Thrombin potently regulates human platelets by the G protein-coupled receptors protease-activated receptor (PAR) 1 and PAR4. Platelet activation by thrombin and other agonists is broadly inhibited by prostacyclin and nitric oxide acting through adenylyl and guanylyl cyclases to elevate cAMP and cGMP levels, respectively. Using forskolin and YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] to selectively activate the adenylyl and guanylyl cyclases, respectively, and the membrane-permeable analogs N(6),2'-O-dibutyryladenosine-3'-5'-cAMP (dibutyryl-cAMP) and 8-(4-parachlorophenylthoi)-cGMP (8-pCPT-cGMP), we sought to identify key antiplatelet steps for cyclic nucleotide actions in blocking platelet activation by PAR1 versus PAR4. Platelet aggregation by PAR1 or PAR4 was inhibited with similar EC(50) of 1.2 to 2.1 microM forskolin, 31 to 33 microM YC-1, 57 to 150 microM dibutyryl-cAMP, and 220 to 410 microM 8-pCPT-cGMP. There was a marked left shift in the inhibitory potencies of forskolin and YC-1 for alpha-granule release and glycoprotein IIbIIIa/integrin alphaIIbbeta3 activation (i.e., EC(50) of 1-60 and 40-1300 nM, respectively) that was not observed for dibutyryl-cAMP and 8-pCPT-cGMP (i.e., EC(50) of 200-600 and 40-140 microM, respectively). This inhibition was essentially instantaneous, and measurements of cyclic nucleotide levels and kinase activities support a model of compartmentation involving the cyclic nucleotide effectors and regulators and the key molecular targets for this platelet inhibition. The different sensitivities of PAR1 and PAR4 to inhibition of calcium mobilization and dense granule release identify key antiplatelet steps for cyclic nucleotide actions and are consistent with the signaling models for these receptors. Specifically, PAR4 inhibition depends on the regulation of both calcium mobilization and dense granule release, and PAR1 inhibition depends predominantly on the regulation of dense granule release.