Assembly of multimeric von Willebrand factor directs sorting of P-selectin

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) tha t synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis an d storage organelles, was transfected with full-length vWF cDNA or a deleti on mutant thereof. Stable transfectants of T24 with full-length vWF cDNA re vealed the generation of WPB-like organelles as demonstrated by colocalizat ion of vWF and P-selectin with double-labeling immunofluorescence. In contr ast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whe reas no indication was found for the presence of WPB-like organelles. The c ontents of the organelles in full-length vWF cDNA-transfected T24 cells wer e released on activation of the protein kinase C pathway, similar to the si tuation with genuine endothelial cells. The expression of vWF did not affec t the biosynthesis of P-selectin, as deduced from the observation that untr ansfected 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.