CRYSTAL STRUCTURE OF VON WILLEBRAND FACTOR A1 DOMAIN

Scripps Research Institute, La Jolla CA

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Abstract

The objectives of this proposal are: (i) to determine the three dimensional structure of the A1 domain of von Willebrand factor (vWF) in complex with botrocetin, a snake venom protein that induces agglutination on platelets by stabilizing the interaction between the A1 domain and the platelet receptor glycoprotein ibalpha; (ii) to determine the crustal structure of three mutants of the vWF A1 domain with abnormal function causing the type IIB Willebrand disease phenotype; and (iii) to evaluate whether the vWF A1 domains forms a complex with the 7th type A domain of collagen type VI and determine the structure of the complex. The binding of the platelet glycoprotein (GP) IBalpha, a component of the GP IB-IX-V receptor complex, to the vWF A1 domain is essential to arrest bleeding. This is the sole interaction capable of mediating the initial adhesion of circulating platelets to wounds in vessels where blood flow is rapid, such as arterioles, allowing the subsequent development of a hemostatic plug. As a first step to understand the molecular bases of the anti-hemorrhagic function of the vWF A1 domain, we have determined the crystal structure of the A1 domain in complex with the function blocking antibody NMC-4. This work has led to the recognition of a putative binding surface for the platelet receptor glycoprotein Ibalpha. The goal of this proposal is to obtain structural information relevant to understand the regulation of vWF A1 domain binding to GP Ibalpha. We have already obtained 0.2 mm long crystals of the complex between the vWF A1 domain and the modulator botrocetin. Success of the preliminary crystallization trials demonstrates the feasibility of the project. The structure of the complex will give insight into how botrocetin restrains the conformation flexibility of the A1 domain such that dissociation from GP IB is slowed down or blocked. Crystal structures of the three anterior mutants causing type IIB Willebrand disease will reveal how these mutations affect the conformation of the loop situated above them ad in turn change the interactions with GP Ibalpha. Finally, investigation of the mechanism of interaction between vWF A1 domain and collagen type VI A7 domain may reveal the structural bases of a key step necessary for immobilizing vWF at a site of vascular injury and initiating hemostasis or thrombosis.