Division of Clinical Pharmacology, General Clinical Research Center, Nanjing First Hospital, China Pharmaceutical University, Nanjing, China; Division of Clinical Pharmacology, General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
Division of Clinical Pharmacology, General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
J Thromb Haemost. 2023 Jan;21(1):117-132. doi: 10.1016/j.jtha.2022.10.010. Epub 2022 Dec 22.
Trimethylamine N-oxide (TMAO), a gut microbe-generated metabolite, elicits thrombotic events by enhancing platelet reactivity; however, no studies have reported the effects of TMAO on the metabolism of and response to clopidogrel.
To determine whether choline and TMAO could significantly impair metabolic activation of and platelet response to clopidogrel in choline- or TMAO-fed mice and the mechanisms involved.
Male mice were fed with vehicle control (Ctrl), TMAO, choline alone or in combination with 3,3-dimethyl-1-butanol, N-acetyl-L-cysteine, or ML385 for 14 days and then treated with Ctrl or a single oral dose of clopidogrel. Plasma TMAO, protein levels of clopidogrel-metabolizing enzymes in the liver, plasma concentrations of clopidogrel and its metabolites, and adenosine diphosphate-induced platelet aggregation and activation were measured. In addition, HepG2 cells were treated with Ctrl or TMAO alone or in combination with N-acetyl-L-cysteine, ML385, or apocynin, and CES1, reactive oxygen species (ROS), and Nrf2 protein levels were measured, respectively.
TMAO significantly increased Ces1 protein expression and activity and clopidogrel hydrolysis in the liver as well as intracellular ROS and CES1 levels and Nrf2 nucleus translocation in HepG2 cells but decreased the formation of clopidogrel active metabolite and impaired platelet response to clopidogrel. Furthermore, concomitant use of 3,3-dimethyl-1-butanol, N-acetyl-L-cysteine, or ML385 effectively reversed choline- or TMAO-induced impairment of inhibition of platelet aggregation by clopidogrel in mice, respectively.
Choline and TMAO impair the metabolic activation of and platelet response to clopidogrel through the activation of the NOX-dependent ROS/Nrf2/CES1 pathway, suggesting novel strategies for overcoming clopidogrel resistance from bench to bedside.
三甲胺 N-氧化物（TMAO）是一种肠道微生物生成的代谢物，通过增强血小板反应性引发血栓事件；然而，尚无研究报道 TMAO 对氯吡格雷的代谢和反应的影响。
确定胆碱和 TMAO 是否会显著损害胆碱或 TMAO 喂养的小鼠中氯吡格雷的代谢激活和血小板反应，并探讨其相关机制。
雄性小鼠连续 14 天给予对照物（Ctrl）、TMAO、胆碱或 TMAO 联合 3,3-二甲基-1-丁醇、N-乙酰-L-半胱氨酸或 ML385 喂养，然后给予 Ctrl 或单次口服氯吡格雷。检测血浆 TMAO、肝脏中氯吡格雷代谢酶的蛋白水平、血浆中氯吡格雷及其代谢物浓度以及二磷酸腺苷诱导的血小板聚集和激活情况。此外，分别用对照物或 TMAO 单独或联合 N-乙酰-L-半胱氨酸、ML385 或 apocynin 处理 HepG2 细胞，检测 CES1、活性氧（ROS）和 Nrf2 蛋白水平。
TMAO 显著增加了肝脏中 Ces1 蛋白表达和活性以及氯吡格雷水解，同时增加了 HepG2 细胞内 ROS 和 CES1 水平以及 Nrf2 核易位，但减少了氯吡格雷活性代谢物的形成并损害了血小板对氯吡格雷的反应。此外，3,3-二甲基-1-丁醇、N-乙酰-L-半胱氨酸或 ML385 的联合使用分别有效地逆转了胆碱或 TMAO 诱导的小鼠氯吡格雷抑制血小板聚集的作用受损。
胆碱和 TMAO 通过激活 NOX 依赖性 ROS/Nrf2/CES1 通路，损害氯吡格雷的代谢激活和血小板反应，提示从实验室到临床克服氯吡格雷抵抗的新策略。
