Abstract 71: G protein-dependent Constitutive and Evoked Endothelial Cell vWF secretion

Abstract

A poorly understood mechanism and yet critically important requirement of hemostasis and thrombosis is von Willebrand Factor (vWF) secretion by endothelial cells (ECs). In the present study, we used genetic approaches in vitro and in vivo to study the role of G proteins, RhoA, and αSNAP in basal and evoked EC-specific vWF secretion. As expected, αSNAP siRNA completely abolished constitutive, Ca2+-induced, as well as thrombin-induced vWF secretion from human umbilical vein endothelial cells (HUVEC), indicating vWF secretion is dependent on fusion and exocytosis of Weibel-Palade Bodies (WPBs). In addition, siRNA-mediated depletion of guanine-nucleotide exchange protein 12 α subunit (GNA12) inhibited both constitutive and thrombin-induced vWF secretion whereas there was no effect of GNA13 siRNA. In addition, exogenous expression of constitutively active GNA12 mutant promoted vWF secretion. In GNAq-, p115 RhoGEF-, and RhoA siRNA-depleted HUVECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal vWF secretion was not affected. Based on these data, we hypothesized that GNA12 and GNAq deficiency, by affecting constitutive and Ca2+-dependent vWF secretion, would cause defects in hemostasis and thrombosis in a mouse model of vascular injury. In GNAq/11-/- mice, plasma levels of vWF were normal although clotting-time was prolonged, whereas GNA12-/- mice showed increased tail snip-clotting time, reduced laser-injury clot formation, and reduced vWF levels in blood as well as buffered-perfused mouse lungs. Importanly, purified human vWF rescued tail snip clot formation in GNA12-/- mice. In vitro binding assays revealed GNA12 N-terminal domain aa 10-15 was sufficient for binding to αSNAP, and when expressed in endothelial cells, αSNAP binding domain minigene-peptide blocked thrombin-induced vWF secretion. Taken together, these studies uncover obligitory and complementary roles of GNA12 and GNAq/11 in constitutive and evoked vWF secretion, thrombosis, and hemostasis.