Despite significant progress over the past several decades, disease of the arterial vasculature continues to be the most important cause of morbidity and mortality in this country. Platelet hyperreactivity has been correlated with ischemic events and mortality in patients with vascular disease of the coronary, cerebral and peripheral and peripheral arteries. Platelet membrane glycoproteins are polymorphic and our preliminary studies demonstrate 1) significant associations between ischemic coronary events and polymorphisms on GPIbalpha and integrin beta 3 and 2) combined effects of these polymorphisms on platelet reactivity. However, relatively little information is available suggesting these genetic changes affect receptor function. Because of the central role played by GPIb-IX and alphaIIbbeta3 in the development of ischemic events in the arterial vasculature, the hypothesis driving out studies is that certain isoforms of these receptors induce conformational changes making them hyper- reactive and that these effects are additive or synergistic. The major goal of this proposal is to characterize functional differences among the different isoforms of GPIba and integrin beta 3 and to assess their additive and synergistic effects.
In Aim 1 we will generate cell lines that express both GPIb-IX and alophaIIbbeta3 and assess the relative effects of the met/thr145 and VNTR polymorphisms on GPIbalpha function and the additive effects of the GPIbalpha and P1/A2 polymorphisms. Using primarily assays employ shear forces, we will test adhesions to appropriate substrates, activation of alphaIIbbeta3, actin cytoskeletal rearrangements, and the relative contribution of the tyrosine kinase, Syk, to signaling by each receptor. GPIb-IX and alphaIIbbeta3 are receptors for thrombin and pro-thrombin, and in Aim 2, the effect of genotype on fibrin generation will be studied by measuring pro-thrombin binding to alphaIIbbeta3, thrombin binding to GPIbalpha, and the conversion of pro- thrombin to thrombin. We have previously identified P1/A genotype- dependent differences in pp125/FAK and MAP kinase phosphorylation and inhibition by indomethacin, and Aim 3 will extend these studies by assessing beta3, phosphorylation, the role of ras proteins, MAP kinase, phospholipase A2, and arachidonic acid metabolism. These studies would greatly enhance our understanding of genetic effects on platelet physiology, provide key mechanistic information to support or refute clinic genetic association studies, and provide a rationale for our long- term goal of tailoring anti-thrombotic therapy according to a patient's thrombotic risk phenotype.
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