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Prepublished online as a Blood First Edition Paper on June 5, 2003; DOI 10.1182/blood-2003-02-0599. This Article Full Text (PDF) All Versions of this Article: 2003-02-0599v1 102/7/2452    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Michaux, G. Articles by Cutler, D. F Search for Related Content PubMed PubMed Citation Articles by Michaux, G. Articles by Cutler, D. F Social Bookmarking                   What's this? Submitted February 24, 2003 Accepted May 12, 2003 Analysis of intracellular storage and regulated secretion of three von Willebrands disease-causing variants of von Willebrand factor Gregoire Michaux, Lindsay J Hewlett, Sarah L Messenger, Anne C Goodeve, Ian R Peake, Martina E Daly, and Daniel F Cutler* MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom Academic Unit of Hematology, Royal Hallamshire Hospital, Sheffield, United Kingdom * Corresponding author; email: d.cutler{at}ucl.ac.uk. Von Willebrand factor (VWF) is stored in the Weibel-Palade bodies (WPB) of endothelial cells, allowing for its rapid exocytosis in response to vascular injury. We describe a system for examining the ability of VWF to drive both the formation of a storage compartment and the function of that compartment with respect to regulated secretion. Transient transfection of HEK293 cells with wild-type human VWF cDNA leads to the formation of numberous elongated organelles that resemble WPB. These "pseudo-WPB" exhibit the internal structure as well as recruiting membrane proteins including P-selectin of bona fide WPB. Finally, VWF was efficiently secreted upon stimulation by phorbol ester. We used this system to examine three VWF mutations leading to von Willebrand disease which affect VWF multimerisation and constitutive secretion. Surprisingly we find that all three mutants can, to some extent, make pseudo-WPB which recruit appropriate membrane proteins and which are secretagogue-responsive. The most striking defects are a delay in formation plus a reduced length and number of pseudo-WPB, in proportion to the clinical severity of the mutation. Studies of pseudo-WPB formation in this system thus yield insights into the structure-function relationships underpinning the ability of VWF to form functional WPB. 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