Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
The Centenary Institute and University of Sydney, Sydney, New South Wales, Australia.
J Thromb Haemost. 2023 Aug;21(8):2137-2150. doi: 10.1016/j.jtha.2023.03.034. Epub 2023 Apr 8.
Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines.
We hypothesized that PDI is sulfenylated during oxidative stress, contributing to the prothrombotic potential of PDI.
Biochemical and enzymatic assays using purified proteins, platelet and endothelial cell assays, and in vivo murine thrombosis studies were used to evaluate the role of oxidative stress in PDI sulfenylation and prothrombotic activity.
PDI exposure to oxidants resulted in the loss of PDI reductase activity and simultaneously promoted sulfenylated PDI generation. Following exposure to oxidants, sulfenylated PDI spontaneously converted to disulfided PDI. PDI oxidized in this manner was able to transfer disulfides to protein substrates. Inhibition of sulfenylation impaired disulfide formation by oxidants, indicating that sulfenylation is an intermediate during PDI oxidation. Agonist-induced activation of platelets and endothelium resulted in the release of sulfenylated PDI. PDI was also sulfenylated by oxidized low-density lipoprotein (oxLDL). In an in vivo model of thrombus formation, oxLDL markedly promoted platelet accumulation following an arteriolar injury. PDI oxidoreductase inhibition blocked oxLDL-mediated augmentation of thrombosis.
PDI sulfenylation is a critical posttranslational modification that is an intermediate during disulfide PDI formation in the setting of oxidative stress. Oxidants generated by vascular cells during activation promote PDI sulfenylation, and interference with PDI during oxidative stress impairs thrombus formation.
氧化应激会导致动脉粥样硬化、炎症、感染、衰老和恶性肿瘤中的血栓形成。氧化剂诱导的半胱氨酸修饰，包括亚磺酰化，可以作为一种氧化还原敏感的开关，控制蛋白质的功能。蛋白二硫键异构酶（PDI）是一种促血栓形成的酶，其活性部位的半胱氨酸具有极其敏感的氧化还原特性。
我们假设 PDI 在氧化应激过程中发生亚磺酰化，从而导致 PDI 的促血栓形成潜能。
使用纯化蛋白、血小板和内皮细胞测定以及体内小鼠血栓形成研究进行生化和酶学测定，以评估氧化应激在 PDI 亚磺酰化和促血栓形成活性中的作用。
PDI 暴露于氧化剂会导致 PDI 还原酶活性丧失，并同时促进亚磺酰化 PDI 的产生。暴露于氧化剂后，亚磺酰化 PDI 会自发转化为二硫键化 PDI。以这种方式氧化的 PDI 能够将二硫键转移到蛋白质底物上。抑制亚磺酰化会损害氧化剂形成二硫键的能力，表明亚磺酰化是 PDI 氧化过程中的一个中间步骤。激动剂诱导的血小板和内皮细胞激活导致亚磺酰化 PDI 的释放。氧化型低密度脂蛋白（oxLDL）也可使 PDI 发生亚磺酰化。在血栓形成的体内模型中，oxLDL 明显促进了小动脉损伤后的血小板聚集。PDI 氧化还原酶抑制阻断了 oxLDL 介导的血栓形成增强作用。
PDI 亚磺酰化是一种关键的翻译后修饰，是氧化应激条件下二硫键 PDI 形成的中间步骤。血管细胞在激活过程中产生的氧化剂促进 PDI 亚磺酰化，而在氧化应激过程中干扰 PDI 会损害血栓形成。
