Introduction

Venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE), is a major contributor to global illness and death. Its incidence is influenced by age, comorbidities, and risk exposure. DVT occurs frequently, with an annual rate of about 1 per 1000 people.^1,2 Multiple VTE risk factors include older age, obesity, major surgery or trauma, hospitalization, cancer, immobility, estrogen therapy, pregnancy / puerperium, sepsis, chronic inflammatory diseases, and genetic predisposition.^1,2 Owing to VTE prevention strongly advocated within the last 20 years, the thrombotic risk following hospitalization, injury, or pregnancy has been markedly reduced.³ In Western populations, roughly 1 in 12 individuals will develop DVT during their lifetime. Notably, 25%–30% of first‑time DVT cases occur without identifiable risk factors. VTE recurrence affects 20%–36% of patients within 10 years.^1,2 Long‑term anticoagulation is highly effective in preventing VTE recurrence, but at the cost of an elevated bleeding risk. Post‑thrombotic syndrome (PTS), a chronic complication, develops in up to half of DVT patients, typically 3–6 months after the initial event. PE is less common than DVT, with 60–120 cases per 100 000 individuals annually, and approximately 370 000 cases per year in the United States.^1,2,4 However, in many countries, PE is still associated with high prevalence and a 30‑day mortality rate of up to 14%–20%,⁴ particularly in elderly patients and those with a high comorbidity burden. Chronic thromboembolic pulmonary hypertension (CTEPH) is a serious long‑term complication affecting 1%–4% of PE patients, with substantial mortality.^1,5

Accumulating evidence indicates that immune cells, particularly neutrophils, are implicated in VTE.^6-17 Neutrophils that serve as frontline effectors of innate immunity can generate neutrophil extracellular traps (NETs) in the process of NETs formation (NETosis), which represents a specialized antimicrobial defense mechanism, discovered by Brinkmann and co‑workers in 2004.¹⁸ It is also well known that NETosis contributes to venous thrombus formation and stabilization, giving rise to the concept of immunothrombosis. Understanding the role of NETosis in VTE not only provides mechanistic insights into disease pathogenesis but also opens new avenues for biomarker development and targeted therapy. In this review, we present available data on the associations between NETosis and venous thrombosis, in particular the mechanisms through which NETs contribute to thrombus formation. We also discuss potential therapeutic strategies targeting NETosis, and critically evaluate methods for the quantification of NET‑associated proteins. Finally, we address practical implications of NETosis in the management of VTE patients.

Role of neutrophil extracellular traps in thrombus formation

The release of NETs in response to strong proinflammatory stimuli, such as infections and damage‑associated molecular patterns (DAMPs), represents a key mechanism of innate immunity. NETosis can be triggered by autoantigens, urate crystals, oxidized low‑density lipoprotein, cholesterol, high‑mobility group bo × 1 protein (HMGB1), and proinflammatory cytokines (eg, interleukin [IL]-1β, IL‑8, and tumor necrosis factor α).¹⁹ Phorbol‑12‑myristate‑13‑acetate (PMA), lipopolysaccharides (LPS), calcium ionophores, and bacteria can induce NETosis both in vitro and in vivo.¹⁹ NETs are 3‑dimensional structures composed of decondensed chromatin decorated with neutrophil granule proteins, including neutrophil elastase (NE), myeloperoxidase (MPO), and proteinase 3.¹⁸ NETosis is a complex, tightly regulated process involving activation of nicotinamide adenine dinucleotide phosphate oxidase regulated by protein kinase signaling cascade Raf‑MEK‑ERK pathway. This pathway plays an important role in the initiation of NETosis, particularly its classical, reactive oxygen species (ROS)-dependent form.²⁰ Subsequent post‑translational histone modifications, such as citrullination, and progressive disintegration of intracellular membranes ultimately result in the extrusion of DNA into the extracellular space.²¹ Peptidyl arginine deiminase 4 (PAD4), localized primarily in the cytoplasm and translocated to the nucleus following calcium influx, catalyzes citrullination of histones (conversion of arginine to citrulline), promoting chromatin decondensation essential for NETosis. Owing to their spatial architecture, NETs act as sticky, protein‑rich scaffolds that immobilize microorganisms, including bacteria, fungi, and certain viruses.²² NETs locally concentrate potent antimicrobial factors, such as proteolytic enzymes, ROS, and cytotoxic histones.¹⁸

