Neutrophil extracellular traps in ischemic stroke thrombi

Objective: Neutrophil extracellular traps (NETs) have been shown to promote thrombus formation. Little is known about the exact composition of thrombi that cause ischemic stroke. In particular no information is yet available on the presence of NETs in cerebral occlusions. Such information is however essential to improve current thrombolytic therapy with t-PA. This study aimed at investigating the presence of neutrophils and more specifically NETs in ischemic stroke thrombi. Methods: Sixty-eight thrombi retrieved from ischemic stroke patients undergoing endovascular treatment were characterized by immunostaining using neutrophil markers (CD66b and neutrophil elastase) and NETs markers (citrullinated histones H3 (H3Cit) and extracellular DNA). Neutrophils and NETs were quantified. In addition, extracellular DNA was targeted by performing ex vivo lysis of retrieved thrombi with DNase 1 and t-PA. Results: Neutrophils were detected extensively throughout all thrombi. Citrullinated histones H3 (H3Cit), a hallmark of NETs, were observed in almost all thrombi. H3Cit-positive area varied up to 13.45% of total thrombus area. Co-localization of H3Cit with extracellular DNA released from neutrophils confirmed the specific presence of NETs. H3Cit was more abundant in thrombi from cardioembolic origin compared to other etiologies. Older thrombi contained significantly more neutrophils and H3Cit compared to fresh thrombi. Interestingly, ex vivo lysis of patient thrombi was more successful when adding DNase 1 to standard t-PA. Interpretation: Neutrophils and NETs form important constituents of cerebral thrombi. Targeting of NETs with DNase 1 might have prothrombolytic potential in treatment of acute ischemic stroke. This article is protected by copyright. All rights reserved.