Neutrophil Extracellular Traps: At the Interface of Thrombosis and Comorbidities

Abstract

Since their discovery in 2004, neutrophil extracellular traps (NETs) have been at the center of multidisciplinary attention. Although a key tool in neutrophil-mediated immunity, these filamentous, enzyme-enriched DNA–histone complexes can be detrimental to tissues and have been identified as an underlying factor in a range of pathological conditions. Building on more than 20 years of research into NETs, this review places thrombosis, the pathological formation of blood clots, in the spotlight. From this point of view, we discuss the structure and formation of NETs, as well as the interaction of their components with the hemostatic system, dissecting the pathways through which NETs exert their marked effect on formation and the dissolution of thrombi. We pay distinct attention to the latest developments in the research of a key player in NET formation, peptidyl-arginine-deiminase (PAD) enzymes: their types, sources, and potential cross-play with the hemostatic machinery. Besides these molecular details, we elaborate on the link between pathological thrombosis, NETs, and widespread conditions that represent a debilitating public health burden worldwide, such as sepsis and neoplasms. Finally, future implications on the treatment of thrombosis-related conditions will be discussed.

 

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Keywords

neutrophil extracellular traps (NETs) - fibrinolysis - thrombosis - cancer - sepsis

Webs Hiding in Plain Sight: An Introduction

Neutrophil extracellular traps (NETs) have been hiding in plain sight for over a 100 years since Metchnikoff and Ehrlich started examining neutrophils under the microscope.[1] [2] Fast forward 150 years, with Zichilinsky's discovery in 2004,[3] these filamentous, decorated DNA–histone webs have become the center of attention across a variety of fields. Although originally identified as a newly discovered defense mechanism in neutrophils' weaponry, it quickly became apparent that their presence contributes to a variety of pathologies, ranging from lung diseases to cancer and cardiovascular disease. Focusing on the intersection of NETs and thrombosis, this paper will provide an overview of how NET components interact with hemostatic pathways and impact thrombosis in a variety of medical conditions, with a special focus on the role of a key NETosis driver enzyme, peptidyl-arginyl deiminase-4 (PAD4).

 

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Of Forms and Formation

Forms

Since their discovery, NETs have been treated with waves of scientific enthusiasm and somewhat smaller waves of skepticism when it comes to their typography and their formation pathways (reviewed in studies by Brinkmann[4] and Nauseef and Kubes[5]). These waves have identified three distinct forms of NETosis so far (with additional nuances in each category). First, suicidal NETosis, a kamikaze weapon in a true sense: NET formation that is coupled with cell death. This form of cell death is neither necrosis nor apoptosis, rather something in between.[6] Second, as a response to Staphylococcus aureus infection, vital NETosis has been identified as a form where rather than one explosive burst, NET particles are secreted in distinct, less intrusive packages while the neutrophil is still functioning.[7] With this elegant move, the neutrophil joins the elite club of cellular players of hemostasis that already have a reputation for functioning without a nucleus: platelets and red blood cells. Third, even thriftier, some neutrophils choose to catapult mitochondrial DNA rather than nuclear.[8] Clearly, the three distinct forms must imply diverging intracellular pathways in NET formation; however, we are not there yet to make a judgment as the role of many intracellular NETosis regulators is under investigation and/or subject to debate.

NETs consist of DNA and various histones decorated with granular proteins. They are fragile, complex structures composed of smooth “threads” of 15 to 25 nm diameter, representing a chain of nucleosomes from unfolded chromatin. Under scanning electron microscope (SEM), NET threads seem to be sprinkled with globuli of 30 to 50 nm[9] that contain multiple cathelicidins originating from the neutrophil granules (or lysosomes). Threads are wound into “cables” that can add up to a diameter of 100 nm ([Fig. 1]) and form complex three-dimensional structures that can be hard to distinguish from fibrin networks with SEM.[10] Under in vitro circumstances, such as in multi-well plates, NETs float within the medium, similar to a spider's web.[11] Their “stickiness” via their electrostatic charge and extent over areas of several microns make them effective at trapping[12] and possibly killing microorganisms.[11]

Fig. 1 Scanning electron microscopy images of neutrophil extracellular traps produced by neutrophils activated with phorbol 12-myristate 13-acetate. Images are taken at 10,000x magnification. Scale bars = 1 μm.

