SYNERGISTIC INTERACTION OF ADENYLATE-CYCLASE ACTIVATORS AND NITRIC-OXIDE DONOR SIN-1 ON PLATELET CYCLIC-AMP

The molecular mechanism of the synergistic platelet inhibition by activators of adenylate cyclase and guanylate cyclase in human platelets was investigated. The adenylate cyclase activators iloprost and prostaglandin E(1) and the guanylate cyclase activator 3-morpholino-sydnonimine (SIN-1) dose-dependently inhibited thrombin-induced aggregation of washed human platelets. Furthermore, SIN-1 at a concentration inhibiting platelet aggregation by only 10% shifted the IC50 values of iloprost and prostaglandin E(1) by one order of magnitude to the left, indicating a synergistic action of adenylate cyclase and guanylate cyclase activators. Iloprost and prostaglandin E(1) dose-dependently elevated platelet cAMP without a significant influence on cGMP. In contrast, the platelet cGMP level was dose-dependently elevated by SIN-1. In addition, SIN-1 markedly increased cAMP level induced by low concentrations of adenylate cyclase activators (0.1-0.3 nM iloprost or 10-150 nM prostaglandin E(1)). In contrast, the rise in cAMP induced by higher adenylate cyclase activator concentrations (3 nM iloprost or 30 mu M prostaglandin E(1)) was significantly reduced in the presence of SIN-1. The same biphasic mode of action of SIN-1 was observed with forskolin, an adenylate cyclase stimulator acting receptor independently, indicating a prostacyclin-receptor independent mechanism. The cAMP elevating effect of SIN-1 in the presence of low prostanoid concentrations was completely abolished by piroximone, a selective inhibitor of phosphodiesterase type III. Therefore, the inhibition of phosphodiesterase III by cGMP seems to be the mechanism for the elevation of cAMP levels by SIN-1 in the presence of low concentration of adenylate cyclase activators in human platelets. In contrast, the mechanism by which SIN-1 reduces platelet cAMP levels induced by higher concentrations of prostaglandin E(1), iloprost, and forskolin, remains to be clarified.