title: Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease-causing variants of von Willebrand factor.
abstract: The rapid exocytosis of von Willebrand factor (VWF) in response to vascular injury can be attributed to the fact that VWF is stored in the Weibel-Palade bodies (WPBs) of endothelial cells. 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 numerous elongated organelles that resemble WPBs. These "pseudo-WPBs" exhibit the internal structure, as well as the ability to recruit membrane proteins including P-selectin, of bona fide WPBs. Finally, VWF was efficiently secreted upon stimulation by phorbol ester. We used this system to examine 3 VWF mutations leading to von Willebrand disease that affect VWF multimerization and constitutive secretion. Surprisingly we find that all 3 mutants can, to some extent, make pseudo-WPBs that recruit appropriate membrane proteins and that are responsive to secretagogues. The most striking defects are a delay in formation and a reduction in the length and number of pseudo-WPBs 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 WPBs.
authors.affiliation: Medical Research Council, Laboratory for Molecular Cell Biology, University College London, United Kingdom.
authors.forename: Grégoire
authors.initials: G
authors.lastname: Michaux
authors.forename: Lindsay J
authors.initials: LJ
authors.lastname: Hewlett
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Messenger
authors.forename: Anne C
authors.initials: AC
authors.lastname: Goodeve
authors.forename: Ian R
authors.initials: IR
authors.lastname: Peake
authors.forename: Martina E
authors.initials: ME
authors.lastname: Daly
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Cutler
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology; D008854:Microscopy, Electron; D017354:Point Mutation; D020302:Weibel-Palade Bodies / Q000378:metabolism / Q000648:ultrastructure; D014842:von Willebrand Diseases / Q000378:metabolism* / Q000503:physiopathology*; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism*
chemical_list: D014841:von Willebrand Factor