Abstract
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 N6,2′-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 EC50 of 1.2 to 2.1 μM forskolin, 31 to 33 μM YC-1, 57 to 150 μM dibutyryl-cAMP, and 220 to 410 μM 8-pCPT-cGMP. There was a marked left shift in the inhibitory potencies of forskolin and YC-1 for α-granule release and glycoprotein IIbIIIa/integrin αIIbβ3 activation (i.e., EC50 of 1–60 and 40–1300 nM, respectively) that was not observed for dibutyryl-cAMP and 8-pCPT-cGMP (i.e., EC50 of 200–600 and 40–140 μM, 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.
Footnotes
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This work was supported by National Institutes of Health Grant HL081009 to H.E.H. and National Heart, Lung, and Blood Institute Grant SCCOR P50 HL081009. M.L.B. is supported by National Institutes of Health Training Grant T32 HL007411 (to Douglas E. Vaughan, Chief of the Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University Medical Center).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.121830.
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ABBREVIATIONS: PAR, protease-activated receptor; GPIIbIIIa, glycoprotein IIbIIIa/integrin αIIbβ3; NO, nitric oxide; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PDE, phosphodiesterase; PAR-AP, protease-activated receptor agonist peptide; YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole; dibutyryl-cAMP, N6,2′-O-dibutyryladenosine-3′-5′-cAMP; 8-pCPT-cGMP, 8-(4-parachlorophenylthoi)-cGMP; IBMX, 3-isobutyl-1-methylxanthine; VASP, vasodilator-stimulated phosphoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AM, acetoxymethyl ester; PLD, phospholipase D.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received February 22, 2007.
- Accepted May 23, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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