Are NETs a Novel, Exciting, Thrombosis Risk Marker?

Neutrophil Extracellular Traps: Potential Prothrombotic State Markers and Therapeutic Targets for Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is a potentially life-threatening condition that can contribute to stroke and thromboembolism. While anticoagulation has reduced the risk of thromboembolism in patients with AF, morbidity and mortality remain unacceptably high, hence the recent move in guidelines toward a more holistic or integrated care approach to improve clinical outcomes.[1] [2]

Understanding the underlying prothrombotic mechanisms in AF patients may lead to better risk stratification and identify novel therapeutic interventions.

In this issue of the journal, Liu and colleagues[3] report the results of a study that combined clinical investigation and animal models and demonstrated that neutrophil extracellular traps (NETs) could serve as a potential prothrombotic marker and therapeutic target in AF.

NETs were first reported in 1996[4] and further detailed by Brinkmann et al who termed the process NETosis.[5] [6] When neutrophils are activated, they can expel nuclear chromatin to form a web-like amalgam that traps and kills bacteria as a first line of defense against infection. In addition, extruded components that include DNA, histones, neutrophil elastase (NE) and myeloperoxidase (MPO) promote inflammation and facilitate thrombosis.[7] This triangulation of innate immune activation, inflammation, and coagulation is increasingly recognized in many human diseases and referred to as immunothrombosis.[8] [9]

Liu et al[3] detected NETs in blood samples from patients with AF and their finding that NETs levels in samples from the left atrial appendage (LAA) were higher than those from the left atrial and peripheral venous samples indicate that NETs are formed in the LAA. They used a rapid atrial pacing (RAP) rat model and demonstrated that RAP increases local NETs formation, which can be reduced by protein-arginine deiminase type 4 (PAD4) inhibition that can block NETosis.[10] These observations suggest a close relationship between NETs formation and AF.

How does RAP induce NETosis? It is now known that NETs also form in response to inflammatory cytokines, histones, and activated platelets.[11] [12] Liu et al performed mRNA sequencing and found that RAP up-regulated genes that are mainly involved in tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-17 signaling pathways and in focal adhesion. Indeed, pro-inflammatory cytokines including IL-6 and TNF-α in the LAA of RAP rats were significantly increased, suggesting that RAP might stimulate NETs formation by promoting the expression of cytokine and adhesion genes. However, this cannot rule out the possibility that RAP might damage atrial endothelium and blood cells to release histones, which are known to stimulate pro-inflammatory cytokine release and initiate the pathological process. Circulating histones have also been shown to cause calcium influx in cardiomyocytes, release cardiac troponin, and induce arrythmia.[13] Histone infusion in mice models leads to AF[14] and their role in patients with AF require further investigation.

Liu and colleagues provided a few lines of evidence to suggest that NETs formation is responsible for thrombosis induced by AF. They first demonstrate that NETs levels were increased significantly in AF patients and positively correlated with spontaneous echo contrast grades; a clinical examination of atrial thrombosis. In addition, they demonstrated that NETs level could significantly enhance the predictivity of CHA₂DS₂-VASc scores for AF thrombogenicity. In a rat model, they demonstrated that RAP could increase the size and incidence of thrombosis in partially ligated LAA, which can be significantly blocked by PAD4 inhibition. This strongly indicates a close relationship between NETs and thrombosis in AF.

NETs promote thrombosis in a number of ways. First, there is the biophysical aspect of forming a scaffold on to which platelets and coagulation factors congregate. This juxtapositioning facilitates and catalyzes thrombin generation.[7] [15] [16] Second, extruded nuclear and cytoplasmic constituents can act as damage-associated molecular patterns (DAMPs). Many DAMPs are procoagulant. For example, extracellular histones activate coagulation by assembling alternative prothrombinase that does not require factor Va or phospholipid surfaces to enhance thrombin generation.[17] In addition, extracellular histones induce platelet aggregation and activation,[18] damage endothelial cells,[19] and reduce thrombomodulin-activated protein C anticoagulant activity.[20] [21] Cell-free DNA is also pro-coagulant by generating thrombin through contact activation of factors XI and XII.[22] Furthermore, the presence of DNAs and histones inside clots increases their resistance to fibrinolysis[23] and stabilizes the clot integrity, which may facilitate the development of long-lasting thrombosis in AF.

Coagulation and inflammation are an intrinsically intertwined processes, which can be self-sustaining and become maladaptive with potential pathogenic consequences.[20] Extracellular histones can enhance inflammation by activating NLRP3 and stimulation of proinflammatory cytokine release, including IL-6, IL-8, and TNF-α.[24] [25] Histones released locally may also activate or damage atrial endothelium, stimulate von Willebrand factor release,[26] and induce more NETs formation to form a vicious cycle,[19] which might enable NETs and thrombosis to anchor to the atrial wall and grow in size to form the pathological basis for stroke and thromboembolism in AF ([Fig. 1]).

