Binding of the A1 domain of von Willebrand factor (VWF A1) to the platelet glycoprotein (GP) Ib-alpha is a central event in the arrest of bleeding but also in the onset of pathological arterial thrombosis. Moreover, GP Ib-alpha binds alpha-thrombin, which in turn activates platelets and regulates clotting. We propose to use crystallography to study the interactions of VWF A1 and alpha-thrombin with the N-terminal domain of GP Ib-alpha (GP Ib-alpha N). We have recently determined the structures of GP Ib-alpha N and its 1:1 complex with alpha-thrombin. The complex exhibits a novel sequential mode of ligand binding, with the self-induction of a second receptor site after occupancy of a first site by the ligand. We have also shown that GP Ib-alpha N and alpha-thrombin can form 1:2 and 2:1 complexes in solution, which we propose to isolate and crystallize for structural analysis as they may represent the physiological mode of a-thrombin interaction with GP Ib-alpha on platelets. VWF A1 and GP Ib-alpha do not interact in solution, but gain-of function mutants in the ligand or receptor lower this barrier and may explain how the interaction is modulated by conformational changes. The structure of the Ile546Val mutant VWF A1 already gave insights into a possible mechanism of activation. We will analyze other mutants in both VWF A1 and GP Ib-alpha to advance our understanding of how the interaction is regulated. VWF binding to collagen in the vessel wall is central to the initiation of thrombus formation. We propose to elucidate the structure of domain A7 of collagen type VI, which contributes to immobilize VWF at sites of injury by binding to the A1 domain. The results of this work will facilitate, in the future, the structural analysis of the complex between collagen VI domain A7 and VWF A1. Our studies will provide a clearer picture of adhesive and activation mechanisms central to platelet function and to the development of diseases such as myocardial infarction and stroke.
