Reflections on targeting neutrophil extracellular traps in deep vein thrombosis

The contribution of neutrophil extracellular traps (NETs) to pathological thrombus formation is one of the earliest identified negative consequences of NET release¹ since their first description in 2004.² A PubMed search for the term “Extracellular traps” yields nearly 6000 results as of late June 2024; of these, over 900 are related to thrombosis - highlighting the degree of investigation of this subject by the research community. In this Perspective article, we provide an update on developments in the last decade since our first review³ of this field, as well our thoughts on future directions related to NET targeting in the treatment and prevention of venous thromboembolism.

NETs in thrombosis: from animal models to human evidence

The scientific literature employing animal models to study deep vein thrombosis (DVT) has provided substantial insight into the contribution of NETs in thrombosis (Figure 1). This has led to a better understanding of the complex cell-cell communication environment in which neutrophils are imbedded, which includes mast cells^{4, 5} recruited to the site and promoting endothelial activation, platelets, monocytes producing tissue factor, red blood cells, and natural killer (NK) cells whose contribution remains to be explained.⁶ An important interaction of NETs and histones with von Willebrand factor (VWF) was established⁷ and observed in vivo,¹ first in a baboon model of DVT (Figure 2) indicating that VWF tethers NET strands and thus thrombus to the vessel wall. Indeed, mice lacking VWF are fully resistant to DVT in the inferior vena cava model.⁸ The crucial interplay between platelets and neutrophils, without which DVT is severely limited in animal models, will be discussed below.

Figure 1. Schematic representation of key molecular and cellular interactions in deep vein thrombosis (DVT) onset.

ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; ASC, apoptosis-associated speck like protein containing a caspase recruitment domain; DNase, deoxyribonuclease; GP1b, glycoprotein 1b; GSDMD, gasdermin D; H, histone; HMGB1, high mobility group box 1; Jak2, Janus-associated kinase 2; NE, neutrophil elastase; NETs, neutrophil extracellular traps; PAD4, peptidylarginine deiminase 4; PSGL1, P-selectin glycoprotein ligand 1; RBC, red blood cell; TF, tissue factor; TGFβ, transforming growth factor β; VWF, von Willebrand factor.

Figure 2. Cross-section of an occluded vessel showing von Willebrand factor (VWF) tethering the thrombus to the vessel wall through interactions with neutrophil extracellular traps (NETs).

Experimental deep vein thrombosis was induced by balloon catheterization of the iliac vein in a baboon model. DNA is stained in green (panels A and B) and VWF in red (panel B). The white dashed line depicts the vessel wall, and panel B highlights the tethering of NET DNA (diffuse green staining pattern) within the thrombus to the vessel wall by VWF. Image reprinted with adaptations from Fuchs et al., PNAS 2010¹

As methods to detect and quantify NETs have improved in specificity and sensitivity, including the quantification of citrullinated histones or complexes of myeloperoxidase or citrullinated histones with DNA, so has our understanding of the relevance of NETs in human venous thromboembolism (VTE) and other severe thrombotic conditions involving vascular occlusion.⁹ These conditions, including DVT, are driven by NETs and consequently by environments promoting NETosis involving sterile inflammation such as hypoxia, cancer, clonal hematopoiesis of indeterminate potential (CHIP), or by infection, which increases the incidence and severity of these occlusive events in humans. Being able to identify which patients would benefit most from NET-targeted therapeutics and the type of drug to be chosen remains an important task for the scientific/medical community and will be discussed next.

Targeting NETs in DVT

Clinical guidelines from the American Society of Hematology for VTE prevention, diagnosis, management, and secondary prevention received an update in 2020.¹⁰ Evident among these was the general shift from vitamin K antagonists toward direct thrombin inhibitors or factor Xa inhibitors. To date there are no studies measuring the impact of these oral anticoagulants on NETs, but it has been demonstrated that NET components can increase protein expression of coagulation factors upstream of factor X¹¹ and NETs were shown to promote contact pathway activation.¹² Future directions indicate promise for inhibition of factors XIa or XII.¹³ Although the result of factor XI or factor XII inhibition has not been tested, there is preclinical evidence that NETs provide a surface for intrinsic coagulation pathway activation¹³ including activation of factor XII.¹⁴ In targeting NETs, we must consider four strategies: preventing neutrophil recruitment and activation, inhibiting NET formation, dislodging NETs from the vessel wall, and degradation of NETs. The importance of these strategies will be explained, and currently-known drugs summarized.

