title: Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide.
abstract: BACKGROUND
  Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca(2+)-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 Ca(2+)-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.
  
  
  RESULTS
  Ca(2+)-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 Ca(2+)-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.
authors.affiliation: Division of Physical Biochemistry, Medical Research Councils National Institute for Medical Research, London, United Kingdom.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Hewlett
authors.forename: Gregor
authors.initials: G
authors.lastname: Zupančič
authors.forename: Gregory
authors.initials: G
authors.lastname: Mashanov
authors.forename: Laura
authors.initials: L
authors.lastname: Knipe
authors.forename: David
authors.initials: D
authors.lastname: Ogden
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Hannah
authors.forename: Tom
authors.initials: T
authors.lastname: Carter
mesh_terms: D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D011498:Protein Precursors / Q000378:metabolism*; D013696:Temperature; D020302:Weibel-Palade Bodies / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D011498:Protein Precursors; D014841:von Willebrand Factor; C067003:von Willebrand factor propolypeptide
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