 

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Formation

Given their intriguing appearance under the microscope, one cannot help but wonder: how does the dense nuclear material turn into such an expansive web-trap? Loosening the DNA–histone interaction is crucial to the process. Reducing the positive charge of histones, the means chosen by the process of NETosis can be accomplished in at least three possible ways: citrullination, chlorination, and proteolysis.

Peptidyl-arginyl Deiminases (PADs)

Citrullination is an irreversible post-translational modification in which peptidyl-arginyl residues are converted to peptidyl-citrulline via deimination of the positively charged guanidino group of arginine turning it to an electrically neutral ureido group of citrulline. This conversion is catalyzed by members of the PAD family of enzymes and results in loss of one positive charge per converted residue and a slight increase in the molecular weight of the respective protein. The process has received particular attention in the field of rheumatoid arthritis for a long time (reviewed in study by Darrah and Andrade[13]). Out of the five PAD isoenzymes expressed in humans (PAD1–4 and PAD6), PAD4 has been recognized as the key molecule of chromatin decondensation in NETosis.[14] Although PADs are principally cytosolic enzymes, PAD2 and PAD4—of which the latter has a nuclear localization signal—were shown to have a nuclear activity that gives them an easy access to histones. Hypercitrullination by PAD4 causes a massive loss of positive charges on histone molecules that loosens their molecular interactions with DNA resulting in subsequent large-scale chromatin decondensation. In addition to various core[14] and linker histones,[15] additional nuclear substrates of PAD4 have been identified: lamin C (in the process of apoptosis[16]) and PAD4 itself.[17] [18] As our knowledge widened, other ways of processing histones have been recognized: chlorination by myeloperoxidase (MPO) and proteolysis by neutrophil elastase.

 

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Chlorination

Chlorination is a somewhat less specific, but similarly fine-tuned process catalyzed by MPO resulting in the disappearance of a positive charge in the chlorinated peptidyl side chains. MPO is one of the most abundantly expressed enzymes in activated neutrophils. Basically, it is released from the azurophil granules into phagosomes and cytoplasm during respiratory burst and up to 30% of cellular MPO can be secreted to the extracellular space.[19] MPO catalyzes the formation of hypohalous acids from H₂O₂, out of which hypochlorous acid (HOCl) is one of the primary products. In addition to the neutrophil-mediated intraphagosomal microbial killing, following migration to the nucleus HOCl modifies histones resulting in a wide range of products out of which N-chloramines are the most abundant.[20] The loss of positive charges may lead to chromatin decondensation, promote fragmentation and aggregation of histones, and alter their cytotoxic functions in a yet unclear manner.[21]

 

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Proteolytic Clipping

The third approach, proteolysis, is more dramatic, leading to removal of whole peptide chains—called N-terminal histone tails—containing positive charges. Performed by serine or cysteine proteases, this posttranslational modification is a from-yeast-to-mammals conserved response to diverse NET stimuli.[22]

Interestingly, although all three pathways are plausible, none of them seems quintessential to the process. Neutrophil elastase deficient mice still produce NETs, inhibition of MPO does not necessarily inhibit NET formation, and organisms without PADs (such as plants) can still form NET-like structures.

Similarly, the pathway leading up to the activation of these enzymes has been much debated. What stimulates these proteins to partake in intranuclear processing of histones? For PAD—nuclear signal and calcium activation or something that sensitizes it? Neutrophil elastase takes matters into its own hand and cleaves its way into the nucleus during NETosis via processing gasdermin.[23] However, if neutrophil elastase is extruded from the cell via degranulation before it could process gasdermin intracellularly, gasdermin is unable to form vesicular pores that help channel neutrophil elastase into the nucleus, which has been shown to be an important step, at least in certain forms of NETosis.[24]

 

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Upstream Happenings

NADPH oxidase was one of the earliest recognized key players in the signal transduction pathway that leads to NETosis: originally deemed crucial for NET release, as NADPH oxidase-deficient patients were shown to be incapable of NET formation,[25] [26] and gene therapy restored their NET-forming ability.[27] However, it has been since shown that certain bacterial stimuli such as ionophores do not require oxidative burst to trigger NETosis[28] and a NETosis-independent antimicrobial action has been suggested via NADPH oxidase-enhanced kynurenine formation.[29] [30]

The key players of NETosis do not finish their lifecycle inside the cell. Instead, they get ejected with forming NETs, and a new chapter of their life begins in the extracellular space. In the next section, we will discuss how these key enzymes along with the main mass of NETs, DNA and histones, continue their life in the extracellular space.

