Abstract
BackgroundWeibel-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 FindingsCa2+-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.ConclusionsWe 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.
Highlights
Weibel-Palade bodies (WPBs) are the principle regulated secretory organelle of endothelial cells (ECs) and contain the haemostatic protein von Willebrand factor (VWF) and the VWFpropolypeptide in a 1:1 stoichiometry [1]
We provide evidence; by comparison of biochemical data for Von Willebrand factor (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
Temperature-dependence of WPB exocytosis Proregion-EGFP expression in human umbilical vein endothelial cells (HUVEC) is localised in WPBs and permits a direct and time-resolved analysis of WPB exocytosis in living cells [11,12]
Summary
Weibel-Palade bodies (WPBs) are the principle regulated secretory organelle of endothelial cells (ECs) and contain the haemostatic protein von Willebrand factor (VWF) and the VWFpropolypeptide (proregion) in a 1:1 stoichiometry [1]. The proregion-VWF tubules give rise to the extended rod-like morphology of the WPB [7] and are known to help facilitate the retention and concentration of P-selectin within the WPB membrane [8]. The regulated exocytosis of high molecular weight VWF multimers and P-selectin from WPBs plays an important role in facilitating platelet capture and regulating the initial attachment of neutrophils to the vessel wall under flow conditions at sites of vascular activation or injury [1]. Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). We quantitatively analyse the temperaturedependence 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
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