Genetic modification of von Willebrand factor function.

Abstract

von Willebrand factor (VWF) is an abundant plasma glycoprotein that is essential for the maintenance of hemostasis. Deficiencies in VWF cause the common bleeding disorder von Willebrand disease (VWD). In addition, abnormalities of VWF processing are thought to contribute to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). The studies comprising this thesis focus on post-translational mechanisms contributing to the regulation of VWF function. The first set of studies expands previous work on the RIIIS/J mouse, a model of type 1 VWD. Prior studies identified a switch in the expression of the <italic>Galgt2</italic> glycosyltransferase gene as the underlying genetic mechanism causing decreased VWF levels in this mouse strain. Ectopic expression of <italic>Galgt2</italic> in endothelial cells, the site of synthesis of VWF, leads to altered post-translational modification of VWF and increased clearance from plasma. We now describe progress toward the identification of the DNA sequences responsible for this unique regulatory switch in gene expression. The <italic>Galgt2</italic> expression pattern is characterized further, and transgenic studies narrow the genetic interval containing the elements necessary for controlling its wild-type expression. In addition, we investigate the role of the hepatic asialoglycoprotein receptor in mediating the clearance of GALGT2-modified VWF. The second series of studies investigates the molecular basis of TTP, a thrombotic disorder associated with altered VWF processing. Using a positional cloning approach, we describe mutations in the <italic>ADAMTS13</italic> gene, encoding a zinc metalloproteinase, as the cause of familial TTP. We also demonstrate that recombinant ADAMTS13 cleaves VWF, and that TTP patient mutations impair this proteolytic activity. Together, these studies shed further light on the post-translational regulation of VWF function and contribute to the understanding of the mechanisms underlying the variable expressivity of VWD and the molecular basis of TTP.