Mechanism of platelet inhibition by nitric oxide: In vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase

Abstract

Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet activation. NO stimulates production of cGMP and activates cGMP-dependent protein kinase (G kinase), which by an unknown mechanism leads to inhibition of Gα q -phospholipase C-inositol 1,4,5-triphosphate signaling and intracellular calcium mobilization for several important agonists, including thromboxane A 2 (TXA 2 ). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA 2 receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (8-Br-cGMP) potently inhibited activation of the TXA 2 -specific GTPase in platelet membranes in a concentration-dependent fashion, suggesting that G kinase catalyzes the phosphorylation of some proximal component of the receptor–G protein signaling pathway. Nanomolar concentrations of G kinase were found to catalyze the phosphorylation of platelet TXA 2 receptors in vitro , but not Gα q copurifying with the TXA 2 receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA 2 receptors by cyclic GMP was demonstrated from 32 P-labeled cells treated with 8-Br-cGMP. Peptide mapping studies of in vivo phosphorylated TXA 2 receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA 2 receptor. G kinase also catalyzed the phosphorylation of peptides corresponding to the cytoplasmic tails of both α and β forms of the receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA 2 receptor. These data identify TXA 2 receptors as cGMP-dependent protein kinase substrates and support a novel mechanism for the inhibition of cell function by NO in which activation of G kinase inhibits signaling by G protein-coupled receptors by catalyzing their phosphorylation.