This project explores the functional and structural alterations of von Willebrand factor with respect to intracellular processing and trafficking and elucidates its role as an intracellular chaperone for factor VIII. While FVIII and vWF have been known to associate extracellularly for years, recently, we have demonstrated the importance of this interaction in intracellular processing as well. Using chimeric molecules comprised of human and canine vWF, the signals for cell sorting, multi-merization, and interaction(s) individually with factor VIII and platelets will be determined and explored in vitro and ex vivo. We will explore the interaction between vWF and FVIII in vivo using a number of specific techniques in mice. The cDNA for FVIII will be used to transduce murine CD34+ cells to demonstrate the co-localization of FVII together with vWF in the alpha-granules of platelets. Since there is some ambiguity about the cell that normally synthesizes FVIII, we will study murine tissues to determine the cells that produce FVIII using in situ hybridization, in situ PCR, and FVIII specific RT-PCR amplification of mRNA from microvascular endothelial cells from multiple tissue beds. In other experiments, the murine promoter elements for FVIII if differential expression of FVIIII is identified through in situ hybridization analysis.
The final aim will employ knockout models of hemophilia A to further study tissue expression of FVIII and vWF. In order to validate the importance of endothelial expression of FVIII, a conditional knockout of FVIII will be developed where FVIII expression by endothelial cells is eliminated. These studies will define the critical elements for the intracellular trafficking of vWF and FVIII and impact on the approach to gene therapy of hemophilia A and vWD.
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