Differential Kinetics of Cell Surface Loss of von Willebrand Factor and Its Propolypeptide after Secretion from Weibel-Palade Bodies in Living Human Endothelial Cells

• Published in: The Journal of biological chemistry Vol. 280; no. 24; pp. 22827 - 22830
• Main Authors: Hannah, Matthew J, Skehel, Paul, Erent, Muriel, Knipe, Laura, Ogden, David, Carter, Tom
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 17.06.2005
• Subjects: Antigens, CD - biosynthesis; Cell Adhesion; Cell Membrane - metabolism; Cells, Cultured; Endothelial Cells - cytology; Endothelium, Vascular - cytology; Exocytosis; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - metabolism; Histamine - chemistry; Humans; Immunohistochemistry; Kinetics; Microscopy, Fluorescence; Peptides - chemistry; Platelet Membrane Glycoproteins - biosynthesis; Protein Binding; Protein Precursors - chemistry; Protein Precursors - metabolism; Tetraspanin 30; Time Factors; Transfection; von Willebrand Factor - chemistry; von Willebrand Factor - metabolism; Weibel-Palade Bodies - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M412547200

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.