Department of Biomedical Laboratory Science, College of Biomedical Science and Engineering and Regional Research Center, Inje University, Gyungnam, Korea.
J Atheroscler Thromb. 2012;19(4):337-48. doi: 10.5551/jat.10363. Epub 2012 Apr 10.
In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation.
Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) (Ca(2+)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation.
EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both Ca(2+) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit Ca(2+) mobilization and TXA(2) production on collagen-induced platelet aggregation.
These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.
在这项研究中，我们研究了 (-)-表没食子儿茶素-3-没食子酸酯（EGCG）对胶原（10μg/ml）刺激的血小板聚集中环核苷酸产生和环化酶底物磷酸化蛋白（VASP）磷酸化的影响。
用胶原（10μg/ml）刺激 5 分钟，然后用于测定细胞内胞浆游离钙([Ca2+]i)、血栓素 A2 (TXA2)、腺苷 3'，5'-环单磷酸（cAMP）、鸟苷 3'，5'-环单磷酸（cGMP）和 VASP 磷酸化。
EGCG 呈剂量依赖性抑制胶原诱导的血小板聚集，抑制[Ca2+]i 动员和 TXA2 产生。在两种抑制聚集的分子中，cAMP 和 cGMP，EGCG 显著增加细胞内 cAMP 水平，但不增加 cGMP 水平。EGCG 升高的 cAMP 水平被腺苷酸环化酶抑制剂 SQ22536 降低，但不被 cAMP 特异性磷酸二酯酶抑制剂 etazolate 降低。此外，EGCG 可升高完整血小板和胶原诱导的血小板中 VASP-Ser(157)的磷酸化，VASP-Ser(157)是 cAMP 依赖性蛋白激酶（A 激酶）的底物，但不升高 VASP-Ser(239)的磷酸化，VASP-Ser(239)是 cGMP 依赖性蛋白激酶的底物，EGCG 诱导的 VASP-Ser(157)磷酸化被腺苷酸环化酶抑制剂 SQ22536 和 A 激酶抑制剂 Rp-8-Br-cAMPS 抑制。我们已经证明，EGCG 通过激活腺苷酸环化酶增加 cAMP，然后通过激活 A 激酶使 VASP-Ser(157)磷酸化，从而抑制胶原诱导的血小板聚集时的[Ca2+]i 动员和 TXA2 产生。
这些结果强烈表明，EGCG 是一种有益的化合物，可在胶原-血小板相互作用中升高 cAMP 水平，从而可能预防血小板聚集介导的血栓性疾病。