The negatively charged DNA backbone binds positively charged coagulation factors, platelets, and red blood cells. The key mechanism of coagulation activation via tissue factor (TF) exposure following vessel injury is closely associated with recognition of pathogens by leukocytes and cellular damage. Pathogen‑associated molecular patterns (eg, LPS) and DAMPs stimulate monocyte receptors, such as toll‑like receptors (TLRs) and CD14, leading to increased TF gene transcription and protein expression.¹⁹ NETs can bind TF derived from activated monocytes, endothelial cells, or microparticles.²³ They also activate the contact pathway via factor XII (FXII), as the negatively charged surfaces of extracellular DNA and histones promote FXII autoactivation, leading to activation of FXI, FIX, and ultimately FX.^24,25 The dual engagement of intrinsic and extrinsic pathways underscores the potent procoagulant capacity of NETs.

NET‑associated enzymes, namely, NE and MPO, also oxidize natural anticoagulants, including thrombomodulin and TF pathway inhibitor, promoting endothelial dysfunction and a prothrombotic state.^26,27 Moreover, NET‑rich thrombi exhibit increased resistance to fibrinolysis.^28,29 This contributes to thrombus persistence and may impair the resolution of venous clots. Experimental degradation of NETs with DNase I enhances thrombolysis, supporting the mechanistic role of chromatin in fibrinolytic resistance.³⁰

Platelets cooperate closely with neutrophils, linking inflammation with thrombosis, mainly through interactions between platelet P‑selectin and neutrophil P‑selectin glycoprotein ligand‑1, resulting in the formation of platelet–neutrophil aggregates.^31,32 These aggregates enhance further neutrophil recruitment, promote leukocyte adhesion to the endothelium, and stimulate NETs release.³³ Also, histones bind to platelet TLR2 and TLR4, inducing calcium influx, granule secretion, and activation of integrin αIIbβ3.³⁴ Platelets, in turn, stimulate NETosis via P‑selectin and HMGB1, creating a self‑amplifying loop.^23,35

Neutrophil extracellular trap formation testing

To our knowledge, no widely adopted, diagnostically approved in vitro diagnostic and fully standardized method for NETs quantification is currently available for routine clinical use. Although several commercial kits and laboratory‑developed assays have been proposed,^36-38 their clinical implementation remains limited due to substantial analytical and preanalytical variability (Table 1).^36,39 Theoretically, the most specific circulating NET markers are MPO‑DNA and NE‑DNA complexes. MPO‑DNA complexes were used in multiple clinical studies,^37,40,41 and were shown to correlate with disease severity in sepsis.⁴² However, their association with cardiovascular risk factors in the general population³⁷ or with acute VTE⁴³ has not been clearly established due to several limitations of the results obtained (Table 1).³⁹ In addition, MPO‑DNA levels are highly sensitive to preanalytical factors, which significantly affects reproducibility (Table 1).⁴⁴ The lack of standardized calibrators and reference materials further limits interstudy comparability. Plasma contains cell‑free DNA (cfDNA) that originates from multiple sources, including apoptosis and necrosis, which may interfere with assays; therefore, citH3 is frequently used as a surrogate marker of NETosis.^45,46 However, histone citrullination is not specific to NETs formation and may occur in other inflammatory conditions or autoimmune diseases.^46-48 Consequently, current evidence suggests that multimarker approaches combining NET‑related proteins and coagulation parameters may better reflect the NETs burden than single biomarkers. Notably, citH3 and NE did not correlate with MPO‑DNA complexes in plasma.^37,39 These problems render NETosis assessment in VTE patients (and in other populations) quite challenging, and contribute to inconsistent results reported by various research groups.

Neutrophil extracellular trap formation and immunothrombosis

Increasing evidence highlights the central role of NETosis in immunothrombosis.^27,35,49

Immunothrombosis refers to the formation of thrombi as part of the innate immune response, aimed at trapping and neutralizing pathogens within microthrombi and limiting their systemic dissemination.⁵⁰ However, excessive or dysregulated NETosis promotes thrombosis.³⁴ NETs structures have been detected in fresh thrombi, where their abundance correlated with thrombus burden and disease severity.^51-53

Of note, serum obtained from patients with eosinophilic granulomatosis with polyangiitis enhanced the capacity of neutrophils isolated from healthy controls to generate NETs.⁵⁴ Peripheral blood eosinophil counts correlated with the percentage of neutrophils undergoing NETs formation, suggesting a potential modulatory effect of eosinophils or eosinophilic inflammation on neutrophil activation and NETosis, which may contribute to disease pathogenesis.⁵⁴

