Towards Targeting of Inflammasome Signaling in Venous Thrombosis

Abstract

Deep vein thrombosis (DVT) is a serious and debilitating vascular condition with sequelae that include post-thrombotic syndrome and pulmonary embolism. Current treatments for DVT are limited by their contraindications and sub-optimal safety profiles. Pharmacological approaches that safely and effectively reduce thrombus formation are being investigated in laboratory and clinical settings, including agents that aim to inhibit the interplay between thrombosis and inflammation. Recent studies have shown that inflammasome complexes-- multiprotein inflammatory complexes activated by pathogen-associated molecular patterns and damage-associated molecular patterns-- play a role in the development and propagation of DVT. Thus, inflammasome complexes have attracted considerable interest as possible therapeutic targets for the treatment of DVT. This review summarizes the current knowledge on inflammasome complexes and discusses studies on their role and therapeutic potential in DVT. An improved understanding of inflammasome-dependent regulation of DVT could lead to the development of safe and effective treatments for patients affected by this condition.

1. Introduction

Deep vein thrombosis (DVT) is a common condition with potentially fatal consequences, especially if it is not identified and treated at an early stage [1]. Consequences of DVT include post-thrombotic syndrome and life- threatening pulmonary embolism [2]. DVT is the third leading cause of cardiovascular-related death and disability after myocardial infarction and stroke [3]. The incidence of DVT is less than 1 per 100,000 per year in childhood, but increases with age to 6–8 per 1,000 per year in those aged 80 years or more [4]. Unfortunately, current treatments for DVT (anti-coagulation, thrombolysis, and thrombectomy) are contraindicated in some patients and anti-coagulation increases bleeding risk by up to 7-fold, and the risk of fatal bleeding by 1.3-fold [5, 6]. Other limitations of current DVT treatments include failure to promote natural thrombus resolution and failure to prevent thrombus recurrence.

Venous thrombosis is induced by blood coagulation, blood stasis, and endothelial injury, and often originates in the valve pockets of large veins [7]. Leukocytes, platelets, tissue factor (TF)-positive microvesicles, and neutrophil extracellular traps (NETs) play major roles in venous thrombosis [7]. The development of novel treatments for DVT relies on a thorough understanding of the regulatory drivers of DVT. One such regulatory driver is inflammasome signaling [8]. Novel therapeutic strategies inhibiting inflammasome-dependent thrombosis could address the limitations of current treatments by inhibiting or reversing thrombosis without increasing the risk of bleeding.

Elevated levels of inflammasome complexes, particularly the NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome, play a crucial role in the development and propagation of DVT [8–10]. Inflammasomes are multiprotein complexes of the innate immune system that regulate cell death and inflammation. They are formed in the cytoplasm of immune cells as a result of pattern recognition receptors sensing pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs) from the host cell, or homeostatic disruptions [11]. In this review, we describe the structure and activation of the inflammasome complexes and the regulation of DVT by the NLRP1, NLRP3, and absent in melanoma 2 (AIM2) inflammasomes. We also suggest topics of future research in inflammasome-dependent regulation of venous thrombosis.

2. Structure of the Inflammasome Complexes

2.1. The nucleotide-binding oligomerization domain-like receptor (NLR) family

All NLR proteins are characterized by a sensor domain at the C-terminal, the leucine-rich repeat (LRR), and by a central NACHT domain that plays a critical role in inflammasome activation [11]. Depending on whether the N-terminal effector domain is a pyrin domain (PYD) or a caspase activation and recruitment domain (CARD) domain, the NLR family is divided into two subfamilies, known as the NLRP and NLRC inflammasomes [11]. The NLRP family comprises NLRP1, NLRP3, NLRP6, NLRP9b, NLRP12, and the NLRC family includes NLRC3, NLRC4, and NLRC5 [11].

2.1.1. NLRP inflammasomes.

Differently from other NLRP inflammasomes, NLRP1 presents two extra domains at the C-terminal: a function-to-find domain (FIIND) followed by a CARD domain [12]. Pro-caspase-1 is a co-effector protein of NLRP1 [12]. Procaspase-1 is recruited via the formation of a CARD-CARD domain complex between the ASC and procaspase-1 [12]. Later, it undergoes autocleavage to become active caspase-1 [12].

The NLRP3 complex assembles via the homotypic interaction of the ASC protein, between the PYD domain of NLRP3 and the PYD domain of ASC [13]. The main function of the LRR domain is to regulate the stability of protein-protein interactions during inflammasome assembly. The LRR domain interacts with another adopter protein of NLRP3 known as NIMA-related kinase 7 (NEK7). Oligomerized NLRP3 recruits ASC and activate caspase-1, leading to the cleavage of pro-inflammatory cytokines (such as IL-1β and IL-18) and triggering of programmed cell death [14]. The functional and structural characteristics of the NLRP family members are summarized in Table 1.

