Elevated levels of circulating DNA and chromatin are independently associated with severe coronary atherosclerosis and a prothrombotic state

Abstract

Objective: Aberrant neutrophil activation occurs during the advanced stages of atherosclerosis. Once primed, neutrophils can undergo apoptosis or release neutrophil extracellular traps. This extracellular DNA exerts potent proinflammatory, prothrombotic, and cytotoxic properties. The goal of this study was to examine the relationships among extracellular DNA formation, coronary atherosclerosis, and the presence of a prothrombotic state.

Approach and results: In a prospective, observational, cross-sectional cohort of 282 individuals with suspected coronary artery disease, we examined the severity, extent, and phenotype of coronary atherosclerosis using coronary computed tomographic angiography. Double-stranded DNA, nucleosomes, citrullinated histone H4, and myeloperoxidase-DNA complexes, considered in vivo markers of cell death and NETosis, respectively, were established. We further measured various plasma markers of coagulation activation and inflammation. Plasma double-stranded DNA, nucleosomes, and myeloperoxidase-DNA complexes were positively associated with thrombin generation and significantly elevated in patients with severe coronary atherosclerosis or extremely calcified coronary arteries. Multinomial regression analysis, adjusted for confounding factors, identified high plasma nucleosome levels as an independent risk factor of severe coronary stenosis (odds ratio, 2.14; 95% confidence interval, 1.26-3.63; P=0.005). Markers of neutrophil extracellular traps, such as myeloperoxidase-DNA complexes, predicted the number of atherosclerotic coronary vessels and the occurrence of major adverse cardiac events.

Conclusions: Our report provides evidence demonstrating that markers of cell death and neutrophil extracellular trap formation are independently associated with coronary artery disease, prothrombotic state, and occurrence of adverse cardiac events. These biomarkers could potentially aid in the prediction of cardiovascular risk in patients with chest discomfort.

Keywords: DNA; atherosclerosis; chromatin; coagulation, blood; neutrophils; nucleosomes; thrombin; thrombophilia.

Figure 1. Circulating DNA, nucleosome fragments and markers of NETs according to the presence and severity of coronary artery disease

Patients were divided in categories, based on the severity of CAD, as assessed with CCTA. Patients who did not undergo CCTA because of a high calcium score were considered as a separate category (“Extremely Calcified”). A total of four markers were measured in all patients (panel A-D). Shaded area demonstrates the range of the measured markers in plasma from healthy controls (n=10 NPP (from n=85 healthy volunteers) is indicated by a horizontal dotted line. CAD = Coronary Artery Disease; CCTA = Coronary Computed Tomographic Angiography; dsDNA = Double Stranded DNA; NPP = Normal Pooled Plasma * = p<0.05; ** = p<0.01; *** = p<0.001.

Figure 2. Plasma levels of markers of leukocyte, platelet, endothelial and coagulation activation according to the presence and severity of coronary artery disease

Patients were divided into categories based on the severity of CAD as assessed with CCTA. Patients who did not undergo CCTA because of a high calcium score were considered as a separate category (“Extremely Calcified”). A total of five markers were measured in all patients (panel A-E). Shaded area demonstrates the range of the measured markers in plasma from healthy controls (n=10). NPP (from n=85 healthy volunteers) is indicated by a horizontal dotted line. CAD = Coronary Artery Disease; CCTA = Coronary Computed Tomographic Angiography; dsDNA = Double Stranded DNA; NPP = Normal Pooled Plasma; MPO = myeloperoxidase; VWF = Von Willebrand Factor; TAT = Thrombin-Antithrombin; PMN = Polymorphonuclear; α1-PI = alpha 1-proteinase inhibitor; PF4 = Platelet Factor 4 * = p<0.05; ** = p<0.01; *** = p<0.001.

Figure 3. Relationship between extracellular DNA generation, NETosis markers and a prothrombotic state

Panels A and B show the relationship between levels of dsDNA, citrullinated histone H4 and mean TAT levels (panel A) or mean VWF levels (panel B), respectively. Panels C and D show the relationship between levels of MPO-DNA, citrullinated histone H4 and mean TAT levels (panel C) or mean VWF levels (panel D). Elevated levels of both extracellular DNA generation (dsDNA) and NETosis markers (MPO-DNA complexes; citrullinated histone H4) are associated with the presence of a prothrombotic state, defined by increased TAT and VWF levels. TAT = Thrombin-Antithrombin; dsDNA = Double Stranded DNA; VWF = Von Willebrand Factor; NPP = Normal Pooled Plasma; T = Tertile.