The NO/cGMP pathway inhibits Rap 1 activation in human platelets via cGMP-dependent protein kinase I.
The NO/cGMP signalling pathway strongly inhibits agonist-induced platelet aggregation. However, the molecular mechanisms involved are not completely defined. We have studied NO/cGMP effects on the activity of Rap 1, an abundant guanine-nucleotidebinding protein in platelets. Rap 1-GTP levels were reduced by NO-donors and activators of NO-sensitive soluble guanylyl cyclase. Four lines of evidence suggest that NO/cGMP effects are mediated by cGMP-dependent protein kinase (cGKI): (i) Rap 1 inhibition correlated with cGKI activity as measured by the phosphorylation state of VASP, an established substrate of cGKI, (ii) 8-pCPT-cGMP, a membrane permeable cGMP-analog and activator of cGKI, completely blocked Rap1 activation, (iii) Rp-8pCPT-cGMPS, a cGKI inhibitor, reversed NO effects and (iv) expression of cGKI in cGKI-deficient megakaryocytes inhibited Rap1 activation. NO/cGMP/cGKI effects were independent of the type of stimulus used for Rap1 activation. Thrombin-,ADP- and collagen-induced formation of Rap 1-GTP in platelets as well as turbulence-induced Rap 1 activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rap 1 activation induced by the Galpha(i)-coupled P2Y12 receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rap 1 activation in human platelets and that this effect is mediated by cGKI. Since Rap1 controls the function of integrin alpha(IIb)beta3, we propose that Rap 1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.
Danielewski
Oliver
O
Institute for Biochemistry II, University of Frankfurt Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany.
Schultess
Jan
J
Smolenski
Albert
A
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D003094:Collagen / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism*; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D003094:Collagen; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; D020827:rap1 GTP-Binding Proteins; D006152:Cyclic GMP