Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

硫氧还蛋白(Thioredoxin, Trx)是一类具有重要生理功能的氧化还原酶。NADPH/硫氧还蛋白还原酶/硫氧还蛋白系统失衡与多种病理状态密切相关，尤以癌症为甚，目前已有多项针对硫氧还蛋白及硫氧还蛋白还原酶抑制剂的临床试验正在推进或已完成。
鉴于氧化还原酶在止血过程中的新兴作用，以及当前临床开发抑制剂的研究热点，我们认为有必要评估NADPH/硫氧还蛋白还原酶/硫氧还蛋白系统抑制是否会影响血小板功能与血栓形成。
我们采用硫氧还蛋白的小分子抑制剂PMX 464与PX-12，探究硫氧还蛋白活性对血小板功能的影响；同时运用无偏蛋白质组学策略，筛选血小板表面可能参与血小板反应性与功能调控的潜在硫氧还蛋白底物。通过液相色谱-串联质谱(LC-MS/MS)分析，我们发现PMX 464与PX-12可改变多种细胞表面蛋白的巯基氧化状态。血小板黏附与活化的关键表面受体均受其调控，包括胶原受体GPVI与血管性血友病因子(von Willebrand factor, vWF)受体GPIb。
为实验验证上述发现，我们分别在PMX 464、PX-12以及芦丁（一种靶向相关蛋白二硫键异构酶的选择性抑制剂）存在的条件下评估血小板功能。结果与蛋白质组学数据一致：硫氧还蛋白小分子抑制剂可选择性抑制GPVI介导的血小板活化，并减弱瑞斯西丁素诱导的GPIb-vWF介导的血小板凝集，从而验证了蛋白质组学研究的结论。
本研究揭示了硫氧还蛋白通过调控血管损伤部位早期血小板反应所需的蛋白（GPVI与GPIb）来调节血小板反应性的全新功能。同时，本研究也为将PMX 464与PX-12重新开发为抗血小板药物提供了潜在机遇。