Platelet GPVI and integrin alpha 2/beta 1 cooperate closely to mediate effective platelet adhesion to collagens and subsequent platelet procoagulant activity. In preliminary findings, we have observed that the levels of platelet GPVI vary in parallel with those of alpha 2/beta 1. Differences in the density of alpha 2/beta 1 or GPVI in themselves may not compromise platelet function in otherwise healthy individuals, but they can influence clinical outcomes in individuals who are at increased risk for thrombosis or bleeding. In order to develop a comprehensive understanding of platelet adhesion to collagens, our goal is to study the molecular biology of both integrin alpha 2/beta 1 and platelet GPVI. The focus of this grant is the latter receptor, platelet GPVI. To accomplish our goal, we have developed new resources: First, we have isolated a BAC plasmid containing the human GPVI gene, and we have assembled the complete human gene sequence. Second, we have cloned and sequenced murine platelet GPVI cDNA, and the knowledge of this sequence was employed to isolate BAC plasmids containing the complete murine GPVI gene. With these resources, then, the Specific Aims of this application are: 1) To analyze the platelet content of GPVI vis-a-vis the density of integrin alpha 2/beta 1 among normal individuals and to assess the effect of receptor variation on platelet procoagulant activity and platelet adhesion to collagens; 2) To characterize the binding sites on GPVI for collagen and convulxin (CVX); 3) To characterize the human GPVI gene and define the molecular basis for variation in platelet GPVI expression; and 4) To develop a mouse model for GPVI deficiency using transgenic technology. The successful completion of the proposed studies will lead to a substantial increase in our understanding of the genetics and physiology of the platelet collagen receptor, GPVI.
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