Markers of neutrophil extracellular traps are associated with adverse clinical outcome in stable coronary artery disease

Abstract

Background Neutrophil extracellular traps, comprising chromatin and granule proteins, have been implicated in atherothrombosis. Design and methods We investigated whether the circulating neutrophil extracellular traps markers, double-stranded DNA and myeloperoxidase-DNA were associated with clinical outcome and hypercoagulability in patients with stable coronary artery disease. Patients with angiographically verified stable coronary artery disease ( n = 1001) were included. Follow-up was 2 years, recording 106 clinical endpoints (unstable angina, non-haemorrhagic stroke, myocardial infarction or death). Serum collected at baseline was used to determine double-stranded DNA and myeloperoxidase-DNA levels. Results The neutrophil extracellular traps markers were weakly intercorrelated ( r = 0.103, P = 0.001). Patients with the highest quartile of double-stranded DNA had weakly but significantly elevated hypercoagulability markers (prothrombin fragment 1+2, D-dimer, free and total tissue factor pathway inhibitor ( P < 0.001 for all)). Men, smokers, patients with metabolic syndrome and patients with a previous myocardial infarction had significantly elevated double-stranded DNA levels ( P ≤ 0.002 for all). Significantly higher double-stranded DNA levels were observed in the group experiencing a clinical endpoint compared to the group without ( P = 0.019). When categorising double-stranded DNA into quartiles, a distinct cut-off between the lowest and upper three quartiles was observed. Adjusting for relevant covariates, patients in the upper three quartiles had an odds ratio of 2.01 (95% confidence interval 1.12, 3.58, P = 0.019) for experiencing a clinical endpoint. Myeloperoxidase-DNA was not significantly associated with clinical outcome or hypercoagulability. Conclusions Double-stranded DNA levels were significantly related to adverse clinical outcome after 2 years, but only weakly associated with hypercoagulability. These observations suggest that the detrimental effects of neutrophil extracellular traps in coronary artery disease might extend beyond those related to hypercoagulability.

Complement Activation in Association with Markers of Neutrophil Extracellular Traps and Acute Myocardial Infarction in Stable Coronary Artery Disease

“…To our knowledge, this is the largest study investigating complement activation in patients with stable CAD and the first to relate complement to circulating NET markers in such a population. We have recently shown the impact of NETs in this population [35], and due to the increasing evidence of reciprocal activation between complement activation and NETs [34], we hypothesized that they 3 Mediators of Inflammation would activate each other, thus, propagating their individual negative effects on the progression of the disease [8,30,37].…”

“…Markers of NETs, MPO-DNA and dsDNA, were analyzed as previously described [35]. In brief, MPO-DNA complexes were quantified in a serum using an enzyme-linked immunosorbent assay (ELISA) technique where anti-MPO monoclonal antibodies (AbD Serotec, Hercules, CA, USA) were coated to plates and incubated overnight at 4°C.…”

“…In addition, the methods used for measuring NETs pose a well-known challenge [40]. As previously discussed [35], dsDNA could in principle be released from any nucleated cell, and the ELISA for measuring MPO-DNA is in need of standardization. Nevertheless, MPO-DNA is at present considered the most specific circulating NET marker [40].…”

“…Serum dsDNA levels were quantified using a fluorescent nucleic acid stain, quant-iT PicoGreen (Invitrogen Ltd., Paisley, UK), and a Fluorskan Ascent fluorometer (Thermo Fisher Scientific Oy, Vantaa, Finland). The interassay CVs for the analyses were 10.5% for MPO-DNA and 7% for dsDNA[35].2.3. Clinical Endpoints.…”

“…Although experimental data have indicated a significant interplay between complement activation and NETs by being potent activators of each other, thus, propagating an inflammatory reaction [34], this has not yet been explored in clinical studies. We recently showed that markers of NETs were associated with adverse clinical outcome in patients with stable CAD [35]. Based on the hypothesis of a positive feedback loop between complement activation and NET release, we now aimed to investigate whether complement activation by levels of TCC and expression of C5aR1 was associated with levels of the NET markers dsDNA and myeloperoxidase-DNA (MPO-DNA) in patients with stable CAD.…”