 

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When Webs Cross Paths: NETosis and Hemostasis

When the integrity of the blood vessel wall is disrupted at points of inflammation, NETs are not the only barrier struggling to restrict the progress of tissue damage. In parallel with NET formation, the contact pathway of blood coagulation generates a fibrin meshwork.[31] Thus, two filamentous structures co-localize in vivo, one primarily originating from plasma components and one from neutrophils. They not only seem to work together structurally, but also their pathways cross overregulating each other's formation. In this section, we will break the process to its elements.

NET–Hemostasis Interactions Promoting Thrombosis

NETs and the Blood Vessel Wall

Located at the interface of blood and other tissues, endothelium serves an integrative role in hemostasis. While intact and healthy endothelium prevents thrombosis by supporting antiplatelet and anticoagulant functions, once endothelium is activated or damaged it promotes thrombosis, both deep vein and arterial. Endothelial cell activation, damage, or even death can be induced by NET-related proteases, defensins, and most likely histones.[32] [33] [34] [35] [36] Upon binding to the phospholipids of the endothelial cell membranes, histones induce pore formation leading to ion influx[37] [38] [39] and elevated calcium levels within the cells with subsequent von Willebrand factor (VWF) release[40] and endothelial activation or even cell death. Activated endothelial cells produce reactive oxygen species (ROS), which in turn contribute to NET formation.[33] Additionally, ex vivo experiments with iliac artery cross sections evince thrombogenic effects of one of the NET-related proteases, neutrophil elastase, on deeper layers of the arterial wall,[41] but further in vivo evidence is needed concerning the role of NET-bound neutrophil elastase at sites of vascular damage. In the subendothelium, NETs have been observed in both murine and human atherosclerotic lesions[42] probably as a consequence of neutrophils infiltrating arteries at early stages of atherosclerosis.[43]

 

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NETs and Cellular Elements of Thrombi

NET fibers bind platelets directly and/or indirectly and support their aggregation.[44] When perfused with blood, NETs bind platelets serving as an alternative scaffold for platelet adhesion and activation.[45] The first step of platelet binding involves either electrostatic interactions between NET histones and platelet surface phospholipids[37]/carbohydrates,[46] or histone binding to Toll-like receptors 2 and 4.[47] Platelets also bind double- and single-stranded DNA in vitro.[48] [49] Adhesion molecules may also play a role in thrombocyte–NET interactions, such as VWF (binding histones through its A1 domain),[50] fibronectin, or fibrinogen.[40] [45] The interaction of histones with platelets results in calcium influx either by pore formation[51] or by opening of existing channels,[52] a process which triggers activation of the integrin αIIbβ3.[53] This chain of events raises the possibility of a sequential histone-induced activation of platelets (first binding to platelet surface, then, following activation, binding to adhesion molecules[44]), which could explain the unsaturable nature of histones binding to platelets.[44] When infused into mice, histones co-localize with platelets and induce thrombocytopenia and thrombosis,[39] [40] [44] possibly partially through potentiation of thrombin-dependent platelet activation.[54]

Red blood cells (RBCs) are no longer considered as passively entrapped elements of thrombi, but cells that may promote thrombosis by exposing phosphatidylserine and altering blood viscosity[55]; furthermore, their presence modulates structural parameters of the forming fibrin meshwork through integrin-mediated fibrin(ogen)–red blood cell interactions.[56] [57] Similarly to platelets, RBCs avidly bind to NETs after perfusion of whole blood,[45] possibly through direct and indirect mechanisms. RBCs can bind DNA, since this was eluted from the surface of isolated RBCs from cancer patients.[58] Activated neutrophils or platelets entrapped in NETs can also recruit RBCs as observed at very low venous shear in vitro.[59] NETs are predominantly found in the red, RBC-rich part of experimental deep vein thrombi of mice, suggesting that NETs could be important for RBC recruitment to venous thrombi.[40]

 

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NETs in Fibrin Formation and Fibrinolysis

NETs have diverse effects on the formation and degradation of the fibrin network: both promotion of coagulation and inhibition of fibrinolysis serve their basically procoagulant nature; however, fibrinolysis supporting effects of some neutrophil-derived NET components have also been reported.