Table 1:

Characteristics of the NLRP family members

NLRP	Unique Domains	Activation Mechanism	Ref
NLRP1	FIIND, CARD	Proteolytic cleavage by pathogen components	[95]
NLRP3	Broad LRR	Potassium efflux, ROS, lysosomal damage	[96]
NLRP6	Similar to NLRP3	Gut microbiota signals	[97]
NLRP9b	Similar to NLRP3	Pathogen components, RNA viruses	[98]
NLRP12	Similar to NLRP3	Inhibition of NF-κB signaling	[99]

2.1.2. NLRC inflammasomes.

NLRCs are characterized by the presence of a CARD effector domain at the N-terminal. In addition to the typical domains, NLRC4 has two helical domains (HDs), HD1 and HD2. These domains provide structural stability to the NACHT domain and assist in maintaining the protein’s conformation [15]. The CARD domain is located at the N-terminus and it acts as a scaffolding element that promotes the assembly of the NLRC4 inflammasome complex by linking caspase-1 CARD domain to the NLRC4 structure [16].

2.2. Absent in melanoma 2 (AIM-2)

AIM-2 contains two domains known as the HIN200 domain and the PYD domain. Under normal conditions, AIM-2 is inactive, with the PYD domain bound to the HIN200 domain [17]. The interaction between AIM2 and DNA is dependent on the HIN200 domain localized at the C-terminal and independent of the DNA sequence [17]. AIM2 belongs to the PYHIN family and contains an N-terminal PYD (1–87) and a C-terminal HIN domain (138–343) connected through a long linker [17]. The stimulators and structural characteristics of the major inflammasome complexes are shown in Figure 1.

Figure 1: Structure of the major inflammasome complex proteins.

Stimulators of inflammasome complexes include lethal toxin, PAMPs, DAMPs, lipoteichoic acid, flagellin, and double-stranded DNA (dsDNA). The major inflammasome family members are NLRP1, NLRP3, NLRP6/9/12, NLRP4-NAIP, and AIM2. For abbreviations see text.

3. Activation of the Inflammasome Complexes

3.1. NLRP1 activation

NLRP1 is activated by direct triggers such as anthrax lethal toxin, which directly targets NLRP1 by cleaving its N-terminal region, leading to the release and activation of its C-terminal domain [18]. This process enhances the formation of the NLRP1-caspase-1 complex. Subsequently, the NLRP1-ASC complex assembles with pro-caspase-1 through the interaction of the ASC-CARD domain with the CARD domain of pro-caspase-1, which converts pro-caspase-1 into its active form [19]. The active form of caspase-1 then cleaves pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18 [19].

3.2. NLRP3 activation

Canonical activation of the NLRP3 inflammasome occurs via two steps. In the first step, priming signal activation of nuclear factor κB (NF-κB) enhances the level of NLRP3 and inflammasome-related cytokines [20]. In the second signal, NLRP3 is triggered by PAMPs or DAMPs [20] through toll-like receptors (e.g., TLR4) or cytokine receptors (e.g., tumor necrosis factor-α receptors, TNF-α receptors), which initiate downstream signaling [20].

Non-canonical NLRP3 activation, marked by caspase 11 activation, leads to pyroptosis [21]. This pathway is activated by gram negative bacteria components, particularly lipopolysaccharide (LPS), through the release of caspase-1 and caspase-11 in mice, and caspase-4 and -5 in humans [21], which cleave and activate their substrate, gasdermin D (GSDMD). Cleaved GSDMD enhances the expression of NLRP3 and increases cytokine release [22]. Canonical and non-canonical NLRP3 pathways are shown in Figure 2. Key similarities in canonical and non-canonical NLRP3 activation include the release of interleukin (IL)-1β and IL-18 [20, 23], while key differences include the stimuli and effector molecules. The relative contributions of canonical versus non-canonical activation of NLRP3 in venous thrombosis, however, require further investigation.

Figure 2: Mechanisms of NLRP3 inflammasome activation.

