title: Differential kinetics of cell surface loss of von Willebrand factor and its propolypeptide after secretion from Weibel-Palade bodies in living human endothelial cells.
abstract: The time course for cell surface loss of von Willebrand factor (VWF) and the propolypeptide of VWF (proregion) following exocytosis of individual Weibel-Palade bodies (WPBs) from single human endothelial cells was analyzed. Chimeras of enhanced green fluorescent protein (EGFP) and full-length pre-pro-VWF (VWF-EGFP) or the VWF propolypeptide (proregion-EGFP) were made and expressed in human umbilical vein endothelial cells. Expression of VWF-EGFP or proregion-EGFP resulted in fluorescent rod-shaped organelles that recruited the WPB membrane markers P-selectin and CD63. The WPB secretagogue histamine evoked exocytosis of these fluorescent WPBs and extracellular release of VWF-EGFP or proregion-EGFP. Secreted VWF-EGFP formed distinctive extracellular patches of fluorescence that were labeled with an extracellular antibody to VWF. The half-time for dispersal of VWF-EGFP from extracellular patches was 323.5 +/- 146.2 s (+/-S.D., n = 20 WPBs). In contrast, secreted proregion-EGFP did not form extracellular patches but dispersed rapidly from its site of release. The half-time for dispersal of proregion-EGFP following WPB exocytosis was 2.98 +/- 1.88 s (+/-S.D., n = 32 WPBs). The slow rate of loss of VWF-EGFP is consistent with the adhesive nature of this protein for the endothelial membrane. The much faster rate of loss of proregion-EGFP indicates that this protein does not interact strongly with extracellular VWF or the endothelial membrane and consequently may not play an adhesive role at the endothelial cell surface.
authors.affiliation: National Institute for Medical Research, Mill Hill, London, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Hannah
authors.forename: Paul
authors.initials: P
authors.lastname: Skehel
authors.forename: Muriel
authors.initials: M
authors.lastname: Erent
authors.forename: Laura
authors.initials: L
authors.lastname: Knipe
authors.forename: David
authors.initials: D
authors.lastname: Ogden
authors.forename: Tom
authors.initials: T
authors.lastname: Carter
mesh_terms: D015703:Antigens, CD / Q000096:biosynthesis; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology*; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006632:Histamine / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry*; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D060149:Tetraspanin 30; D013997:Time Factors; D014162:Transfection; D020302:Weibel-Palade Bodies / Q000378:metabolism*; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C554586:CD63 protein, human; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011498:Protein Precursors; D060149:Tetraspanin 30; D014841:von Willebrand Factor; C050818:prepro-von Willebrand factor; D049452:Green Fluorescent Proteins; D006632:Histamine