von Willebrand Factor (vWF) is responsible for the formation of its secretory granules, involving both the selective aggregation of secreted proteins and the recruitment of membrane proteins. To better understand the origin of von Willebrand disease, we will look at trafficking defects induced by mutant forms of vWF. This study of clinical variants of vWF will include transfection of these mutant forms in three different cell types, Human epithelioid H.Ep.2, AtT20 anterior pituitary cells, and primary Human Umbilical Vein Endothelial Cells (HUVECs) which are the physiological model system used in the Cutler lab. Morphological analyses at the light (including video) and electron-microscopic levels, plus biochemical steady-state (by Elisa and Western Blotting) and pulse-chase analyses (using immunoprecipitations) on whole cell extracts plus following sub cellular fractionation will be used to determine WPB trafficking. P-selectin recruitment will be assayed using a chimera with horseradish peroxidase attached to P-selectin, allowing for the detection of very few expressed molecules in both biochemical and EM analyses. The combination of morphological plus biochemical analysis should provide new general insights on vWF role in granule core formation and membrane protein recruitment, a central problem in membrane trafficking. This postdoc project will give me the occasion to learn all the above classical cell biology techniques which I will be able to use in the future to study cell biology.