NLRP3 is activated by endogenous DAMPs and PAMPs. Ion flux (K+ efflux, Ca2+ influx) imbalances affect mitochondria, lysosomes, and Golgi, which release mediators such as mitochondrial ROS, cathepsin B and ATP; this triggers canonical NLRP3 inflammasome activation. NEK7 promotes the oligomerization of the NLRP3 inflammasome complex, forming NLRP3, ASC, and pro-caspase-1. Activated caspase-1 induces the inflammatory form of programmed cell death, known as pyroptosis, and cleaves the precursor cytokines pro-IL-1β and pro-IL-18, generating biologically active cytokines, IL-1β and IL-18. Non-canonical NLRP3 inflammasome activation is triggered by cytosolic LPS sensed by caspase 4/5/1, which cleave their substrate, GSDMD, resulting in the formation of GSDMD membrane pores. This causes potassium efflux, which promotes activation of the NLRP3 inflammasome. LPS also triggers NLRP3 activation through TRIF, RIPK1, FADD, and caspase-8, leading to inflammasome activation independent of pyroptosis. The release of pro-inflammatory cytokines enhance thrombin generation, platelet aggregation, and fibrin clot formation. For abbreviations see text.

3.3. Activation of the AIM2 inflammasome

Upon stimulation of double stranded DNA from viral and bacterial infections, the PYD domain of AIM-2 detaches from the HIN-200 domain and interacts with the PYD domain of ASC, leading to AIM-2 complex oligomerization and caspase-1 activation [24]. This enhances the release of IL-1β and IL-18 and facilitates pore formation-mediated inflammatory cell death [24].

4. Roles of the Inflammasomes in DVT

4.1. Contributions of NLRP3 to venous thrombosis

4.1.1. Canonical and noncanonical NLRP3 activation in venous thrombosis.

Canonical NLRP3 activation by LPS enhances TF release and pyroptosis in endothelial cells by disrupting the cytoskeleton [25]. TF binds with Factor VIIa, culminating in fibrin formation [25]. GSDMD deficiency suppresses TF release without altering IL-1β and IL-18 receptor signaling following caspase-11-induced activation [25]. Treatment with resveratrol reduces the level of prothrombin fragments, IL-1β, caspase-1, TF, and NLRP3 in thrombosed mice [26]. Thrombotic burden depends on the level of NLRP3 activation in a HIF-1α-dependent manner [11]. Following inflammasome activation, TF expression is increased, triggering caspase-1-dependent coagulation, which is prevented by GSDMD or caspase-1 deficiency [25]. In response to hypoxia, NLRP3 contributes to DVT by promoting caspase-1 activation and release of IL-1β in a HIF-1α-dependent manner [11]. NLRP3, caspase-1, and IL-1β expression are increased in humans with altitude-induced venous thrombosis [11], highlighting the clinical relevance of this pathway in the pathogenesis of venous thrombosis. Future studies could attempt to elucidate the environmental and genetic factors that predispose to venous thrombosis via NLRP3.

Alternatively, noncanonical NLRP3 activation has also been implicated in venous thrombosis. In a mouse model of EprJ-induced thrombosis, both canonical (caspase-1) and noncanonical (caspase-11) pathways stimulated the release of TF-rich extracellular vesicles by macrophages and promoted coagulation, while deletion of GSDMD prevented coagulation [25]. Caspase-1, caspase-11, and GSDMD are downstream mediators of transmembrane protein 173, which drives pyroptosis and TF release [27]. LPS-induced TF activity and thrombin-antithrombin complex formation are reduced in TLR4-, caspase 11- and NLRP3-deficient mice [28]. In contrast, deficiency of caspase-1 and GSDMD, but not caspase-11, inhibited thrombosis in a mouse DVT model [29]. Although evidence in humans is limited, NLRP3/caspase-1 and caspase-4/5 contribute to inflammation in patients with Kawasaki Disease, a condition with increased risk of venous thrombosis [30]. Venous thrombosis is characterized by fibrin deposition, NET formation, TF expression, and NLRP3 activation through NF-κB [31], but the relative contributions of canonical versus non-canonical NLRP3 activation to venous thrombosis remain to be determined.

4.1.2. Oxidative stress and NLRP3 activation in venous thrombosis.

In venous stasis, hypoxia leads to ROS generation and NLRP3 activation [11]. Conversely, NLRP3-driven inflammation is associated with inflammatory cell recruitment and increased ROS, establishing a positive feedback loop between oxidative stress and NLRP3 [29]. The oxidative stress regulator, thioredoxin-interacting protein (TXNIP), also participates in thrombosis [32]. TXNIP interacts with the NLRP3 inflammasome complex to increase the level of IL-18 and IL-1β and thrombus formation [32]. Conversely, inhibition of TXNIP attenuates DVT formation, possibly via NLRP3 deactivation [33]. However, direct evidence linking the TXNIP/NLRP3 axis to DVT is lacking. Future studies could aim to uncover redox-sensitive mechanisms of thrombus formation that represent novel targets for antioxidant or inflammasome-based DVT therapies.

