The NO/cGMP pathway inhibits Rap1 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 Rapl, an abundant guanine-nucleotide-binding protein in platelets. Rapl-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) Rapl 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 Rapl activation, (iii) Rp8pCPT-cGMPS, a cGKI inhibitor, reversed NO effects and (iv) expression of cGKI in cGKI-deficient megakaryocytes inhibited Rapl activation. NO/cGMP/cGKI effects were independent of the type of stimulus used for Rapl activation. Thrombin-,ADP-and collagen-induced formation of Rapl-GTP in platelets as well as turbulence-induced Rapl activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rapl activation induced by the Galpha(i)-coupled P2Y(12) receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rapl activation in human platelets and that this effect is mediated by cGKI. Since Rapl controls the function of integrin alphaIIbbeta3, we propose that Rap I inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.