Abstract 1656: Vegf Induces vWF Release via A VEGFR2/Tyr1175-PLC- gamma1 Pathway

Abstract

Many endothelial inflammatory and prothrombotic mediators are stored in and rapidly released from Weibel-Palade bodies (WPBs), endothelium-specific storage organelles upon stimulation. The von Willebrand factor (vWF), a major component inside WPBs, mediates the initial contact of platelets with the injured vessel wall and thus plays an important role in haemostasis and thrombosis. It has previously been shown that vascular endothelial growth factor (VEGF) triggers a rapid release of vWF. However, specific VEGF receptors and their potential downstream pathways involved in this process have not been carefully determined. To dissect the role of VEGF receptors in vWF release activation, we utilized two approaches: one is to use receptor-specific ligands and the other is to use a chimeric receptor approach. The ligands for VEGF receptor 2 (VEGFR2), but not VEGF receptor 1 (VEGFR1), stimulated vWF release. The predominant role of VEGFR2 in vWF release regulation was further confirmed by using a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR1 or VEGFR2, and was then expressed in early-passage HUVECs. Further, the knockdown of phospholipase C-γ1 (PLCγ1) suppressed VEGF-triggered intracellular calcium increase and blocked VEGF-induced vWF release. In addition, the two products of PLCγ1 hydrolysis, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), are both required for VEGF-induced vWF release. Finally, combined with point mutagenesis, the responsible binding sites for PLCγ1 and their importance in VEGFR2-activated vWF release have been determined. Point mutation of a single tyrosine residue Tyr1175, a putative binding site for PLCγ1 on the C-terminus, abolished VEGFR2-activated vWF release. This study presents the first evidence that the PLCγ1 is essential for VEGF-triggered vWF release mediated through a VEGFR2/Tyr1175 pathway.