Fig. 1 The potential pathogenic mechanism of neutrophil extracellular traps in atrial fibrillation.

Targeting NETosis or degrading existing NETs (or associated mechanistic pathways) has been extensively investigated in many disease models associated with NETs and thrombosis.[27] [28] [29] [30] NETosis starts from the activation of NADPH oxidase (NOX) complex via protein kinase C (PKC)-Raf/MERK/ERK by external stimuli, followed by activation of MPO, NE, and PAD4. PAD4 is an enzyme that catalyzes citrullination of histones and promotes chromatin de-condensation with reactive oxygen species (ROS) to induce gradual separation and loss of the nuclear membrane.[31] [32] NETs can be cleared by enzyme cleavage, such as DNaseI to cleave DNAs, and phagocytosis.[33] [34] Therefore, the development of therapeutic tools to target NETosis and NETs is mainly based on known pathways, as mentioned above. Current therapeutic strategies mainly include inhibiting DNA decondensation by PAD4 inhibitors, reducing ROS by inhibiting NOX and using ROS scavengers, and degrading NETs using DNase I.[27] Others, such as toll-like receptor inhibitors and calcineurin inhibitors, are also candidates for further trials. However, no drug for targeting NETosis and NETs has been approved for clinical use. Some might become available in the near future and could be considered in AF if there is mounting evidence of a NETs-dependent immunothrombotic pathogenesis.

The results of the study by Liu et al must be interpreted with caution because it is not clear whether NETs initiate thrombosis or are just involved in the thrombotic event. Further evidence is needed, such as whether NETs exist within clots in partially ligated LAA and how clots or NETs are anchored to the wall of the LAA. Development of thrombosis is a complicated process and many different factors are involved, especially in AF whereby detailed molecular mechanisms are still far from clear. Patients with AF are heterogeneous and some may have underlying pro-inflammatory states that enable NETs to play a more significant role. In the pro-inflammatory state of sepsis where involvement of NETs has been established, AF is a common complication that increases risks of adverse outcomes.[13] [35] These patients tend to have a higher risk of bleeding and might be a group that could potentially benefit from anti-NETs treatment rather than anticoagulant management. Short episodes of AF in older people are relatively common and may link to a transient inflammatory stimulus but large-scale clinical studies would need to be performed to examine the role of NETs in this and other settings.[36] For such studies, there is also a need for standardized assays of NETs, which are simple, rapid, and robust in performance.[37] This remains a translational development that requires timely investment from the diagnostics industry. With some way to go before we fully comprehend the role of NETs as a marker for both risk stratification and therapeutic targeting, this research article by Liu et al has laid important new foundations in the field of AF.

