Von Willebrand Factor (VWF) is synthesized as a pre-proVWF precursor in which the pre-sequence directs the protein into the secretory pathway in endothelial cells and megakaryocytes-platelets. The proVWF contains an extremely large propeptide, VWFpp, that is comprised of 741 amino acids. VWFpp directs many of the important intracellular processes, regardless of whether it is a contiguous covalent part of the proVVVF (cis construct) molecule or is synthesized in trans as a separate polypeptide protein. Work done under this grant in previous years has defined many of the subtle aspects of the role of this propeptide and during this past funding cycle, some of the obligate domains and/or sequences have been more clearly delineated. These intracellular processes include multimerization, storage, and release - the latter being under the control of a regulatory system that is initiated by ligand-induced cellular activation. Within the endothelial cell there appears to be no secretory granule (WeibeI-Palade body) formed in the absence of VWF synthesis. We therefore will study further the induction of granule formation by VWF and the structural requirements necessary for this assembly. In platelets, however, in the absence of VWF, alpha granule formation is maintained and the contents of the alpha granule are much more complex than the relatively limited protein composition of the WeibeI-Palade body. Natural and site directed mutations will explore the necessary components of the full proVWF molecule and initial studies will be undertaken using plasmid and viral vectors to induce VWF synthesis, multimerization, storage, and release in cells and animals deficit in VWF synthesis ? ?
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