Neutrophil extracellular trap formation in patients with venous thromboembolism

Available studies on NETosis and its association with the risk of VTE or its complications^6-17 are summarized in Table 2. In 2013, van Montfoort et al⁶ analyzed 150 adult patients with acute symptomatic DVT and 195 individuals with a clinical suspicion of DVT, and observed that higher (>80th percentile) levels of circulating nucleosomes and elastase-α1‑antitrypsin complexes were associated with a 2–3‑fold greater risk of thrombosis (adjusted odds ratio [OR], 3; 95% CI, 1.7–5 and adjusted OR, 2.3; 95% CI, 1.4–3.9, respectively). The Michigan Research Venous Group reported about 220% higher levels of circulating DNA in 47 symptomatic DVT patients, as compared with those free of DVT.⁷ Moreover, elevated plasma cfDNA levels correlated positively with the DVT Wells score, D‑dimer, MPO, and von Willebrand factor levels in patients with DVT.⁷

Table 2. Clinical findings regarding neutrophil extracellular traps in venous thromboembolism

Study population	Main findings	References
Abbreviations: cfDNA, cell‑free DNA; CTEPH, chronic thromboembolic pulmonary hypertension; dsDNA, double‑stranded DNA; DVT, deep vein thrombosis; FXI, factor XI; IQR, interquartile range; OR, odds ratio; PAD4, peptidylarginine deiminase 4; PE, pulmonary embolism; PTS, post‑thrombotic syndrome; Q, quartile; others, see Table 1
Patients with acute symptomatic DVT (n = 150) vs patients with suspected DVT (n = 195)	Circulating nucleosomes and elastase–α1‑antitrypsin complexes >80th percentile associated with a 2–3‑fold higher DVT risk	van Montfoort et al6
Patients with symptomatic DVT (n = 47) vs patients with excluded DVT (n = 28)	Circulating DNA higher in the DVT than the non‑DVT group (mean [SD], 57.7 [6.3] vs 17.9 [3.5] ng/ml; P <⁠0.01)	Diaz et al7
Patients with VTE (n = 11)	Extracellular DNA, MPO, citH3, and PAD4 visualized within venous thrombi	Savchenko et al8
Patients with acute PE (n = 126) vs healthy controls (n = 25)	Three‑fold higher citH3 levels in PE associated with hypofibrinolysis and higher simplified PE severity index; citH3 >4.11 ng/ml positively associated with 1‑year PE‑related death	Ząbczyk et al9
Patients with CTEPH (n = 141) vs controls (n = 60)	citH3 expression within fresh red thrombi; about 2‑fold higher MPO and dsDNA levels in CTEPH	Sharma et al10
Patients with VTE (n = 37) vs patients with no VTE history (n = 37)	Median (IQR) MPO‑DNA fold increase, 1.05 (0.96–1.13) vs 1 (0.94–1.05); P = 0.04	Zapponi et al11
Patients with DVT (n = 52) vs healthy volunteers (n = 51)	Three to 5‑fold higher plasma levels of MPO‑DNA, NE‑DNA, and citH3 in DVT	Li et al12
Patients with trauma‑related VTE (n = 10) vs trauma patients without VTE (n = 29)	Four‑fold higher citH3 levels in the patients who developed VTE as compared with those without VTE (median [IQR], 12.8 [7.1–30.8] vs 3 [1.8–6.8] ng/ml; P = 0.02)	Navarro et al13
Patients with hyperhomocysteinemia‑related DVT (n = 71) vs non‑DVT patients (n = 323)	Higher levels of MPO‑DNA (by 44%), citH3 (by 65%), and cfDNA (by 67%) in DVT patients (all P <⁠0.001)	Shao et al14
Patients with prior DVT followed for 53 months (n = 179)	PTS (n = 43) associated with 68.8% higher baseline citH3 levels as compared with no PTS ( P <⁠0.001); Recurrent VTE corresponded to 37.8% higher citH3 concentrations compared with nonrecurrence	Krupa‑Zabiegała et al15
Patients with acute VTE (n = 611)	cfDNA and nucleosome abundance was about 25% higher in PE vs proximal DVT and about 50% vs distal DVT; cfDNA predicted VTE‑related death during 3‑year follow‑up	Jiménez‑Alcázar et al16
Patients with DVT/DVT+PE (n = 172)	FXI correlated with citH3 levels ( R = 0.359; P <⁠0.001) The highest (Q4) FXI and citH3 levels were associated with an elevated risk of PTS (OR, 25.1; 95% CI, 6.88–91.54)	Michel et al17

