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 A₂ (TXA₂). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA₂ receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (8-Br-cGMP) potently inhibited activation of the TXA₂-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 TXA₂ receptors in vitro, but not Gα_q copurifying with the TXA₂ receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA₂ receptors by cyclic GMP was demonstrated from ³²P-labeled cells treated with 8-Br-cGMP. Peptide mapping studies of in vivo phosphorylated TXA₂ receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA₂ 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 TXA₂ receptor. These data identify TXA₂ 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.