Neutrophil Extracellular Traps Enhance the Hypercoagulation State in t-PA Thrombolysis in Acute Ischemic Stroke

Background: Neutrophil extracellular traps (NETs) have been studied as crucial components of thrombi in stroke patients. Little is known about the dramatic changes of circulating NETs in the process of thrombolysis treatment. Our study aimed to investigate the potential role of NETs and their interaction with endothelial dysfunction in the process of thrombolysis treatment. Methods: NET markers were detected by ELISA in 13 healthy subjects and 25 stroke patients who received thrombolysis treatment. NETting neutrophils and neutrophil-platelet (PLT) aggregates were detected in blood samples from healthy subjects and stroke patients analyzed by confocal microscopy and flow cytometry. Endothelial cells (ECs) were treated with NETs, and the expression of vascular endothelial (VE)-cadherin, zonula occludens (ZO)-1, occludin, Claudin-5, tissue factor (TF), phosphatidylserine (PS) and plasminogen activator inhibitor-1 (PAI-1) was detected. Findings: The plasma levels of NETs showed no significant differences before and after thrombolysis treatment, but neutrophils from patients showed a stronger ability to generate NETs after treatment. TLR9-PF4 signaling was upregulated in platelets from patients and induced NET formation. NETs enhanced PCA though tissue factor and phosphatidylserine and   by activating platelets. NETs also induced the disruption of the endothelial barrier and drove ECs toward a procoagulant phenotype. Interpretation: NET formation mediated by activated platelets enhances thrombogenicity, which could result in tissue plasminogen activator (t-PA) resistance.  Funding Information: This work was supported by grants from the National Natural Science Foundation of China (grant nos. 61575058, 81903144, 81970415). Declaration of Interests: Nothing to report. Ethics Approval Statement: This study was approved by the Ethics Committee of Harbin Medical University and was conducted following the specifications of the Declaration of Helsinki. Blood from healthy subjects and patients was obtained with informed consent.