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Abstract-We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA-transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA-transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules. Key Words: von Willebrand factor Ⅲ P-selectin Ⅲ Weibel-Palade bodies Ⅲ protein sorting M ultimeric von Willebrand factor (vWF) is an obligatory glycoprotein for the formation of a hemostatic plug. It connects the platelet to the subendothelium that is exposed on injury of the vessel wall. vWF is synthesized by megakaryocytes and vascular endothelial cells. Endothelial cells display 2 secretory routes for newly synthesized vWF, namely, a constitutive and a regulated pathway. 1 Constitutively secreted vWF is rapidly transported in secretory vesicles toward the plasma membrane. This material consists of vWF dimers and low-molecular-weight multimers. Another portion of the newly synthesized vWF is encountered in specific storage organelles, denoted Weibel-Palade bodies (WPBs). These organelles fuse with the plasma membrane on activation of the protein kinase C signal transduction pathway, followed by release of their contents (regulated secretion). 2,3 vWF routed by this pathway consists of high-molecular-weight multimers that are Ϸ2 orders of magnitude more effective in mediating adhesion of platelets to the injured vessel wall than are vWF dimers or low-molecular-weight multimers. 4 The WPBs are considered to be endothelial cell-specific, rod-shaped, electron-dense storage organelles. 5 Apart from vWF multimers and the vWF propolypeptide, 6 -8 the WPB contains a number of other proteins, namely CD63, a transmembrane glycoprotein that is also present in lysosomal membranes of many cell types 9,10 ; interleukin-8 11 ; tissue plasminogen activator 12 ; and the transmembrane receptor P-selectin (also called GMP-140 or PADGEM). 13,14...