Genetic and Pharmacological Modifications of Thrombin Formation in Apolipoprotein E-deficient Mice Determine Atherosclerosis Severity and Atherothrombosis Onset in a Neutrophil-Dependent Manner

Background: Variations in the blood coagulation activity, determined genetically or by medication, may alter atherosclerotic plaque progression, by influencing pleiotropic effects of coagulation proteases. Published experimental studies have yielded contradictory findings on the role of hypercoagulability in atherogenesis. We therefore sought to address this matter by extensively investigating the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, and plaque phenotype determination. Methodology/principal findings: We generated transgenic atherosclerosis-prone mice with diminished coagulant or hypercoagulable phenotype and employed two distinct models of atherosclerosis. Gene-targeted 50% reduction in prothrombin $(FII^{−/WT}:ApoE^{−/−})$ was remarkably effective in limiting disease compared to control $ApoE^{−/−}$ mice, associated with significant qualitative benefits, including diminished leukocyte infiltration, altered collagen and vascular smooth muscle cell content. Genetically-imposed hypercoagulability in $TM^{Pro/Pro}:ApoE^{−/−}$ mice resulted in severe atherosclerosis, plaque vulnerability and spontaneous atherothrombosis. Hypercoagulability was associated with a pronounced neutrophilia, neutrophil hyper-reactivity, markedly increased oxidative stress, neutrophil intraplaque infiltration and apoptosis. Administration of either the synthetic specific thrombin inhibitor Dabigatran etexilate, or recombinant activated protein C (APC), counteracted the pro-inflammatory and pro-atherogenic phenotype of pro-thrombotic $TM^{Pro/Pro}:ApoE^{−/−}$ mice. Conclusions/significance: We provide new evidence highlighting the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. Our findings reveal that thrombin-mediated proteolysis is an unexpectedly powerful determinant of atherosclerosis in multiple distinct settings. These studies suggest that selective anticoagulants employed to prevent thrombotic events may also be remarkably effective in clinically impeding the onset and progression of cardiovascular disease

New Insights into Modulation of Thrombin Formation

Thrombin is a pleiotropic enzyme that regulates hemostasis and nonhemostatic functions, including an array of actions within and on the vasculature. Physiologically, thrombin generation serves mainly to protect against thrombosis, but also to maintain vascular endothelial integrity. This protective effect is mediated in part through generation of anticoagulant enzymes, including activated protein C, formed on the action of thrombin on the endothelial receptor thrombomodulin. Partly, thrombin's vascular effects are effectuated through interaction with protease-activated receptors on various cell types. Pathophysiologically, downregulation and shedding of anticoagulant-acting receptors such as thrombomodulin and endothelial protein C receptor may contribute to a shift in activities of thrombin towards thrombogenic and proinflammatory actions. This shift may typically occur in the process of atherosclerosis, leading to a proatherogenic direction of the effects of thrombin. Therapeutically, the long-terminhibition of thrombin may create new ways of reducing atherosclerosis burden, altering the plaque phenotype

Accelerated In Vivo Thrombin Formation Independently Predicts the Presence and Severity of CT Angiographic Coronary Atherosclerosis

ObjectivesThis study sought to investigate the association between thrombin generation in plasma and the presence and severity of computed tomography angiographically defined coronary atherosclerosis in patients with suspected coronary artery disease (CAD).BackgroundBesides its pivotal role in thrombus formation, experimental data indicate that thrombin can induce an array of pro-atherogenic and plaque-destabilizing effects. Although thrombin plays a role in the pathophysiology of atherosclerosis progression and vascular calcification, the clinical evidence remains limited.MethodsUsing 64-slice coronary computed tomographic angiography, we assessed the presence and characteristics of CAD in patients (n = 295; median age 58 years) with stable chest pain. Coronary artery calcification was graded as absent (Agatston score 0), mild (Agatston score 1 to 100), moderate (Agatston score 101 to 400), and severe (Agatston score >400). Calibrated automated thrombography was used to assess endogenous thrombin potential in plasma in vitro. Thrombin-antithrombin complex (TATc) levels were measured as a marker for thrombin formation in vivo.ResultsTATc plasma levels were substantially higher in patients with CAD versus patients without CAD (p = 0.004). Significant positive correlations were observed between steadily increasing TATc levels and the severity of CAD (r = 0.225, p < 0.001). In multinomial logistic regression models, after adjusting for established risk factors, TATc levels predicted the degree of coronary artery calcification: mild (odds ratio: 1.56, p = 0.006), moderate (odds ratio: 1.56, p = 0.007), and severe (odds ratio: 1.67, p = 0.002). Trends were comparable between the groups when stratified according to the degree of coronary luminal stenosis.ConclusionsThis study provides novel clinical evidence indicating a positive independent association between enhanced in vivo thrombin generation and the presence and severity of coronary atherosclerosis, which may suggest that thrombin plays a role in the pathophysiology of vascular calcification and atherosclerosis progression

VWF-mediated leukocyte recruitment with chromatin decondensation by PAD4 increases myocardial ischemia/reperfusion injury in mice

Innate immune cells play a major role in the early response to myocardial ischemia/reperfusion (MI/R) injury. Recombinant human ADAMTS13 (rhADAMTS13), cleaving von Willebrand factor (VWF), reduces leukocyte recruitment in mice. Death of cardiomyocytes and the possible formation of neutrophil extracellular traps (NETs) may result in chromatin release that is prothrombotic and cytotoxic. We investigated the pathophysiological role of extracellular chromatin during MI/R to evaluate the therapeutic potential of targeting extracellular DNA and VWF by using DNase I with/without rhADAMTS13. Finally, we examined the impact of histone citrullination and NETosis by peptidylarginine deiminase 4 (PAD4) on MI/R. We used a 24-hour MI/R mouse surgical model. MI/R injury caused an increase in plasma nucleosomes, abundant neutrophil infiltration, and the presence of citrullinated histone H3 at the site of injury. Both monotherapies and coadministration of DNase I and rhADAMTS13 revealed a cardioprotective effect, resulting in subsequent improvement of cardiac contractile function. PAD4(−/−) mice, which do not produce NETs, were also significantly protected from MI/R and DNase I treatment had no further beneficial effect. We demonstrate that extracellular chromatin released through NETosis exacerbates MI/R injury. Targeting both VWF-mediated leukocyte recruitment and chromatin removal may be a new therapeutic strategy to reduce ischemia-related cardiac damage

Elevated Levels of Circulating DNA and Chromatin Are Independently Associated With Severe Coronary Atherosclerosis and a Prothrombotic State

OBJECTIVE: Aberrant neutrophil activation occurs during the advanced stages of atherosclerosis. Once primed, neutrophils can undergo apoptosis or release neutrophil extracellular traps (NETs). This extracellular DNA exerts potent pro-inflammatory, prothrombotic and cytotoxic properties. The goal of this study was to examine the relationships between 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 (CAD), we examined the severity, extent, and phenotype of coronary atherosclerosis by using coronary computed tomographic angiography (CCTA). Double-stranded DNA, nucleosomes, citrullinated histone H4 and myeloperoxidase (MPO)-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 MPO-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 (OR: 2.14, 95% CI 1.26-3.63; p=0.005). Markers of NETs, such as MPO-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 NET formation are independently associated with CAD, prothrombotic state and occurrence of adverse cardiac events. These biomarkers could potentially aid in the prediction of cardiovascular risk in patients with chest discomfort