Specific Aims: To continue to explore the mechanisms of von Willebrand factor (vWf) biosynthesis by further study of the cultures of endothelial cell (EC) obtained from individuals with von Willebrand's disease. To explore some specific aspects of vWf biosynthesis which may relate to formation of large multimers. To compare vWf biosynthesis in EC with that in HEL cells. To use the expression of our Vwf cDNA clone as a tool in these studies. Methods: Further studies will be conducted on the Type IIA von Willebrand's endothelial cells to try to determine the molecular nature of the defect and to more precisely localize the mechanism of its expression. Reasons for the increased level of vWf mRNA expression in these cells will be sought. We shall also attempt to develop the technique of direct cloning of a given segment from a patient's genomic DNA. A patient with circulating provWf will be the prototype for these experiments which we hope may prove to have more general applicability in the future. Certain aspects of vWf biosynthesis which we feel might be related to the formation of large vWf multimers will be explored in EC cultures. We will also compare vWf biosynthesis in HEL cells with the EC process. The metabolic labeling, immunoisolation and gel electrophoretic techniques which we have used successfully in the past will continue to be the mainstay of our experimental approach in these areas. In HEL cells, the vWf mRNA will be carefully searched for any differences from its EC counterpart which might result from alternative splicing. Finally, we will attempt to develop cell lines expressing our full length vWf cDNA clone. Such recombinant lines would be used as an adjunct to the studies described above, with a particular emphasis on factors responsible for the formation and maintenance of large vWf multimers.
