Molecular Mechanisms of vWF Secretion

von Willebrand factor (vWF) secreted by endothelial cells (EC) is essential for hemostasis and thrombosis. However the molecular mechanism of vWF secretion is poorly understood. Here, we employed genetic approaches in vitro and in vivo to determine the role of G proteins, RhoA, and alpha-SNAP in constitutive vs. evoked vWF secretion. SiRNA-mediated depletion of heterotrimeric G protein subunit G alpha 12 or alpha-SNAP inhibited both constitutive and thrombin-induced vWF secretion, while over-expression of activated G alpha 12 promoted vWF secretion. In G alpha q, p115 RhoGEF, and RhoA-depleted HUVECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Increased blood loss in cEC-GNA13/GNA12-/- mice was normalized by infusion of human vWF, whereas G alpha 12-/- mice exhibited reduced blood vWF, impaired arteriolar thrombus formation, and reduced basal and thrombin-induced EC vWF secretion in isolated buffered-perfused mouse lungs. Plasma vWF level was normal in G alpha q/11-/- mice although hemostasis was defective. In vitro binding assays showed G alpha 12 N-terminal domain (aa 10-15) mediated the binding to alpha-SNAP whereas an engineered alpha-SNAP binding domain mini-gene peptide blocked constitutive and evoked vWF secretion. Thus, discovery of obligatory and complementary roles of G alpha 12 and G alpha q/11 in constitutive vs. evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.