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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1763.)
© 2000 American Heart Association, Inc.

Vascular Biology

Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin

Caroline Hop; Andrea Guilliatt; Martina Daly; Hubert P. de Leeuw; Herm-Jan M. Brinkman; Ian R. Peake; Jan A. van Mourik; Hans Pannekoek

From the Department of Biochemistry (C.H., H.P), Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands; the Department of Medicine and Pharmacology (A.G., M.D., I.R.P.), Section of Molecular Genetics, University of Sheffield, Sheffield, UK; and the Department of Blood Coagulation (H.P.d.L., H.-J.M.B., J.A.v.M.), CLB, Sanquin Blood Supply Foundation, Amsterdam, Netherlands.

Correspondence to Dr H. Pannekoek, Academic Medical Center, Department of Biochemistry (K1-159), Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail h.pannekoek{at}amc.uva.nl

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 • P-selectin • Weibel-Palade bodies • protein sorting

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