Molecular determinants of glycoprotein Ib/vonWillibrand factor interaction

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The first step in the recognition of arterial lesions by the hemostatic system is an interaction between a large glycoprotein (GP) complex on the platelet surface, the GP Ib-IX-V complex, and an even larger glycoprotein affixed to the vascular matrix, von Willebrand factor (vWF). Within the GP Ib-IX-V complex, GP Ibalpha contains all of the known vWF-interacting sequences. VWF also circulates in the plasma but under circumstances does not bind platelets. That interaction requires that vWF be immobilized onto the subendothelium or the presence of extremely high fluid shear stresses, such as may be found in regions of artery narrowing. Once the platelets bind vWF by either means, signals are transmitted across the plasma membrane that have a number of consequences, the most important being the activation of the integrin alphaIIbbeta3 and the platelet aggregation, and the generation of pro- coagulant activity that promotes consolidation of the thrombus. We have three Specific Aims to continue our work to define the molecular nature of the GP Ib-IX-V-vWF interaction and the mechanisms by which shear stress induces it and leads to platelet activation. In Specific Aim 1, we will focus on the leucine-rich repeats region of GP Ibalpha-which we hypothesize is the mot important vWF-binding region of GP Ibalpha, to precisely define the sequences involved in binding vWF. We will use several approaches, including studies of canine-human GP Ibalpha chimeras to define specific species sequences involved in the interaction, mutagenesis of charged residues conserved in three mammalian species for which the GP Ibalpha sequence is known, and molecular modeling to identify potentially interactive residues. In the second Specific Aim, we will examine the disulfide loops that flank the GP Ibalpha leucine-rich repeats on both the and C-termini for their role in either directly facilitating vWF binding or regulating exposure of binding sites. In the third Specific Aim, we will test the hypothesis that shear stress not only induces vWF to bind GP Ibalpha but not it may also exerts a traction force to initiate signal transduction. We expect that successful accomplishment of these aims will yield insight into the very first step in the formation of an arterial thrombus, and will further the overall goal of this SCOR applications-to develop improved and specific therapies for arterial thrombosis.