Endothelial activation and platelet-neutrophil interactions

Hypoxia is an important activator of endothelium and an initiating factor for DVT It can be caused by disturbed blood flow behind a venous valve, poor blood circulation to extremities, or at high altitude.¹⁵ Similarly, local inflammation caused by the presence of activated mast cells or platelets results in endothelial activation and the important Weibel-Palade body (WPB) release of von Willebrand factor (VWF) and P-selectin. This recruits platelets and neutrophils (Figure 1), providing them with activating intracellular signaling. Secretion of WPBs can be inhibited by increasing NO (nitric oxide), thus reducing inflammation.¹⁶ Activated platelets are a well-established inducer of NETosis, and NETs in turn also promote further platelet recruitment and thrombosis.¹ Approaches that would block this vicious cycle by preventing the key adhesion interactions may therefore reduce NET-mediated thrombosis. Some endothelial adhesion molecule, such as E-selectin, are induced by the cytokine interleukin (IL-1β) produced by inflammasome activation. Hypoxia may activate inflammasome through the engagement of HIF-1α (hypoxia-inducible factor 1-alpha).¹⁷ Inhibition of P- and E-selectin¹⁸ would not only diminish neutrophil recruitment but also signaling via PSGL-1 (P-selectin glycoprotein ligand) including NF-κB (nuclear factor kappa-light-chain enhancer of activated B cells) activation. Recombinant ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) that cleaves and dislodges ultra-large VWF, and nanobodies against the A1 domain of VWF that block interactions with platelet Gp1b (glycoprotein 1b),¹⁹ neutrophil integrins, and that of histones on NETs. Conversely on the neutrophil side, PSGL-1 blockade reduces NET release by activated platelets,²⁰ and protects from DVT formation in animal models of antiphospholipid syndrome.^{21, 22}

Preventing NET release

The last decade of research into NET formation has also revealed several key intracellular processes that are important for NET release and may be viable drug targets. Peptidylarginine deiminase 4 (PAD4), a protein-citrullinating enzyme that citrullinates histones and likely other substrates implicated in NETosis, has been the subject of extensive preclinical research and development of multiple inhibitors.⁶ Recently an orally available PAD4 inhibitor was successfully tested in a mouse arthritis model²³ and could be possibly used for DVT prevention on a long-term basis. Insights have also been gained from studying myeloproliferative neoplasms where a constitutively active Jak2 (Janus kinase 2) mutant is linked to high thrombotic risk. The Jak2V617F mutation in neutrophils results in increased PAD4 expression and NET release. Jak2 inhibition may also be an avenue to explore for its NET-inhibitory effect.

Another contributor to venous thrombosis is the inflammasome, already mentioned for its generation of IL-1β. This was first demonstrated in mice deficient in CD39 (cluster of differentiation 39) that removes ATP, a prominent NLRP3 inflammasome activator and indeed, deficiency of CD39 is accompanied by dysregulated thrombosis, NETs, and IL-1β production.²⁴ The reduced NET generation can now be explained by the recent finding that NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome activation is upstream of NETosis.²⁵ Thus, NLRP3 inhibitors, such as MCC950, may have an interesting side-effect, of hindering NETosis. Indeed, the murine DVT model performed in NLRP3−/− mice results in lower density of NETs within thrombi.²⁵ PAD4-deficient neutrophils have reduced ASC speck (inflammasome activation marker) formation, showing that PAD4 activity and NLRP3 activity are tightly and reciprocally linked. Deficiency of PAD4 or NLRP3 prevents both nuclear and plasma membrane rupture, thus arresting NETosis at a very early stage. With the importance of the inflammasome in IL-1β activation and recent evidence of protection from thromboembolic events in the CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) trial (NCT01327846), this is a highly relevant direction to keep an eye on in the coming years with regard to endothelial activation and NET involvement (Figure 1). Caspase 1, the main enzyme product of NLRP3, is also implicated in NETosis.²⁵ It cleaves gasdermin D (GSDMD), a pore forming protein, linked to pyroptosis and NETosis. The role of GSDMD in NETosis became recently controversial, but we can confirm that GSDMD−/− neutrophils abort NETosis (unpublished observation, Wagner lab), and thus GSDMD inhibitors remain a possibility.²⁶ Caspase 1 and gasdermin D inhibitors also reduced thrombosis in the venous setting.²⁷ As we learn more about NET biology, other targets may also be revealed in the future.

NETs as thrombolytic targets

Inhibiting NET release is a therapeutic approach most relevant for reducing risk of VTE or in preventing secondary thrombotic events. In patients with ongoing or existing thrombi, NET targeting should shift to lysis or removal of NETs that will already be present in the thrombi. DNases (deoxyribonucleases) are enzymes endogenously present in circulation (DNase 1 and DNase 1L3) as well as in cells that are involved in NET clearance including macrophages (DNase 2). Defective DNase activity or inhibition by antibodies results in an exacerbation of NET-related pathologies including vascular occlusion and autoimmune diseases such as lupus and rheumatoid arthritis. DNase 1 has been an FDA (Food and Drug Administration)-approved medication in use for cystic fibrosis for decades as an inhaled therapeutic. Recent clinical trials are testing the safety and efficacy of recombinant DNases administered intravenously in sepsis and in stroke (NCT05453695, NCT05880524, NCT05203224). The recent approval of recombinant ADAMTS13 for use in congenital thrombocytopenic thrombocytopenic purpura (TTP) and the successful clinical trial just reported for immune TTP²⁸ may also pave the way for future application in broader thrombotic disease such as DVT and even post-thrombotic syndrome. Since NETs specifically interact with VWF, ADAMTS13 dislodges NETs from the vessel wall (Figure 2). ADAMTS13 treatment may not only reduce thrombotic burden but could also further prevent neutrophilic involvement as it has anti-inflammatory properties.²⁹ Finally, the role of PAD4 in thrombosis is not limited to NET release but it also promotes thrombosis through its citrullinating action on coagulation contributors such as tissue factor pathway inhibitor (resulting in loss of factor Xa inhibition),³⁰ SERPINS (serine protease inhibitors) (eliminating their inhibitory activity)³¹ and ADAMTS13 (rendering it inactive).³² In addition, citrullinated fibrin appears more resistant to fibrinolysis.³³ Pairing PAD4 inhibitors with NET-degrading approaches may thus be needed to promote optimal thrombolysis.