The majority of the NET-derived procoagulant factors originate from neutrophils. Neutrophil elastase and cathepsin G activate platelets,[60] [61] [62] and some coagulation factors—FV, FVIII, and FX.[63] [64] [65] Neutrophil-derived proteases including several matrix metalloproteases (MMP1, -7, -9, and -12) cleave and inactivate the tissue factor pathway inhibitor (TFPI).[66] [67] Nucleosomes expelled by neutrophils bind TFPI, and thus they serve as a platform for TFPI degradation by neutrophil elastase; this process is also supported by activated platelets.[68] MPO inactivates thrombomodulin.[69] Histones induce platelet aggregation either directly or via recruitment of fibrinogen and VWF[44] [45] and inhibit thrombomodulin-mediated protein C activation.[70] Finally, DNA enhances the activation of the contact pathway of blood coagulation accelerating thrombus formation and serves also as a scaffold that holds together erythrocytes and activated platelets.[45] [71] In addition to DNA, a broad spectrum of factors available at sites of NET formation could contribute to FXII activation—damaged endothelial cells, negatively charged surfaces (e.g., short chain polyphosphates released from activated platelets), or entrapped pathogens (live bacteria and bacterial wall–derived pathogen-associated molecular patterns).[47] [72] Tissue factor has also been identified as a NET component,[73] [74] and protein disulfide isomerase originating from damaged or activated endothelial cells probably participates in its activation.[75]

The effects of histones and DNA—the main components of NETs—on fibrinolysis and clot stability reinforce the prothrombotic characteristics of NETs outlined so far: adding purified histone and DNA to the forming clot in vitro results in increased mechanical stability and higher enzymatic resistance presumably due to the observed alterations in clot structure.[76]

Besides the predominantly procoagulant and antifibrinolytic properties of NET components, some of them support the balance of formation and removal of fibrin by promoting fibrinolysis in different ways. For example, together with cathepsin G, neutrophil elastase can degrade fibrin,[77] possibly serving as an alternative, neutrophil-based mechanism to compensate for any impairment of plasmin-mediated fibrinolysis.[78] [79] Neutrophil elastase also inactivates the fast-acting major plasmin inhibitor α2-antiplasmin and contributes to the formation of miniplasmin, the fibrinolytic activity of which is higher on crosslinked fibrin than that of plasmin.[80] The presence of histone 2B in NETs may provide support for the abovementioned profibrinolytic effects via recruiting plasminogen on the cell surfaces.[81]

Due to its ternary web-like structure, consisting of expelled chromatin, VWF, and fibrin,[38] [40] [73] dissolution of a thrombus seems to be much more complicated than simple fibrinolysis alone; therefore, the principal VWF-degrading protease ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13)[82] and DNases are also needed in addition to plasmin. DNase I and DNase I-like 3 act together during in vitro chromatin breakdown,[83] while histone degradation may be accomplished by plasmin,[84] neutrophil elastase, activated protein C, or thrombin.[85] The activity of ADAMTS13 is restrained by numerous NET-derived factors. Generated upon MPO action, HOCl oxidizes VWF A2 peptide and plasma VWF multimers, thus turning them to less susceptible substrates of ADAMTS13,[86] which is also oxidized and therefore inactivated.[87] Alpha defensins in vitro inhibit the cleavage of VWF by ADAMTS13[88]; finally, citrullination of ADAMTS13 by PAD4 leads to a pronounced decline of its activity toward VWF.[89] Thereby, similar to the NET-related prothrombotic shift in the balance of fibrin generation and lysis, NETs favor the formation of more stable thrombi through their effects on the multimerization and proteolysis of VWF.

 

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Citrullination, the Great Shapeshifting by PADs

Citrullination of plasma proteins has been extensively researched in fields unrelated to thrombosis, such as rheumatology, for decades, where the significance of citrullinated plasma proteins in the pathogenesis of rheumatoid arthritis has been under investigation. The discovery of NETs gave a boost to these efforts and widened the interest in this particular form of posttranslational protein modification.

