Bilodeau Matthew L, Hamm Heidi E
Department of Pharmacology, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 442 Robinson Research Building, Nashville, TN 37232-6600, USA.
J Pharmacol Exp Ther. 2007 Aug;322(2):778-88. doi: 10.1124/jpet.107.121830. Epub 2007 May 24.
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.
凝血酶通过G蛋白偶联受体蛋白酶激活受体（PAR）1和PAR4对人类血小板进行有效调节。凝血酶和其他激动剂引起的血小板激活可被前列环素和一氧化氮广泛抑制，它们分别通过腺苷酸环化酶和鸟苷酸环化酶发挥作用，从而提高cAMP和cGMP水平。我们分别使用福司可林和YC-1 [3-(5'-羟甲基-2'-呋喃基)-1-苄基吲唑] 来选择性激活腺苷酸环化酶和鸟苷酸环化酶，并使用可透过细胞膜的类似物N(6),2'-O-二丁酰腺苷-3'-5'-环磷酸腺苷（二丁酰-cAMP）和8-(4-对氯苯基硫代)-环磷酸鸟苷（8-pCPT-cGMP），试图确定环核苷酸在通过PAR1与PAR4阻断血小板激活过程中的关键抗血小板作用步骤。PAR1或PAR4诱导的血小板聚集受到抑制，福司可林、YC-1、二丁酰-cAMP和8-pCPT-cGMP的半数有效浓度（EC(50)）相似，分别为1.2至2.1微摩尔、31至33微摩尔、57至150微摩尔和220至410微摩尔。福司可林和YC-1对α-颗粒释放和糖蛋白IIbIIIa/整合素αIIbbeta3激活的抑制效力有明显的左移（即EC(50)分别为1至60纳摩尔和40至1300纳摩尔），而二丁酰-cAMP和8-pCPT-cGMP则未观察到这种情况（即EC(50)分别为200至600微摩尔和40至140微摩尔）。这种抑制基本上是瞬时的，环核苷酸水平和激酶活性的测量结果支持一种区室化模型，该模型涉及环核苷酸效应器和调节剂以及这种血小板抑制的关键分子靶点。PAR1和PAR4对钙动员和致密颗粒释放抑制的不同敏感性确定了环核苷酸作用的关键抗血小板步骤，并且与这些受体的信号传导模型一致。具体而言，PAR4的抑制取决于对钙动员和致密颗粒释放的调节，而PAR1的抑制主要取决于对致密颗粒释放的调节。
