Differential kinetics of cell surface loss of von Willebrand factor and its propolypeptide after secretion from Weibel-Palade bodies in living human endothelial cells.
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.
Hannah
Matthew J
MJ
National Institute for Medical Research, Mill Hill, London, UK.
Skehel
Paul
P
Erent
Muriel
M
Knipe
Laura
L
Ogden
David
D
Carter
Tom
T
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
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