PADs in the Wild: Extracellular Consequences

As discussed in the section “Peptidyl-arginyl Deiminases,” PAD4 remains the dominant isoform in neutrophils and more interestingly, the only member of the PAD family with a nuclear localization signal. The hypothesis that PAD4 therefore must be released into the extracellular space during the expulsion of NETs has been proven and studies show that PAD4 is able to citrullinate proteins in the extracellular space too. Furthermore, PAD4 has been detected on the plasma membrane of neutrophils, and PAD2 has been shown to be spontaneously released from neutrophils independently of NETosis.[90] [91] Interestingly, the millimolar concentration range of Ca²⁺ in the extracellular space brings PADs much closer to their optimal activity than intracellular Ca²⁺ concentrations.

In the extracellular space, NETs, complement, and coagulation factors combat hemorrhage and infection in a joint effort (reviewed in a study by de Bont et al[92]). However, the potential citrullination of the complement and hemostatic proteins by PADs, and the possibility of PAD inhibition by a PAD4 binding protein (PTPN22)[93] further complicate the situation and may modify the outcome. For example, citrullination of C1q inhibitor by PAD1, -2, or -4 has been shown to reduce its inhibitory activity.[94] This proteoglycan also exerts potent anticoagulant effects which are abolished by citrullination.[95] Another defense antimicrobial peptide cathelicidin (LL-37) has prothrombotic properties[96] in addition to its antimicrobial and immunomodulatory functions that are dampened upon citrullination by PAD2 and -4.[97]

Modifying the arginyl residues of hemostatic proteins is a phenomenon of much interest since thrombin, a master regulator of clotting, predominantly cleaves peptide bonds next to arginine residues. Once turned into citrulline, a range of thrombin substrates could be annihilated. Studies so far show that that is indeed the case. Citrullinated antithrombin loses its ability to inhibit thrombin—a phenomenon that has been identified as a candidate for driving the genesis of the pannus in rheumatoid arthritis joint spaces even before the discovery of NETs.[98] The reactivity of some other serpin and non-serpin inhibitors involved in the control of hemostasis is also downregulated upon action by PADs.[94] Citrullinated TFPI[99] together with citrullinated antithrombin may promote pathological thrombus formation.

Given the central role of the cleavage of fibrinopeptide A (at Arg-16 of the Aα-chain) and fibrinopeptide B (at Arg-14 of the Bβ-chain) by thrombin[100] in the conversion of fibrinogen to fibrin and that fibrinogen is the precursor of the primary scaffold of thrombi, its citrullination might be of central importance with regards to the properties of the formed thrombus. Citrullinated fibrinogen has been long identified in rheumatoid arthritis synovial fluids and, to a much lesser extent, in patient sera.[101] [102] However, the possibility of citrullinated fibrinogen being present in thrombi that have been shown to be neutrophil-rich has only been examined very recently.[103] In our murine deep vein thrombosis studies, we have shown the massive presence of citrullinated fibrinogen in thrombi formed at stenosis sites of the inferior cava vein. Having demonstrated the in vivo citrullination of fibrin in thrombi, we evaluated the consequences of citrullination in terms of biomechanical behavior of fibrin clots. Clots formed from citrullinated fibrinogen show reduced clot elasticity, decreased plastic stability, and decreased resistance to shear stress.[103] This in part could be a result of defective fibrin polymerization; however, direct inhibition of thrombin by the citrullinated form of fibrinogen has also been proposed.[104] Because of the critical role of thrombin-susceptible arginyl-peptide bonds, citrullination by PADs is expected to affect profoundly the rate of fibrin polymerization and consequently the polymerization pattern and final structure of the fibrin matrix. In a recent study we have provided direct evidence for the structural alterations in citrullinated fibrin.[105] Citrullinated fibrinogen forms thinner fibrin fibers with higher density that result in a more compact space-filling pattern of the clots. These structural changes explain the modified viscoelasticity of citrullinated fibrin as earlier studies have shown that thinner fibrin fiber networks are associated with decreased elastic modulus and an increase in the loss tangent.[106] [107] The structural findings that citrullination of fibrinogen results in fibrin with thinner fibers and decreased porosity also add a structural background explaining the observed lytic resistance of citrullinated fibrin,[103] because earlier studies have shown that fibrin matrix with similar ultrastructure induced by other factors is more difficult to resolve.[108]

