Pathogenetic role of neutrophil extracellular traps in coronary flow impairment following percutaneous coronary intervention in acute myocardial infarction patients -A multicenter study-
Authors: Takeuchi S , Nishimiya K , Wada K , Adachi A , Oyama K , Shindo T , Godo S , Hao K , Shiroto T , Namiuchi S , Takahashi J , Yasuda S 
Published: November 2023

Abstract

Background
A suicidal function of neutrophils to ensnare and kill microbes by expelling neutrophil extracellular traps (NETs) via histone citrullination and chromatin decondensation has emerged as it enhances local inflammation and thrombogenicity at the culprit lesion of acute myocardial infarction (AMI).

Purpose
This multicenter study was aimed to examine 1) the histopathological feature of coronary thrombus with the NETs formation in AMI patients, and 2) the association of NETs with impaired coronary flow after percutaneous coronary intervention (PCI) for AMI patients.

Methods
Coronary thrombi were obtained from 44 patients with AMI [male; 77%, age; 69±12(SD) y-o)]who underwent PCI within 24 hours from the onset. Aspirated thrombi were immediately bisected and frozen, and 5-µm thickness unstained sections were processed for polarized light microscopy (PLM) imaging and immunofluorescence staining. NETs were immunohistochemically defined when objects were co-stained with anti-myeloperoxidase and anti-citrullinated histone H3 antibodies (Figure A,B). Adjacent sections were stained with Masson’s trichrome to measure the whole thrombus area. The ImageJ1 software was utilized for the automatic segmentation on the digitized histology images. Segmented NETs area was divided by the thrombus area as a measure of NETs. Clinical characteristics, including AMI subset, post PCI final TIMI flow grade and TIMI frame count, were collected.

Results
All aspirated thrombi exhibited NETs (Figure A), and then subjects were split into 2 groups with a median value of NETs (62.8 x10-4 um2/um2); high-NETs and low-NETs groups. Patient characteristics were all comparable between the 2 groups. PLM results showed that birefringent crystals, suggestive of cholesterol crystals, were more frequently noted in the thrombi with high-NETs compared with those with low-NETs (50.0% vs. 13.6%, P=0.010). In the immunofluorescence, NETs were seen in the proximity of the crystals (Figure B). The multivariable analysis with covariates of age, sex, major coronary risk factors, MI related to left anterior descending artery and the onset to aspiration time revealed that the presence of birefringent crystals remained an independent predictor for high-NETs formation [OR=5.78, 95%CI=1.13-29.66, P=0.035]. In the high-NETs group, the prevalence of the final TIMI flow grade 3 was significantly lower than the low-NETs group (63.6% vs. 90.9 %, P=0.031). Likewise, there was a significant positive correlation between NETs area and TIMI frame count, defined as the number of cineframes required for contrast to reach a standardized distal coronary landmark (R=0.681, P&lt;0.001).

Conclusion
The present study demonstrated that NETs were highly expressed in coronary thrombus aspirated from AMI patients in association with crystalline components, relating to coronary flow impairment following PCI in AMI patients.