Abstract
Abstract 698
Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein essential for homeostasis, also involved in inflammation and angiogenesis. The majority of VWF is synthesised by endothelial cells (EC) and is either constitutively secreted or stored in Weibel-Palade bodies (WPB), ready to be released in response to endothelial stimulation. Several studies have shown that formation of WPB is dependent on the presence of VWF, and deletion of VWF in human umbilical vein EC (HUVEC) results in loss of WPB. Amongst the other proteins shown to co-localise to WPB is angiopoietin-2 (Ang2), a ligand of the receptor tyrosine kinase Tie-2. Ang2 regulates endothelial cell survival, vascular stability and maturation, by destabilizing quiescent endothelium and facilitating the response to inflammatory and angiogenic stimuli. VWF is required for storage of Ang2, and release of Ang-2 from EC is increased in VWF-deficient HUVEC. Recently, we have shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by Ang-2. In the present study we investigated the interaction between Ang2 and VWF. Binding analysis demonstrated that recombinant human Ang2 bound to purified plasma-derived VWF in a pH and calcium dependent manner, with optimal binding occurring at pH 6.5 and 10mM calcium, indicative of binding within the Golgi body. Generation of binding isotherms established that Ang2 bound to VWF with high affinity (KD∼3nM); furthermore binding affinity was not dependent on VWF conformation. Using an array of VWF constructs we determined that Ang2 bound predominantly to the VWF A1 domain, which also contains binding sites to the platelet receptor GPIb and extracellular matrix proteins. Co-immunoprecipitation experiments performed on TNFα- and ionomycin-stimulated HUVECs, to induce WPB exocytosis, confirmed that a portion of Ang2 remained bound to secreted VWF. Moreover, immunofluorescence staining of histamine-stimulated HUVECs to induce VWF release demonstrated the presence of Ang2 on VWF strings secreted from ECs. Finally we demonstrated that Ang2 bound to VWF was still able to interact with Tie-2. These data demonstrate that binding of Ang2 to VWF occurs within the cell; we propose that this is the mechanism mediating storage of Ang2 in WPB. Moreover, the finding that the Ang2-VWF interaction is preserved following secretion raises the intriguing possibility VWF may affect Ang2 function, possibly by localising Ang2 to the Tie 2 receptor under the shear forces experienced in flowing blood. Similarly, Ang-2 binding to VWF may modulate its interaction with receptors and extracellular matrix proteins, and ultimately influence the role of VWF in the angiogenic processes.
Disclosures:
No relevant conflicts of interest to declare.
Intracellular receptor EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation
