Frampton, JA; (2013) Synaptotagmins and Weibel-Palade Exocytosis in Human Endothelial Cells. Doctoral thesis , UCL (University College London).

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Abstract

Weibel‐Palade bodies (WPBs) are large rod‐shaped secretory organelles found exclusively in endothelial cells. In response to the activation of the endothelium by chemical or mechanical stimulation they undergo regulated exocytosis and release their contents into the bloodstream. Their primary cargo proteins are the large pro‐thrombotic glycoprotein von Willebrand Factor (VWF) and the leukocyte adhesion receptor P‐selectin. Hence, the principal function of WPBs is to act as a storage compartment for molecular mediators of thrombosis and inflammation. It is known that the exocytosis of WPBs is induced by increasing levels of free Ca2+ ions in the cytosol. However, the molecular machinery which drives exocytosis is currently unclear. The aim of my PhD was to determine the identity of the Ca2+ sensor responsible for the regulation of WPB secretion and it is my hypothesis that this process is driven by one or more members of the synaptotagmin (SYT) family. SYTs are the most well-characterized family of calcium sensors and have established roles in mediating the Ca2+-driven exocytosis of large secretory organelles from a range of cell types. However the expression, subcellular distribution, and function of SYTs in endothelial cells remains largely unknown. Here, I show by RT-PCR and Western blotting that SYTs I, III, V, VI, VII, VIII, XI, and XVII are expressed in Human Umbilical Vein Endothelial Cells (HUVECs). Additionally, I constructed fluorescent fusion proteins of these endothelial-expressed SYTs and have demonstrated by over-expression that SYTs V, VII, VIII, and XVII are trafficked to the WPBs in HUVECs. The use of a novel dose-response assay has revealed that the up-regulation of SYT VII modulates the calcium sensitivity of WPB exocytosis and thus this raises the intriging possibility that SYT VII may be a principle component of the Ca2+-sensing complex on the organelle membrane.

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