The first evidence of NETs involvement in VTE was provided by Savchenko et al,⁸ who detected cfDNA, MPO, citH3, and PAD4 within venous thrombi obtained from 11 patients with VTE. Importantly, NETs were localized mainly within organizing rather than organized thrombi, though venous thrombi contained considerably less NET‑related components than coronary thrombi.³⁰

Regarding acute PE, 370% higher baseline citH3 concentrations were associated with simplified PE severity index and correlated with reduced plasma clot susceptibility to fibrinolysis.⁹ Of note, citH3 levels greater than 4.11 ng/ml (sensitivity, 67% and specificity, 83%) were associated with a greater risk of 1‑year PE‑related death.⁹

Jiménez‑Alcázar et al¹⁶ showed that among patients with acute VTE, those with PE, as compared with those with proximal or distal DVT, had about 25%–50% higher nucleosome and cfDNA levels, without any differences in DNA‑histones‑MPO complexes. Moreover, cfDNA levels, in contrast to DNA‑histones‑MPO complexes, predicted VTE‑related death (hazard ratio [HR], 2.57; 95% CI, 1.59–4.15) but not VTE recurrence during 3‑year follow‑up. Sharma et al¹⁰ provided evidence on NETs involvement in CTEPH by showing abundant citH3 expression within fresh red thrombi excised during pulmonary endarterectomy, along with elevated concentrations of cfDNA and MPO (both P <⁠0.001). Interestingly, enhanced neutrophil adhesion and chemotactic activity, together with 5% increased circulating MPO‑DNA levels, were detected even 2 years after an acute VTE episode, suggesting that activated neutrophils and endothelium mutually stimulate each other via endothelial injury and NET release, likely promoting thrombosis.¹¹

In 2024, Li et al¹² compared 52 DVT patients with healthy individuals and showed about 3–5‑fold higher plasma levels of MPO‑DNA, NE‑DNA, and citH3, along with a substantial platelet contribution to NETs generation and their procoagulant activity. Control neutrophils stimulated using plasma of DVT patients showed enhanced expression of TF and MPO, while NETs presented increased binding of coagulation factors, including fibrinogen, prothrombin, and FX. Moreover, it has been observed that NETosis is mediated by platelet factor 4, whose genetic inhibition reduced thrombosis in the inferior vena cava ligation model by modulating NETs formation.¹²

About 4‑fold higher baseline citH3 levels were also observed in trauma patients who developed VTE during 90‑day follow‑up, as compared with non‑VTE trauma patients.¹³ Similarly, levels of NET‑related proteins, namely, MPO‑DNA, citH3, and cfDNA were approximately 40%–60% higher in 71 individuals with hyperhomocysteinemia‑associated DVT than in non‑DVT patients.¹⁴

Recently, our group showed NETs as a contributor to PTS in a long‑term follow‑up study conducted in 179 individuals with prior DVT. The patients who developed PTS, as compared with the non‑PTS cohort, had nearly 70% higher citH3 levels, which correlated with the Villalta score.¹⁵ Baseline citH3 levels were associated with PTS (by 1‑ng/ml increase, OR, 5.5; 95% CI, 2.52–12.01). Moreover, recurrent VTE was found to correspond to almost 38% higher citH3 concentrations, as compared with no recurrence.¹⁵ This study suggests that NET‑related markers are predictive of serious VTE complications. Intriguingly, increased FXI activity was also shown to be associated with enhanced NETosis in DVT patients, which suggests an interaction between FXI and inflammation.¹⁷ This finding might be relevant for clinical practice given the studies on FXI/FXIa inhibitors.⁷⁰

Collectively, experimental data from animal models, in vitro systems, and translational studies strongly implicate NETs as structural and functional mediators of VTE. These findings have opened new perspectives on the immunothrombotic mechanisms underlying VTE and suggest that targeting NETosis or promoting NETs degradation may represent a promising therapeutic strategy. The mechanisms linking NETosis and VTE are summarized in Figure 1.

