Objective—: Atrial fibrillation (AF) patients experience adverse cardiovascular events (ACEs) despite anticoagulant therapy. We reported that rs2431697 of miR-146a, a negative regulator of inflammation, predicts ACEs in patients with AF. The relationship between neutrophil extracellular traps and thrombogenesis is known. Thus, our aim was to evaluate the role of neutrophil extracellular trap compounds as prognostic markers of ACEs in AF and to study whether miR-146a affects NETosis. Approach and Results—: We included 336 steadily anticoagulated AF patients with a median follow-up of 7.9 years (interquartile range, 7.3–8.1) and 127 healthy subjects. The reviewed ACEs included stroke (ischemic/embolic), acute coronary syndrome, acute heart failure, and global or vascular death. We quantified cell-free DNA and NE (neutrophil elastase) at diagnosis. Rs2431697 was genotyped. Neutrophils from human and mice were seeded to analyze shed cell-free DNA and H3cit (citrullinated histone 3) after activation. In human plasmas, higher NE levels (>55.29 ng/mL), but not cell-free DNA, were independently associated with higher risk of all-cause mortality (hazard ratio, 2.24; 95% CI, 1.36–3.68), cardiovascular mortality (hazard ratio, 4.77; 95% CI, 1.11–20.47), and composite cardiovascular events (hazard ratio, 1.84; 95% CI, 1.01–3.76). In patients, NE levels were associated with rs2431697 (TT: 51.82±2.73 versus CC: 40.01±3.05 ng/mL; P =0.040). In vitro, both human (TT for rs2431697) and miR-146a ^−/− mice neutrophils yielded higher levels of cell-free DNA and H3cit than CC or wild-type cells, respectively. Conclusions—: NE activity can provide new ACE prognostic information in AF patients. These findings provide evidence of a potential role of miR-146a in neutrophil extracellular trap generation and cardiovascular risk in AF.

No biological predictors for the increased risk of thrombosis in patients with immune thrombocytopenia (ITP) have been identified. The aim of the study was to investigate platelet and neutrophil activation as well neutrophil extracellular trap (NET) formation in 63 ITP patients and 30 healthy volunteers. Platelet and neutrophil activation was assessed during steady state using flow cytometry analysis, and NETs were evaluated by quantitation of cell free DNA (cfDNA), and citrullinated histone-3-DNA (CitH3-DNA). Patient platelets and neutrophils showed increased CD62 and CD11b expression compared to controls (p = .038, and p = .022, respectively). In patients, platelet activation inversely correlated with platelet count and platelet size (p < .001), and positively correlated with neutrophil degranulation (p = .024). More NET formation, both CitH3-DNA (p = .025) and cfDNA(p < .001), were present in ITP patients compared to controls. CitH3-DNA inversely correlated with CD62 expression on platelets (p = .042), but higher levels of cfDNA were observed in individuals with classical cardiovascular risk factors for thrombosis, and in those with a previous history of thrombotic events. In this disease, the increased platelet activation and plasma NET levels seem to be separable processes that associate (either positively or inversely in the case of CitH3-DNA or platelet degranulation, respectively) to platelet mass.

Neutrophil extracellular traps (NETs) are formed after neutrophils expelled their chromatin content in order to primarily capture and eliminate pathogens. However, given their characteristics due in part to DNA and different granular proteins, NETs may induce a procoagulant response linking inflammation and thrombosis. Unraveling NET formation molecular mechanisms as well as the intracellular elements that regulate them is relevant not only for basic knowledge but also to design diagnostic and therapeutic tools that may prevent their deleterious effects observed in several inflammatory pathologies (e.g., cardiovascular and autoimmune diseases, cancer). Among the potential elements involved in NET formation, several studies have investigated the role of microRNAs (miRNAs) as important regulators of this process. miRNAs are small non-coding RNAs that have been involved in the control of almost all physiological processes in animals and plants and that are associated with the development of several pathologies. In this review, we give an overview of the actual knowledge on NETs and their implication in pathology with a special focus in cardiovascular diseases. We also give a brief overview on miRNA biology to later focus on the different miRNAs implicated in NET formation and the perspectives opened by the presented data.

Neutrophil extracellular traps (NETs) represent an important link between inflammation and thrombosis. Here, the present study aimed to investigate the influence of the NET components, DNA and histone H4, on hemostatic gene expression. A further aim was to confirm the influence of H4 on the expression of tissue factor (TF) and investigate a potential effect of DNA, and to test the involvement of miR‑17/92 and its paralog miR‑106b‑25 in this regulation. In HepG2 cells, the mRNA levels of factor VII and factor XII, which are crucial in the activation of the coagulation cascade, and of serpin family F member 2 (encoding α2‑antiplasmin) were significantly upregulated by DNA and H4; while the mRNA levels of factor V, which is essential for thrombin generation of protein S, a cofactor of protein C that also has the ability to inhibit the factor X activation pathway, and of serpin family C member 1 (encoding antithrombin, the main endogenous anticoagulant) were significantly upregulated only by H4. H4 and DNA also provoked an increase in hepatocyte nuclear factor 4α (HNF4A) mRNA expression that could be responsible for the increase in the expression of certain coagulant factors. In THP‑1 cells, it was also demonstrated that H4 caused an increase in TF mRNA while decreasing several of the microRNAs (miRNA/miRs) of the cluster miR‑17/92, which may in part explain the increase in the expression of TF. The present results suggest the ability of NET components to alter the hemostatic balance and a possible involvement of HNF4α and miRNAs in this regulation.

Although a growing number of studies suggest that microRNAs (miRNAs) play a relevant role in platelet biology, their implications in bleeding diatheses are starting to be investigated. Indeed, several studies have shown that alterations in the intracellular levels of highly expressed platelet miRNAs provoke a thrombotic phenotype. On the other hand, primary immune thrombocytopenia (ITP), which is considered the hallmark of acquired bleeding disorders, has been recently associated with altered levels of miRNAs in peripheral blood mononuclear cells, plasma, and platelets. In this review, we will focus on miRNAs that may affect the hemostatic and thrombotic functions of platelets, and we will discuss the different studies that have attempted to associate miRNAs with regulatory mechanisms of ITP.