von Willebrand factor (vWf) is a large, multimeric plasma protein that functions in hemostasis as the carrier of Factor VIII and, through interaction with the platelet glycoprotein (GP)Ib/IX receptor complex, as the initiator of platelet adhesion at the site of vascular damage. Defects in the structure and/or function of vWf result in von Willebrand disease (vWd), the most common human hereditary bleeding disorder. Despite intensive study, the mechanisms that regulate the physiological interaction of vWf with the GPIb/IX complex are not clear. Normally, circulating vWf and platelets do not interact. After vascular damage, vWf binds to components of the subendothelial matrix, such as collagen, and in the presence of high sheer forces undergoes a conformational change within the A1 domain that exposes the GPIb-binding site. The long-term objective of this proposal is to further define the structure and function of the vWf A1 domain, specifically focusing on the GPIb- binding site and the residues responsible for the physiological regulation of GPIb-binding activity.
The specific aims of this proposal are: (1) To investigate the structure and function of vWf using monoclonal antibodies to full-length vWf and the isolated A1 domain. These mAb will be used to map functional sites of interaction within vWf responsible for the physiological regulation of the vWf/GPIb interaction. (2) To investigate the structure and function of the vWf A1 domain using site-directed mutagenesis and expression of full-length vWf in mammalian cells. Deletions and single-amino acid substitutions will be targeted to the Cys509 -Cys695 loop within this region, and full-length vWf will be expressed both by transient expression in COS-7 cells and by stable expression in CHO cells. The relative effects of mutations on vWf function will be determined using ELISA-based assays to measure the agonist-induced binding of vWf to platelets, and the binding of Vwf to collagen, and heparin. Direct measurements of the changes in the affinity of vWf for GPIb will be made using standard equilibrium binding techniques and with the Pharmacia BIAcore Biosensor, which is capable of real-time kinetic analyses of protein/protein interaction. The subunit structure and multimeric composition of expressed protein will be determined by SDS/PAGE and SDS/agarose electrophoresis, respectively. (3) To analyze the structure and function of the vWf A1 domain in a genetic system that allows random mutagenesis and selection of reversion mutations. The system being developed is termed phage-display, where the A1 domain is expressed as a coat protein fusion protein on the head of bacteriophage fd. Overall, these studies will further define the complex interaction between vWf and platelets in normal hemostasis, and may provide mutants useful for analysis of the intracellular processes involved in the biosynthesis and storage of vWf. In addition, it is apparent that thrombosis plays an important role in the pathogenesis of accelerated atherosclerosis and acute coronary syndromes. Because of the essential role of vWf in primary platelet adhesion, a better understanding of this process will contribute to understanding the development of these pathological conditions, and may lead to the development of proteins with therapeutic value that prevent the deposition of platelets onto natural or implanted surfaces.
