Circulating neutrophil extracellular traps (NETs) resistant to t-PA have not been studied completely although NETs in thrombi may contribute to tissue plasminogen activator (t-PA) resistance. This research intended to elucidate whether circulating NETs are associated with t-PA resistance and the underlying mechanism. The levels of NETs were detected in the circulating neutrophils, ischemic brain tissue of acute ischemic stroke (AIS) patients, and transient middle cerebral artery occlusion (tMCAO) models. NET formation in blood, thrombi, and ischemic brain tissue of mice were analyzed by immunofluorescence. Exposed phosphatidylserine (PS) was assessed using flow cytometry and confocal microscopy. Procoagulant activity (PCA) was evaluated using fibrin formation assays, thrombin, and purified coagulation complex. The plasma levels of NETs in AIS patients were significantly higher than those in healthy individuals. After thrombolysis, a significant increase was noted in NET markers in no-improvement patients, while the changes in improvement patients were not significant. Importantly, NETs were decorated with von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) in the blood and thrombi, which could reverse the fibrinolytic effects. In addition, NETs activated platelets (PLTs) and endothelial cells (ECs), stimulating a procoagulant phenotype and facilitating vWF and PAI-1 release. DNase I, activated protein C (APC), and sivelestat markedly inhibited these effects. Furthermore, targeting NETs protected mice from tMCAO-induced cerebral ischemia, possibly by regulating vWF and PAI-1. In summary, NETs may contribute to t-PA resistance in AIS through activation of PLTs and ECs. Strategies against NETs may present a promising therapeutic approach to improve the thrombolysis efficiency of t-PA in AIS patients.

中文翻译：

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中性粒细胞胞外陷阱导致急性缺血性卒中组织纤溶酶原激活剂抵抗

尽管血栓中的 NET 可能导致组织纤溶酶原激活剂 (t-PA) 抗性，但对 t-PA 具有抗性的循环中性粒细胞胞外陷阱 (NET) 尚未得到完全研究。这项研究旨在阐明循环中的 NET 是否与 t-PA 耐药性及其潜在机制有关。在循环中性粒细胞、急性缺血性卒中 (AIS) 患者的缺血性脑组织和短暂性大脑中动脉闭塞 (tMCAO) 模型中检测到 NETs 水平。通过免疫荧光分析小鼠血液、血栓和缺血脑组织中的 NET 形成。使用流式细胞术和共聚焦显微镜评估暴露的磷脂酰丝氨酸 (PS)。使用纤维蛋白形成试验、凝血酶和纯化的凝血复合物评估促凝血活性 (PCA)。AIS 患者血浆 NETs 水平显着高于健康个体。溶栓后，未改善患者的NET标志物显着增加，而改善患者的变化不显着。重要的是，NETs 在血液和血栓中被血管性血友病因子 (vWF) 和纤溶酶原激活剂抑制剂-1 (PAI-1) 修饰，可以逆转纤溶作用。此外，NETs 激活血小板 (PLTs) 和内皮细胞 (ECs)，刺激促凝表型并促进 vWF 和 PAI-1 的释放。DNase I、活化蛋白 C (APC) 和西维来司他显着抑制了这些作用。此外，靶向 NETs 保护小鼠免受 tMCAO 诱导的脑缺血，可能是通过调节 vWF 和 PAI-1。总之，NETs 可能通过激活 PLTs 和 ECs 导致 AIS 中的 t-PA 抗性。针对 NET 的策略可能提供一种有前景的治疗方法，以提高 AIS 患者 t-PA 的溶栓效率。