Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT.
Circ Res. 2022 Aug 5;131(4):290-307. doi: 10.1161/CIRCRESAHA.121.320530. Epub 2022 Jul 18.
Unfolded protein response (UPR) is a multifaceted signaling cascade that alleviates protein misfolding. Although well studied in nucleated cells, UPR in absence of transcriptional regulation has not been described. Intricately associated with cardiovascular diseases, platelets, despite being anucleate, respond rapidly to stressors in blood. We investigate the UPR in anucleate platelets and explore its role, if any, on platelet physiology and function.
Human and mouse platelets were studied using a combination of ex vivo and in vivo experiments. Platelet lineage-specific knockout mice were generated independently for each of the 3 UPR pathways, PERK (protein kinase RNA [PKR]-like endoplasmic reticulum kinase), XBP1 (X-binding protein), and ATF6 (activating transcription factor 6). Diabetes patients were prospectively recruited, and platelets were evaluated for activation of UPR under chronic pathophysiological disease conditions.
Tunicamycin induced the IRE1α (inositol-requiring enzyme-1alpha)-XBP1 pathway in human and mouse platelets, while oxidative stress predominantly activated the PERK pathway. PERK deletion significantly increased platelet aggregation and apoptosis and phosphorylation of PLCγ2, PLCβ3, and p38 MAPK. Deficiency of XBP1 increased platelet aggregation, with higher PLCβ3 and PKCδ activation. ATF6 deletion mediated a relatively modest effect on platelet phenotype with increased PKA (protein kinase A). Platelets from diabetes patients exhibited a positive correlation between disease severity, platelet activation, and protein aggregation, with only IRE1α-XBP1 activation. Moreover, IRE1α inhibition increased platelet aggregation, while clinically approved chemical chaperone, sodium 4-phenylbutyrate reduced the platelet hyperactivation.
We show for the first time, that UPR activation occurs in platelets and can be independent of genomic regulation, with selective induction being specific to the source and severity of stress. Each UPR pathway plays a key role and can differentially modulate the platelet activation pathways and phenotype. Targeting the specific arms of UPR may provide a new antiplatelet strategy to mitigate thrombotic risk in diabetes and other cardiovascular diseases.
未折叠蛋白反应（UPR）是一种多方面的信号级联反应，可减轻蛋白质错误折叠。尽管在有核细胞中研究得很好，但在没有转录调节的情况下，UPR 尚未被描述。血小板与心血管疾病密切相关，尽管没有细胞核，但对血液中的应激源反应迅速。我们研究了无核血小板中的 UPR，并探讨了其在血小板生理学和功能中的作用（如果有的话）。
使用体外和体内实验的组合研究了人类和小鼠血小板。分别为 UPR 的 3 条途径（PERK（蛋白激酶 RNA [PKR]-样内质网激酶）、XBP1（X 结合蛋白）和 ATF6（激活转录因子 6））生成血小板谱系特异性敲除小鼠。前瞻性招募糖尿病患者，并在慢性病理生理疾病条件下评估血小板 UPR 的激活情况。
衣霉素诱导了人源和鼠源血小板中的 IRE1α（肌醇需求酶 1α）-XBP1 途径，而氧化应激主要激活了 PERK 途径。PERK 缺失显著增加血小板聚集和凋亡以及 PLCγ2、PLCβ3 和 p38 MAPK 的磷酸化。XBP1 缺陷增加了血小板聚集，同时激活了更高水平的 PLCβ3 和 PKCδ。ATF6 缺失对血小板表型的影响相对较小，增加了 PKA（蛋白激酶 A）。糖尿病患者的血小板表现出疾病严重程度、血小板激活和蛋白聚集之间的正相关，只有 IRE1α-XBP1 激活。此外，IRE1α 抑制增加了血小板聚集，而临床批准的化学伴侣钠 4-苯丁酸盐减少了血小板的过度激活。
我们首次表明，UPR 的激活发生在血小板中，并且可以独立于基因组调节，其选择性诱导取决于应激的来源和严重程度。每个 UPR 途径都起着关键作用，并可以通过不同的方式调节血小板激活途径和表型。针对 UPR 的特定分支可能为减少糖尿病和其他心血管疾病中的血栓形成风险提供新的抗血小板策略。