Looking towards the future

Recent clinical trials in DVT that focus on dosing and timing of approved VTE treatments highlight the need for improved treatment for both efficacy in VTE prevention and in terms of avoiding disastrous bleeding complications. The ANT-005 TKA (Total Knee Arthroplasty) Phase II trial showed great promise in VTE prevention with FXI (factor XI) inhibition (abelacimab)³⁴, and the LILAC-TIMI 76 Phase III trial results with this drug are eagerly anticipated.

Beyond generally long clinical development times, bringing novel NET-targeting treatments to the clinic is complicated by the need for specific targeting without having broader detrimental immune consequences, for example on neutrophil phagocytosis or ROS (reactive oxygen species)-mediated bacterial killing. Even repurposing of existing DNase 1, on the market since 1993 and previously tested for intravenous use in a phase 1b trial,³⁵ has only recently been started to be tested in clinical trials with the intention of addressing NET-mediated pathology in stroke (Improving Early Reperfusion With Adjuvant Dornase Alfa in Large Vessel Ischemic Stroke) (EXTEND-IA DNase, NCT05203224) and (Reduction of SystemiC Inflammation After Ischemic Stroke by Intravenous DNase Administration) (ReSCInD, NCT05880524) and in sepsis (Intravenous DNase I for the Treatment of Sepsis) (IDEALSepsis I, NCT05453695). From cystic fibrosis we learned that inhaled DNase 1(Pulmozyme) is safe in patients with high NET burden, and initial evidence demonstrated safety and efficacy from a randomized clinical trial on COVID19.³⁶

We have little doubt that targeting NETs will be a part of future DVT prevention and treatments. The question is when to treat and how. We still don’t know what the relative contribution of NETs to thrombus initiation and thrombus stability are. The likely answer is that they contribute to both. NETs generate local toxicity and possibly promote detrimental fibrotic remodeling in veins as post-thrombotic syndrome. DVT also has negative systemic effects on the patient, such as lung inflammation and cardiac problems. New evidence is accumulating that NETs impact this as well; therefore, NET removal could be beneficial. We no longer believe that destruction of NETs would make a person generally susceptible to infection, especially intravascular NETs. Experimental evidence in systemic infection models shows no impact on bacterial load and some protection from detrimental effects of NETs in PAD4−/− mice^{37, 38} or in mice receiving therapeutic DNase treatment.³⁹ On the contrary, NETs can form biofilms which harbor bacteria, and digestion of NETs improves antibiotic efficacy in infection.⁴⁰ However, we still wonder whether producing many small fragments of DNA with histones and citrullinated neoantigens could have unexpected consequences.

We need to determine whether production of NETs is needed throughout the life of a thrombus, i.e. would NET-inhibitory treatment be beneficial with a mature thrombus. We think having fewer NETs in thrombi and less citrullination may promote thrombus resolution. Reducing inflammation to the vessel wall may reduce incidence of post-thrombotic syndrome. NETs also promote fibrotic remodeling in chronic thromboembolic pulmonary hypertension, a deadly consequence of chronic VTE.⁴¹ Interestingly, DNase 1 promotes wound healing by degrading NETs, and which may help healing after thrombus removal or dissolution.⁴² NET targeting also prevented perivascular collagen deposition in a mouse model of pressure-overload cardiac injury,⁴³ so there could be additional benefit in fibrotic remodeling. Reduction of neutrophil-mediated cardiac fibrosis was also evident with PAD4 pharmacological targeting.⁴⁴

Of note, the approaches we have highlighted here should be evaluated for potential off-target effects. PAD4, although enriched in neutrophils, is present in other blood cells, as are the NLRP3 inflammasome and GSDMD. Neutrophil-specific drug delivery is not yet possible despite promising ongoing work to specifically target activated neutrophils including by, for example, CD177-recognizing nanoparticles.⁴⁵ PAD4-deficiency, although very sustainable in mice and a plus in murine aging,⁴³ must have some side effects. Recently it was determined that over 4000 proteins are citrullinated in activated neutrophil-like human cells. Most of them reside in the nucleus such as histones and nuclear lamins;⁴⁶ their citrullination likely supports NETosis. There are a lot of molecular and cellular relationships that we still need to understand, but substantial progress towards discovery of new targets to treat DVT has been made.