The activated endothelial cell surface: structure and function

The endothelial-specific secretory organelles called Weibel-Palade Bodies (WPBs) play an important role in haemostasis and in the initiation of the inflammatory response. Activation of the endothelium induces fusion of WPBs at the plasma membrane releasing the pro-thrombotic cargo protein von Willebrand Factor (VWF) and the leukocyte receptor P-selectin, which is involved in the early stages of the inflammatory cascade. In addition, WPBs contain the ubiquitously expressed tetraspanin CD63, which is recruited to the mature organelles in an AP-3 dependent manner. Although CD63 is a long established component of WPBs, the functional significance of this protein within WPB was unknown. This thesis primarily concerns the role of CD63 in inflammation and haemostasis, attempting to determine its function. Using siRNA to knockdown CD63 in Human Umbilical Vein Endothelial Cells (HUVECs), no phenotype was observed in the haemostatic functions nor biogenesis maturation or exocytosis of WPBs. However, CD63 depletion resulted in a failure of P-selectin dependent recruitment of leukocytes to endothelial monolayers. CD63-deficient mice also displayed a reduction in leukocyte recruitment and a decrease in leukocyte extravasation using a peritonitis model. High-resolution scanning electron microscopy and proximity-ligation assays revealed CD63 co-clusters with P-selectin on the surface of activated endothelial cells and that CD63 acts to cluster the receptor, retaining it at the plasma membrane. In addition, scanning electron microscopy of the activated endothelium was carried out to study the structure of VWF exit sites and string formation in static conditions and under shear stress, to recreate physiological conditions. The results reveal new insights into the behaviour of VWF strings and the mechanism of WPB exocytosis at the membrane, involving the actin cytoskeleton