MECHANISM OF PLATELET INHIBITION BY NITRIC-OXIDE - IN-VIVO PHOSPHORYLATION OF THROMBOXANE RECEPTOR BY CYCLIC GMP-DEPENDENT PROTEIN-KINASE

Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet ac tivation. NO stimulates production of cGMP and activates cGMP-dependen t protein kinase (G kinase), which by an unknown mechanism leads to in hibition of G alpha(q)-phospholipase C-inositol 1,4,5-triphosphate sig naling and intracellular calcium mobilization for several important ag onists, 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-brom o-cyclic GMP (S-Br-cGMP) potently inhibited activation of the TXA(2)-s pecific GTPase in platelet membranes in a concentration-dependent fash ion, suggesting that G kinase catalyzes the phosphorylation of some pr oximal component of the receptor-G protein signaling pathway, Nanomola r concentrations of G kinase were found to catalyze the phosphorylatio n of platelet TXA(2) receptors in vitro, but not G alpha(q) copurifyin g with the TXA(2) receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA(2) receptors by cyclic GMP wa s demonstrated from P-32-labeled cells treated with 8-Br-cGMP, Peptide mapping studies of in vivo phosphorylated TXA(2) receptors demonstrat ed cGMP mediates phosphorylation of the carboxyl terminus of the TXA(2 ) receptor. G kinase also catalyzed the phosphorylation of peptides co rresponding to the cytoplasmic tails of both alpha and beta forms of t he receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA(2) receptor. These data identif y TXA(2) receptors as cGMP-dependent protein kinase substrates and sup port a novel mechanism for the inhibition of cell function by NO in wh ich activation of G kinase inhibits signaling by G protein-coupled rec eptors by catalyzing their phosphorylation.