Another aspect of the thrombus lytic stability modified by citrullination is the alteration of pathways that regulate fibrin dissolution. First of all, plasmin, the master conductor of fibrinolysis, cleaves select peptide bonds not only next to lysine, but also next to arginine in fibrin, some of which are located at critical positions at the boundary of the D and E domains of the monomers (arginine 105 and 111 of the α-chain) or between the D domain and the C-terminal tail (arginine 197 and 200 of the α-chain).[100] The arginine–citrulline conversion at these sites may inhibit the fibrinolytic pathway in its terminal step catalyzed by plasmin. Second, a range of serpins associated with this pathway have been shown to undergo citrullination according to proteomic analysis of rheumatoid arthritis patient plasma samples.[109] At the level of serpins, the consequences of citrullination appear to be rather balanced: the loss of activity in citrullinated α2-antiplasmin and plasminogen activator inhibitor 1 is counteracted by the inactivation of citrullinated antithrombin discussed above. Despite these balancing effects, given the complex consequences of extracellular citrullination, it appears that PADs expelled in NETs reinforce the stability of thrombi in parallel with the effects of DNA and histones discussed in the section “NETs in Fibrin Formation and Fibrinolysis.” The key steps of the interplay of NETs and hemostasis discussed so far are summarized in [Fig. 2].

Fig. 2 Aspects of thrombus formation and resolution related to neutrophil extracellular traps (NETs). At sites of intravascular thrombus formation, the interplay of activated platelets and endothelium triggers the release of NETs, whose two major components are DNA and histones. When fibrinogen (Fgn) is converted to fibrin (F), the co-polymerization of fibrin with NET components results in altered fibrin structure with modified functionality such as a template of plasminogen (Pgn) activation by tissue-type plasminogen activator (tPA) and susceptibility to plasmin-mediated proteolysis to generate various polymeric (F', F'') or soluble (→→→) degradation products. NET formation is accompanied by the release of peptidyl-arginyl deiminase isoform 2 and 4 (PAD2, PAD4), which convert arginine residues in several proteins—including fibrinogen—to citrulline. Citrullinated fibrinogen (citFgn) is converted to citrullinated fibrin (citF) present in the structure of murine model of venous thrombi. The citF is composed of thinner fibers and smaller pores. The modified structure of citF results in impaired mechanical stability and enhanced lytic resistance compared with non-citrullinated fibrin. (The figure is reproduced from our illustration of a symposium talk published in Aleman et al[159] under CC BY-NC-ND license [http://creativecommons.org/licenses/by-nc-nd/4.0/].)

 

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PADs from Another World—the Role of Microbes

The conversation is not complete if one does not touch upon the importance of microbial PADs, often-forgotten players in immunothrombosis. Microbes clearly benefit from cleaving NH₄ ⁺ off host proteins by bacterial PADs as this might help neutralize the microenvironment of the pathogen and also serves metabolic purposes as it may provide ATP under anaerobic conditions via further conversion of citrulline.[110] [111] Another perspective of microbial PADs action is the altered function of the citrullinated proteins. For example, PAD secreted by Porphyromonas gingivalis is prone to downsize immune reactions.[112] [113] This form of PAD citrullinates gingipain, histone H3, lysozyme-derived peptide LP9, and the complement protein anaphylatoxin C5a causing loss of their function in the control and execution of immune responses. However, the fact that Porphyromonas gingivalis-derived PAD seems more effective in citrullinating the NET-associated H3 histone than human PADs secreted during NETosis[112] begs the question why a bacterial mechanism would evolve that supports a hallmark process of the supposedly antibacterial NET formation. To this point, in an earlier review we offer an educated speculation on the possibility of different roles for intracellular versus extracellular citrullination.[114] It is possible that intracellular citrullination of histones merely opens up the nuclear material for easier release, and this might not require full citrullination. Many of the histones eventually released as NETs could remain non-citrullinated and exhibit more of the histone-related toxicity than their citrullinated counterparts. Indeed, positively charged histones seem to be more effective in a range of extracellular functions. Extracellular citrullination might be a self-defense mechanism both for the host and the pathogen. Bacterial PAD production could protect them against non-citrullinated histone-mediated killing by the host. Neutrophils themselves express PAD4 on their surface as well,[91] which may shield against self-cytotoxicity. Considering the above-mentioned beneficial effects of citrullination on bacteria, it is not surprising that in their provocative review, Konig and Andrade further evolve the hypothesis that extracellular citrullination might be a defense mechanism to reduce toxicity of histones.[115] They propound that citrullination is a downright bacterial tactic rather than antimicrobial. According to this concept, bacterial toxins cause the so-called leucotoxic hypercitrullination of histones, which is different from classical NETosis, but it also leads to neutrophil cell death.[115] However, this remains unclear since PAD4 has been proved to be essential in the antimicrobial function of NETs.[116]

 

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Wonderful NETs and Where to Find Them

NETs have been identified in a variety of pathological conditions. For the purposes of the current discussion, we will focus on a couple of conditions where an elevated risk for thrombosis is traditionally known and discuss the potential contribution of NET.

