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.

中文翻译：

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新开发的AMP激活的蛋白激酶激活剂YLF-466D的抗血小板作用。

AMP激活的蛋白激酶（AMPK）是细胞能量稳态的主要调节剂。在血小板中，AMPK激活会刺激内皮一氧化氮合酶（eNOS）及其下游信号传导，从而抑制血小板聚集。在这项研究中，新开发的AMPK活化剂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]-苯甲酸测试了乙酸（YLF-466D）的抗血小板活性。用YLF-466D处理分离的血小板以50-150μM的浓度依赖性方式导致AMPK活化。在相同的实验条件下，YLF-466D有效抑制凝血酶，ADP和胶原蛋白等血小板激动剂诱导的聚集。通过使用AMPK抑制剂化合物C（CC）和ara-A进行预处理，取消了此类AMPK活化和聚集抑制作用，表明YLF-466D的抗聚集作用是由AMPK介导的。YLF-466D诱导了Ser1177处的激活依赖性eNOS磷酸化，环核苷酸cGMP和cAMP的升高，以及随后在Ser239和Ser157处血管扩张剂刺激的磷蛋白（VASP）的磷酸化。所有这些事件均由CC和ara-A阻止。除分离的血小板外，YLF-466D还可减弱胶原蛋白诱导的全血聚集。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凝集。这项研究证实了AMPK激活剂的抗血小板活性，并暗示了YLF-466D在抗血小板治疗中的潜在应用，