Publication History

Received: 05 October 2023

Accepted: 05 October 2023

Accepted Manuscript online:
06 October 2023

Article published online:
02 November 2023

• References

• 1 Chao TF, Joung B, Takahashi Y. et al. 2021 Focused Update Consensus Guidelines of the Asia Pacific Heart Rhythm Society on Stroke Prevention in Atrial Fibrillation: executive summary. Thromb Haemost 2022; 122 (01) 20-47
• 2 Romiti GF, Pastori D, Rivera-Caravaca JM. et al. Adherence to the ‘Atrial Fibrillation Better Care’ pathway in patients with atrial fibrillation: impact on clinical outcomes-a systematic review and meta-analysis of 285,000 patients. Thromb Haemost 2022; 122 (03) 406-414
• 3 Liu X, Li X, Xiong S. et al. Neutrophil extracellular traps: potential prothrombotic state markers and therapeutic targets for atrial fibrillation. Thromb Haemost 2024; 124 (05) 441-454
• 4 Takei H, Araki A, Watanabe H, Ichinose A, Sendo F. Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis. J Leukoc Biol 1996; 59 (02) 229-240
• 5 Brinkmann V, Reichard U, Goosmann C. et al. Neutrophil extracellular traps kill bacteria. Science 2004; 303 (5663) 1532-1535
• 6 Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity the second function of chromatin?. J Cell Biol 2012; 198 (05) 773-783
• 7 Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014; 123 (18) 2768-2776
• 8 Shaw RJ, Abrams ST, Austin J. et al. Circulating histones play a central role in COVID-19-associated coagulopathy and mortality. Haematologica 2021; 106 (09) 2493-2498
• 9 Shaw RJ, Bradbury C, Abrams ST, Wang G, Toh CH. COVID-19 and immunothrombosis: emerging understanding and clinical management. Br J Haematol 2021; 194 (03) 518-529
• 10 Rohrbach AS, Slade DJ, Thompson PR, Mowen KA. Activation of PAD4 in NET formation. Front Immunol 2012; 3: 360
• 11 Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 2018; 18 (02) 134-147
• 12 Kenny EF, Herzig A, Krüger R. et al. Diverse stimuli engage different neutrophil extracellular trap pathways. eLife 2017; 6: 6
• 13 Alhamdi Y, Abrams ST, Cheng Z. et al. Circulating histones are major mediators of cardiac injury in patients with sepsis. Crit Care Med 2015; 43 (10) 2094-2103
• 14 Alhamdi Y, Zi M, Abrams ST. et al. Circulating histone concentrations differentially affect the predominance of left or right ventricular dysfunction in critical illness. Crit Care Med 2016; 44 (05) e278-e288
• 15 Fuchs TA, Brill A, Duerschmied D. et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 2010; 107 (36) 15880-15885
• 16 Laridan E, Martinod K, De Meyer SF. Neutrophil extracellular traps in arterial and venous thrombosis. Semin Thromb Hemost 2019; 45 (01) 86-93
• 17 Abrams ST, Su D, Sahraoui Y. et al. Assembly of alternative prothrombinase by extracellular histones initiates and disseminates intravascular coagulation. Blood 2021; 137 (01) 103-114
• 18 Alhamdi Y, Abrams ST, Lane S, Wang G, Toh CH. Histone-associated thrombocytopenia in patients who are critically ill. JAMA 2016; 315 (08) 817-819
• 19 Abrams ST, Zhang N, Manson J. et al. Circulating histones are mediators of trauma-associated lung injury. Am J Respir Crit Care Med 2013; 187 (02) 160-169
• 20 Yong J, Abrams ST, Wang G, Toh CH. Cell-free histones and the cell-based model of coagulation. J Thromb Haemost 2023; 21 (07) 1724-1736
• 21 Ammollo CT, Semeraro F, Xu J, Esmon NL, Esmon CT. Extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein C activation. J Thromb Haemost 2011; 9 (09) 1795-1803
• 22 Swystun LL, Mukherjee S, Liaw PC. Breast cancer chemotherapy induces the release of cell-free DNA, a novel procoagulant stimulus. J Thromb Haemost 2011; 9 (11) 2313-2321
• 23 Varjú I, Longstaff C, Szabó L. et al. DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment. Thromb Haemost 2015; 113 (06) 1289-1298
• 24 Huang H, Chen HW, Evankovich J. et al. Histones activate the NLRP3 inflammasome in Kupffer cells during sterile inflammatory liver injury. J Immunol 2013; 191 (05) 2665-2679
• 25 Beltrán-García J, Osca-Verdegal R, Pérez-Cremades D. et al. Extracellular histones activate endothelial NLRP3 inflammasome and are associated with a severe sepsis phenotype. J Inflamm Res 2022; 15: 4217-4238
• 26 Lam FW, Cruz MA, Parikh K, Rumbaut RE. Histones stimulate von Willebrand factor release in vitro and in vivo. Haematologica 2016; 101 (07) e277-e279
• 27 Mutua V, Gershwin LJ. A review of neutrophil extracellular traps (NETs) in disease: potential anti-NETs therapeutics. Clin Rev Allergy Immunol 2021; 61 (02) 194-211
• 28 Blasco A, Coronado MJ, Vela P. et al. Prognostic implications of neutrophil extracellular traps in coronary thrombi of patients with ST-elevation myocardial infarction. Thromb Haemost 2022; 122 (08) 1415-1428
• 29 Bianchini EP, Razanakolona M, Helms J. et al. The proteolytic inactivation of protein Z-dependent protease inhibitor by neutrophil elastase might promote the procoagulant activity of neutrophil extracellular traps in sepsis. Thromb Haemost 2022; 122 (04) 506-516
• 30 Chen JW, Hsu CC, Su CC. et al. Transient bacteremia promotes catheter-related central venous thrombosis through neutrophil extracellular traps. Thromb Haemost 2022; 122 (07) 1198-1208
• 31 Wang Y, Li M, Stadler S. et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 2009; 184 (02) 205-213
• 32 Parker H, Winterbourn CC. Reactive oxidants and myeloperoxidase and their involvement in neutrophil extracellular traps. Front Immunol 2013; 3: 424
• 33 Farrera C, Fadeel B. Macrophage clearance of neutrophil extracellular traps is a silent process. J Immunol 2013; 191 (05) 2647-2656
• 34 Englert H, Göbel J, Khong D. et al. Targeting NETs using dual-active DNase1 variants. Front Immunol 2023; 14: 1181761
• 35 Kuipers S, Klein Klouwenberg PM, Cremer OL. Incidence, risk factors and outcomes of new-onset atrial fibrillation in patients with sepsis: a systematic review. Crit Care 2014; 18 (06) 688
• 36 Wallenhorst C, Martinez C, Freedman B. Risk of ischemic stroke in asymptomatic atrial fibrillation incidentally detected in primary care compared with other clinical presentations. Thromb Haemost 2022; 122 (02) 277-285
• 37 Abrams ST, Morton B, Alhamdi Y. et al. A novel assay for neutrophil extracellular trap formation independently predicts disseminated intravascular coagulation and mortality in critically ill patients. Am J Respir Crit Care Med 2019; 200 (07) 869-880