NETs and Thrombosis in Neoplasms

Hypercoagulability is frequently seen in cancer patients,[117] which contributes to the fact that the second most common cause of death among them is thrombosis. The intertwined history of cancer and abnormal clotting goes way back in time. The first official reports of Trousseau's syndrome date back to 1865. Since then, much has been uncovered in terms of the weaponry that tumors use to hijack the hemostatic system. From accelerated thrombin generation[118] to tissue plasminogen activator-like action on plasminogen activation,[119] tumors seem to possess an eerie ability to shapeshift and utilize the hemostatic system in a way that fully aligns with their goals—be it fibrin production to evade immune recognition or plasmin generation to facilitate their release from their origin and let them migrate to form metastases.

On the basis of Trousseau's observations, it was believed for a long time that basically venous thrombosis could be associated with malignant diseases (cancer patients have a 4- to 7-fold higher risk of venous thromboembolism compared with the general population[120]), but maybe less commonly, arterial thrombosis can also be the first sign of a previously undiscovered tumor.[121] [122] Cancer cells are able to promote thrombosis not only through direct alteration of hemostasis. In tumor states, increased NET formation can be observed.[123] [124] [125] [126] However, it should be noted that certain tumor cell lines are not able to induce NET formation.[123] [124] Tumor-associated neutrophils in the tumor microenvironment or infiltrating directly the tumor tissue may exert both anti-tumor and pro-tumor activities[127] through secretion of proteases or recruitment of other immune cells by cytokines and chemokines, ROS release, or NET formation.[128] Emerging data show that the primary link between cancer and NET formation is granulocyte colony-stimulating factor (G-CSF). Tumor cells overexpress G-CSF,[129] which is known to prime neutrophils toward NETosis.[130] [131] Evidence for a two-way interaction of NETs and tumors is also available: NETs increase tumor growth and metastasis.[132] Given this complex NET–tumor interplay and the prothrombotic effects of NETs discussed in the section “NET–Hemostasis Interactions Promoting Thrombosis,” it is plausible to hypothesize that NETs form a link between tumors and thrombosis. Accumulating data support the validity of this hypothesis.[45] [133] Ex vivo experiments show that G-CSF receptors are present on the platelet surface and their activation induces platelet aggregation.[134] In our recent study[135] using a murine model of human pancreatic cancer, we observed a higher thrombus burden in tumor-bearing mice that was associated with increased circulating levels of G-CSF and neutrophil counts. The thrombi of these tumor-bearing mice contained significantly higher amounts of DNA and citrullinated histone H3, consistent with more intensive NETosis.

In addition to G-CSF, there are several other tumor-related cytokines that promote the production and mobilization of neutrophils, e.g., chemokine (C-X-C motif) ligand 1 (CXCL1) or transforming growth factor β (TGF-β).[126] [136] As a result, overproduced mature neutrophils leave the bone marrow with increased propensity toward NETosis.[137] Similarly to G-CSF, some cytokines can directly promote NETosis (e.g., TGF-β, CXCL1).[126] [138] Last but not least, independently of the cytokine effects, tumor-derived extracellular vesicles can also directly activate neutrophils to form NETs. Extracellular vesicles from a human breast cancer cell line accelerated the formation of laser-induced venous thrombosis or ferric chloride-induced arterial thrombi in a mouse model of thrombosis in parallel with enhanced NET formation.[139]

Altogether, it appears that cancer cells develop an environment that favors neutrophils to form NETs, which contributes to a systemic prothrombotic state through the mechanisms described in the section “When Webs Cross Paths: NETosis and Hemostasis” above.

