experiments it will be possible to begin to reduce the complex inter-and intracellular processes which characterize primary hemostasis to the critical events linking platelet adhesion to platelet activation and aggregation.