Platelet adhesion to the blood vessel wall under both physiological and pathological conditions is initiated by the interaction of the glycoprotein (GP) Ib-IX-V complex on the platelet surface with von Willebrand factor (vWf) in the subendothelium. This interaction is also induced in the fluid phase by high shear forces and may result in platelet aggregation in regions of arterial stenosis. The vWf-binding site within the GP ib-IX-V complex is contained in the N-terminus of GP Ib-alpha, one of four homologous polypeptides that constitute the complex. In this application, we propose detailed studies to clearly elucidate how sequences in this region are involved in the interaction with vWf. We will test the hypothesis that GP Ib-alpha and vWf interact at two sites and that both contain an exposed sequence ready to bind its counterpart sequence and a cryptic sequence that must first be exposed by modulators or shear forces. Our first Specific Aim is to carefully map the binding sites of a variety of protein reagents (antibodies and snake venom proteins) that bind GP Ib-alpha and block vWf binding. Studies using these reagents have provided interesting insights into the ligand- receptor interaction. Some selectively block the interaction induced by only one modulator while others are able to block the interactions induced by both. At least one of the reagents activates platelets; most do not. Our second Specific Aim is to evaluate the effect of these reagents on shear-induced platelet aggregation. We expect that most will behave as they do in the modulator-induced binding assays, but also expect that we will identify some reagents that do not inhibit shear- induced platelet aggregation, or that paradoxically lower the shear threshold at which this phenomenon occurs. In our third Specific Aim, we propose to carefully dissect by in vitro mutagenesis three regions of the GP Ib-alpha ligand-binding domain that have been implicated as important for the interaction with vWf: 1) the acidic region encompassing Asp-269 to Asp-287, which contains three tyrosine residues that become posttranslationally sulfated; 2) the region between Asp-225 and Thr-240 that contains the gain-of-function mutations of platelet-type von Willebrand disease; and 3) the leucine-rich repeat region that contains seven tandem repeats of a sequence also present in the other polypeptides of the complex and that is affected by several mutations that produce the bleeding disorder, Bernard-Soulier syndrome. This work will elucidate specific regions within the ligand-binding domain of GP Ib-alpha that are involved in the interaction with vWf and will provide clues on the nature of the interaction and how it differs when induced by different modulators or by shear. This knowledge should prove useful for the design of anti-thrombotic agents that work by blocking primary platelet adhesion.