Cancer‑associated thrombosis

Malignancy is a major factor predisposing to VTE, increasing its incidence by approximately 5‑fold through different mechanisms.⁷¹ Multiple studies using neutrophils and plasma from cancer patients indicate that cancer increases the tendency for NETosis in vitro, though in vivo role of NETs in cancer‑associated thrombosis is less defined.^72-77 NET‑related markers, namely citH3 and cfDNA, have been identified within thrombi obtained from cancer patients, including cerebral, coronary, and pulmonary microthrombi.⁷² In an observational study by Mauracher et al,⁷³ increased circulating citH3 levels have been associated with VTE in lung and pancreatic cancer, but not in breast, brain, or colorectal cancer, suggesting that NET markers may help assess thrombotic risk in selected malignancies. NET‑related markers also correlated with thrombin‑antithrombin complexes and D‑dimer, while DNase I effectively reduced NET‑related thrombin generation and subsequent fibrin formation in healthy control plasma, stimulated with neutrophils isolated from gastric cancer patients.⁷⁴ It has also been shown that tumor‑derived growth factors stimulate NETs release, and the interaction between NETs and platelets represents a potential target for the prevention and treatment of tumor‑related thrombosis.⁷⁵ Interestingly, in patients with a history of VTE, increasing levels of circulating citH3‑DNA independently predicted occult cancer during 1‑year follow‑up (per 500‑ng/ml increase; HR, 2.1; 95% CI, 1.23–3.61).⁷⁴ Recently, Li et al⁷⁷ found that cancer cell–derived extracellular vesicles induced NETosis and histone H3 citrullination, augmenting the risk of VTE in cancer patients independently of TF‑mediated blood coagulation activation.

Although NETs have been shown to promote tumor progression, under specific conditions they may also exert tumor‑suppressive effects and mechanically restrict the hematogenous spread of malignant cells.^78-80 Cools‑Lartigue et al⁷⁸ demonstrated that NETs can bind tumor cells within the microcirculation. However, in their experimental model, this interaction facilitated metastasis; the authors emphasized that NET‑mediated sequestration of tumor cells could, in alternative biological contexts, promote their elimination by immune effector cells. Enhanced antitumor properties of NETs through tumor antigen exposure and activation of antigen‑presenting cells have also been proposed by Papayannopoulos.⁷⁹ Additionally, Mousset et al⁸⁰ showed that NET‑associated proteases may induce tumor cell damage and inhibit proliferation in vitro, indicating a potential direct cytotoxic effect of NETs against malignant cells.

In summary, although the predominant body of evidence supports a prothrombotic and prometastatic role of NETs in cancer‑associated thrombosis, there is also evidence that NETs may exert tumor‑suppressive activity through physical sequestration of tumor cells within the vasculature, direct cytotoxic effects mediated by NET‑associated enzymes, and augmentation of antitumor immune responses.

Future directions and challenges

The future of research on NETosis appears quite promising, yet it also demands a cautious assessment that would allow clinical translation. At present, several parallel research directions are developing intensively, including the standardization of NETs measurements, deeper elucidation of the molecular mechanisms underlying NETs formation, and development and testing of therapeutic strategies targeting NETs or promoting their degradation.⁸¹ There has been a marked increase in the number of studies developing enzymatic or immunological approaches to NETs degradation, such as DNase‑based therapies or antihistone antibodies. In parallel, growing attention is being directed toward mapping the heterogeneity of NETosis, as it is becoming increasingly evident that no single, universal pattern of NETs formation exists.^48,82 Instead, this process appears to be highly dependent on the nature of the stimulus, tissue microenvironment, and activation state of neutrophils.⁸²

Conclusions

Accumulating experimental and clinical evidence indicates that NETs play an important role in the pathogenesis of VTE, particularly in the development and progression of DVT. Animal and in vitro studies consistently demonstrate that NETs provide a structural scaffold that promotes platelet adhesion and activates blood coagulation, leading to enhanced thrombin generation and the formation of dense fibrin networks resistant to fibrinolysis. Through interactions with platelets, endothelial cells, and components of the complement and coagulation systems, NETs integrate inflammatory and hemostatic pathways, thereby reinforcing the concept of immunothrombosis in venous disease. Clinical studies, although still limited, support the translational relevance of these findings by demonstrating elevated circulating NET‑related protein levels in acute VTE and long‑term complications, especially PTS and CTEPH. Despite these promising insights, several important challenges need to be addressed before NET‑related biomarkers can be incorporated into routine clinical practice. Current assays for NETs quantification suffer from substantial preanalytical and analytical variability, a lack of standardized calibration, and limited specificity. Furthermore, many commonly used surrogate markers, such as citH3 or cfDNA, are not exclusive to NETosis and may reflect other forms of cell death or inflammation. Future research should focus on large, prospective studies to clarify the prognostic value of NET‑related biomarkers in VTE and to determine whether therapeutic strategies targeting NETosis and promoting NETs degradation or inhibition of factors associated with NETosis can improve clinical outcomes.