This proposal will focus on studies of the regulated secretion of von Willebrand factor (vWf) from the Weibel-Palade bodies of endothelial cells, with emphasis on the polarity of this secretory pathway and on the fate of vWf following release. Polarity studies using a variety of secretagogues will be conducted using endothelial cells cultured on porous membrane filters. Pharmacologic agents will be used to block cell retraction and migration, which can occur concomitant with secretagogue stimulation, and to study the resulting effect on the apparent polarity of release of vWf. Microscopic techniques will be employed to directly visualize the process of Weibel-Palade body release. Preliminary studies reveal that some vWf released from Weibel-Palade bodies remains associated with the external cell surface. The nature of this interaction and its reorganization with time will be explored, using double-label immunofluorescence staining and fluorescence labeling of live cells. Metabolic-labeling studies will be conducted to determine if proteoglycan molecules are constituents of Weibel Palade bodies, if they remain cell-associated following release, and if they function in the reorganization of vWf at the cell surface following release. Endothelial cells will be cultured on an extensive extracellular matrix produced by bovine corneal endothelial cells, and the fate of vWf and its cleaved propolypeptide following release from Weibel-Palade bodies, with regard to its compartmentation between cell lysate, extracellular matrix and culture medium will be studied using this culture system. The proposed studies will provide a greater understanding of the functional role of the Weibel-Palade body pool of vWf and of the cellular processes directing polarized secretion and directing secreted proteins to their site of action.
