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Temperature-Dependence of Weibel-Palade Body Exocytosis and Cell Surface Dispersal of von Willebrand Factor and Its Propolypeptide

Hewlett, L; Zupančič, G; Mashanov, G; Knipe, L; Ogden, D; Hannah, MJ; Carter, T (2011) Temperature-Dependence of Weibel-Palade Body Exocytosis and Cell Surface Dispersal of von Willebrand Factor and Its Propolypeptide. PLOS ONE, 6 (11). e27314. ISSN 1932-6203 https://doi.org/10.1371/journal.pone.0027314
SGUL Authors: Carter, Thomas David

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Abstract

Background: Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca2+-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca2+-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs. Principal Findings: Ca2+-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s−1 (37°C) to 0.80±0.14 s−1 (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca2+-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself. Conclusions : We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.

Item Type: Article
Additional Information: Copyright: 2011 Hewlett et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: Cells, Cultured, Enzyme-Linked Immunosorbent Assay, Exocytosis, Humans, Immunoblotting, Immunohistochemistry, Protein Precursors, Temperature, Weibel-Palade Bodies, von Willebrand Factor, Science & Technology, Multidisciplinary Sciences, Science & Technology - Other Topics, MULTIDISCIPLINARY SCIENCES, HUMAN-ENDOTHELIAL-CELLS, VONWILLEBRAND-FACTOR, BODIES, RELEASE, PROTEIN, SECRETION, MOLECULES, MECHANISM, STORAGE
SGUL Research Institute / Research Centre: Academic Structure > Molecular and Clinical Sciences Research Institute (MCS)
Academic Structure > Molecular and Clinical Sciences Research Institute (MCS) > Cell Sciences (INCCCS)
Journal or Publication Title: PLOS ONE
ISSN: 1932-6203
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Dates:
DateEvent
11 November 2011Published
Web of Science ID: WOS:000297553900032
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URI: http://openaccess.sgul.ac.uk/id/eprint/103284
Publisher's version: https://doi.org/10.1371/journal.pone.0027314

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