Liu Yingqiu, Park Jung-Min, Oh Seok-Jeong, Chang Kyung-Hwa, Lee Moo-Yeol
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea.
Eur J Pharmacol. 2015 Aug 5;760:81-7. doi: 10.1016/j.ejphar.2015.03.084. Epub 2015 Apr 22.
AMP-activated protein kinase (AMPK) acts as a major regulator of cellular energy homeostasis. In platelets, AMPK activation stimulates endothelial nitric oxide synthase (eNOS) and its downstream signaling, and thereby inhibits platelet aggregation. In this study, a newly developed AMPK activator 3-[[(3E)-3-[(4-chlorophenyl)phenylmethylene]-2,3-dihydro-2-oxo-1H-indol-1-yl]methyl]-benzoic acid (YLF-466D) was tested for its antiplatelet activity. Treatment of isolated platelets with YLF-466D resulted in AMPK activation in a concentration-dependent manner in a range of 50-150 μM. Under the same experimental condition, YLF-466D effectively inhibited aggregation induced by platelet agonists including thrombin, ADP and collagen. Such AMPK activation and aggregation inhibition were abolished by pretreatment with the AMPK inhibitors compound C (CC) and ara-A, indicating that antiaggregatory effect of YLF-466D is mediated by AMPK. YLF-466D induced an activation-dependent eNOS phosphorylation at Ser1177, an elevation of cyclic nucleotides cGMP and cAMP, and subsequent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at Ser239 and Ser157. All these events were prevented by CC and ara-A. In addition to isolated platelets, YLF-466D attenuated whole blood aggregation induced by collagen. Taken together, YLF-466D is capable of inhibiting platelet aggregation by activating AMPK and its downstream eNOS-cGMP-PKG signaling axis. This study reconfirms the antiplatelet activity of AMPK activators and suggests the potential application of YLF-466D to antiplatelet therapy, although the in vivo and clinical validation remains to be assessed.
AMP激活的蛋白激酶（AMPK）是细胞能量稳态的主要调节因子。在血小板中，AMPK激活可刺激内皮型一氧化氮合酶（eNOS）及其下游信号传导，从而抑制血小板聚集。在本研究中，对新开发的AMPK激活剂3-[[(3E)-3-[(4-氯苯基)苯基亚甲基]-2,3-二氢-2-氧代-1H-吲哚-1-基]甲基]苯甲酸（YLF-466D）的抗血小板活性进行了测试。用YLF-466D处理分离的血小板，在50-150μM范围内以浓度依赖性方式导致AMPK激活。在相同实验条件下，YLF-466D有效抑制由凝血酶、ADP和胶原等血小板激动剂诱导的聚集。用AMPK抑制剂化合物C（CC）和阿糖腺苷预处理可消除这种AMPK激活和聚集抑制，表明YLF-466D的抗聚集作用是由AMPK介导的。YLF-466D诱导Ser117位点的eNOS磷酸化激活依赖性增加、环核苷酸cGMP和cAMP升高，随后Ser239和Ser157位点的血管舒张刺激磷蛋白（VASP）磷酸化。所有这些事件均被CC和阿糖腺苷阻止。除分离的血小板外，YLF-466D还可减弱胶原诱导的全血聚集。综上所述，YLF-466D能够通过激活AMPK及其下游eNOS-cGMP-PKG信号轴来抑制血小板聚集。本研究再次证实了AMPK激活剂的抗血小板活性，并提示YLF-466D在抗血小板治疗中的潜在应用，尽管其体内和临床验证仍有待评估。
