G Protein-Dependent Basal And Evoked Endothelial Cell Vwf Secretion

vonWillebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-G alpha 13(-/-);G alpha 12(-/-) mice that could be normalized by infusion of human vWF. Blood from G alpha 12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that G alpha 12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in G alpha 12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-G alpha q(-/-);G alpha 11(-/-) and G alpha 12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of G alpha 12 and soluble N-ethylmaleimide- sensitive-fusion factor attachment protein alpha (alpha-SNAP), but not G alpha 13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated G alpha 12 promoted vWF secretion. In G alpha q, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that G alpha 12 N-terminal residues 10-15 mediated the binding of G alpha 12 to alpha-SNAP, and an engineered alpha-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of G alpha 12 and G alpha q/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.