von Willebrand factor (vWF) is a multimeric plasma glycoprotein which plays a central role in hemostasis, serving both as a carrier for factor VIIIC and mediator of platelet adhesion to the subendothelium. Hereditary abnormalities in vWF result in von Willebrand's Disease (vWD), probably the most common inherited bleeding disorder in man. The molecular defects responsible for vWD are not understood, and a bewildering number of clinical and laboratory subtypes and variants of the disease have been described. The functional activities of vWF are equally complex, involving multiple discrete functional domains responsible for binding to several receptors on platelets, subendothelial collagen, heparin, and factor VIIIC. The studies outlined in this proposal will investigate the molecular genetics of vWD, and characterize in detail structure/function relationships within the vWF molecule. In preliminary studies, the porcine model of vWD has been shown to be due to a molecular defect within the porcine vWF gene. Studies in humans indicate that deletions within the vWF gene are rarely the mechanism for vWD and that other molecular defects are the basis for the majority of cases. A panel of restriction fragment length polymorphisms (RFLPs) has been identified in the human vWF gene to permit genetic linkage analysis of the multiple vWD subtypes. Also, in preliminary work, an approach has been devised using the polymerase chain reaction (PCR) to amplify and directly sequence vWF mRNA from platelets beginning with as little as 40 cc of peripheral blood. This should allow characterization of the precise mutation responsible for the various subtypes of vWD and eventually permit rapid and accurate diagnosis at the DNA level. In preliminary structure/function studies, a system has been developed to precisely map immune epitopes and functional domains within the vWF molecule by a bacterial expression approach, and a panel of new monoclonal antibody reagents has been developed. A detailed """"""""epitope map"""""""" of the vWF molecule will be constructed using several large panels of monoclonal antibody reagents. Preliminary localization of functional domains on the basis of antibody data will be confirmed by functional studies using recombinant protein material. The primary amino acid structural requirements for these various functions will be analyzed in detail using recombinant mutagenesis approaches. New monoclonal antibodies will be developed to recognize potentially important regions of vWF not present in the current panel of available reagents. The studies outlined in this proposal should greatly advance our understanding of the molecular basis for vWD, and structure/function relationships within the vWF molecule. In addition, molecular tools will be developed to accurately and rapidly diagnose, and precisely categorize the various subtypes of vWD. The latter should prove of major clinical importance for the care of patients with this disease.
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