 

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NETs and Thrombosis in Sepsis

Neutrophils are at the frontline of defense to combat infections, and NETs are an essential tool in this defense to eliminate pathogens as discussed in the section “Of Forms and Formation.” Not surprisingly, increased blood levels of the key NET components DNA, citrullinated histone H3, and MPO are detected in sepsis patients.[140] High levels of circulating cell-free DNA were observed in sepsis even before the discovery of NETs.[141] Enhanced NETosis in sepsis has been documented by measurements of additional circulating biomarkers of NETs—MPO–DNA complex and nucleosome-high mobility group box 1 protein (HMGB1).[142] The association of sepsis with hemostatic derangements that contribute to localized deep venous thrombosis or dispersed microvascular thrombosis accompanied by consumption of platelets and coagulation proteins and disseminated intravascular coagulation (DIC) has been known for a long time.[143] Hypercoagulability provokes serious complications in sepsis through microthrombi development in small vessels and subsequent organ ischemia. NETs cooperate with platelets to induce the formation of these microthrombi as platelet aggregates have been observed within NETs in vivo.[144] The association of NET levels with sepsis-induced coagulopathy (septic patients with DIC exhibit higher plasma levels of neutrophil elastase and citrullinated H3 than those without DIC) is an indicator of their role in coagulation abnormalities observed in septic patients.[145] Patients with elevated NET levels are prone to more frequent thrombotic and bleeding events and prolonged recovery in sepsis.[146] Critically ill septic patients with vasoplegic shock show elevated plasma levels of NET biomarkers, which correlate with organ dysfunction, as well as D-dimers, which suggest a prothrombotic state.[147] [148]

Given the interactions of NET components with platelets and coagulation proteins to promote thrombosis as discussed in the section “When Webs Cross Paths: NETosis and Hemostasis,” NETs could provide a mechanistic link between sepsis and thrombosis, and in addition, NETs and thrombosis are reciprocally coupled. Following exposure of neutrophils to platelets treated with plasma from septic patients, TLR4-dependent platelet–neutrophil interactions trigger NET formation.[32] NETs activate the stimulator of interferon genes (STING) adaptor protein, which induces tissue factor production in the endothelial cells enriching the NETs with tissue factor and triggering the extrinsic pathway of blood coagulation in a murine model of sepsis-associated lung injury.[149] [150] Cell-free DNA in vivo also induces tissue factor expression in endothelial cells and VWF release, resulting in thrombin generation and platelet activation.[151] As a result of this DNA-induced platelet activation, in sepsis the circulating levels of the chemokine platelet factor 4 (PF4) are elevated,[152] and a reciprocal circuit of PF4-induced NET stabilization is initiated, because the electrostatic interaction of PF4 with NETs renders them resistant to DNase treatment.[153] Platelet activation also causes release of ATP from their δ-granules,[154] which in turn amplifies the formation of NETs and the growth of thrombi.[155] [156] This ATP-dependent self-amplifying platelet/NET circuit can be interrupted by DNase treatment, because these enzymes not only degrade the DNA scaffold of NETs but also hydrolyze ATP to adenosine, which does not exert the pro-inflammatory and pro-thrombotic effects of extracellular ATP.[157]

 

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Conclusion

The multiple circuits of two-way interactions between thrombosis and NETosis in cancer and sepsis highlight the potential benefits from therapeutic approaches based on NET destruction to help the resolution of thrombi. For example, administration of DNases could be an efficient strategy to destabilize thrombi. We have provided sound in vitro evidence that DNase facilitates the lysis of fibrin that is stabilized by DNA and histones.[76] Preclinical studies also confirm the utility of DNases in thrombus resolution. In mice with staphylococcal infection larger thrombi are formed in inferior vena cava ligation model that contain more neutrophils and NETs, and DNase treatment promotes the lysis of these thrombi.[158] Clinical trials (Reg.# NCT04785066, NCT05203224 at https://clinicaltrials.gov) are in progress to test the therapeutic efficacy of DNase administration as adjuvant modality for arterial recanalization in the management of ischemic stroke with thrombectomy or thrombolysis. The results of such clinical trials will provide guidelines for safer and more efficient treatment of thrombosis based on tools targeting NETs.

 

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Conflict of Interest

None declared.

^* These authors share first authorship and have contributed equally to the article.

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Publication History

Received: 31 January 2025

Accepted: 27 February 2025

Accepted Manuscript online:
28 February 2025

Article published online:
28 March 2025