4.1.3. Cell-specific contributions of NLRP3 in venous thrombosis.

Neutrophils, a major source of NLRP3, release IL-1β and IL-18, resulting in thrombo-inflammatory disorders, while NLRP3 deficiency reduces NET density in thrombi [34]. NLRP3 participates in NET formation in a peptidyl arginine deiminase 4 (PAD4)-dependent manner, and genetic ablation of NLRP3 impaired NET formation in venous thrombi [34]. These findings suggest that NLRP3 acts with PAD4 to regulate NETosis. PAD4 also modulates NLRP3 protein levels [34], which could regulate neutrophil activation during DVT. While NLRP3 activation can drive NETosis, it is also important to consider the bidirectional relationship between NETosis and NLRP3, given that NETs activate the NLRP3 canonical pathway, triggering pyroptosis in neutrophils, monocytes, and platelets, thus forming a positive feedback loop that potentiates thrombosis [35].

Stimulation and polarization of macrophages plays an important role in the development of venous thromboembolism through NF-κB [36]. Thus, NLRP3/IL-1β/NF-κB signaling could be a target for treatments that aim to suppress DVT [36]. IL-1β is a pro-inflammatory cytokine produced as a byproduct of inflammasome activation and plays a critical role in DVT. Stimulation of endothelial cells and monocytes by LPS enhances TF expression on endothelial cells and thrombin generation and fibrin formation, which is mediated by IL-1β [37]. In cancer-associated DVT, activated peripheral blood mononuclear cells release IL-1β via NF-κB signaling [38].

Unlike immune cells that rely on pathogen recognition receptors and cytokine priming for inflammasome activation, platelets can activate NLRP3 without classical priming [39]. Stimulation with thrombin and collagen leads to upregulation of NLRP3, ASC, Bruton’s tyrosine kinase (BTK, an important mediator of NLRP3), and caspase-1, and induces the release of IL-1β and IL-18 [40]. Conversely, NLRP3-dependent effects on thrombosis may be regulated by IL-1β from activated platelets, potentially establishing a priming loop involving IL-1 receptors and platelets [41]. There is evidence of NLRP3-dependent and -independent release of IL-1β from platelets [42–44], e.g., human platelets release IL-1β independently of NLRP3 [45]. Pyroptosis could also play a role in thrombosis in a manner that is dependent or independent of NLRP3 and IL-1β [46, 47].

Platelet NLRP3 may affect thrombus formation through caspase-1-dependent generation of thrombo-inflammatory microparticles [48]. NLRP3 not only contributes to platelet activation, aggregation, and thrombus formation, but is upregulated in activated platelets via BTK [49]. Pharmacological inhibition or genetic ablation of BTK in platelets led to decreased platelet activation, aggregation, and thrombus formation [49]. Thus, the NLRP3-BTK signaling axis in platelets represents a therapeutic target for thrombo-inflammatory diseases. However, BTK inhibition is associated with increased bleeding risk [50], so these strategies should be carefully considered. NLRP3 modulates the downstream signaling triggered by αIIbβ3 through mediation of the inflammatory responses associated with platelet activation [43]. A selective inhibitor of NLRP3, CY-09, reduces clot formation in human platelets [51]. The addition of recombinant human IL-1β restored clot retraction in CY-09-treated platelets, suggesting that NLRP3 plays a role in regulating αIIbβ3 signaling in platelets.

4.2. Contribution of other inflammasome complexes to venous thrombosis

While NLRP3 is the most thoroughly characterized inflammasome in DVT, NLRP1 and AIM2 also seem to be involved. As mentioned above, NETs are implicated in DVT pathogenesis [31]. The inflammasome proteins NLRP1 and AIM-2 are elevated in NETs from human thrombi samples, and their presence correlates with enhanced release of IL-18 and IL-1β and poor outcomes following stoke [52]. Systemic lupus erythematosus is a chronic autoimmune disease that leads to a hypercoagulable state. NLRP1 polymorphism and IL-1β expression are associated with the progression of this disease [53]. The NLRP1 inflammasome contributes to inflammation following thromboembolic stroke, releasing IL-1β and IL-18, and activating ASC and caspase-1; NLRP1 proteins are also present in cerebral stroke thrombi and localize with NETs that release IL-1β [52]. The NET-dependent release of prothrombotic factors by inflammasome activation should be investigated.

5. Targeting Inflammasome Complexes in DVT

Therapies that target the NLRP3 pathway to mitigate thrombosis are summarized in Table 2 and Figure 3. The anti-inflammatory drug, colchicine, is a non-selective NLRP3 inhibitor that prevents adverse cardiovascular outcomes in patients with coronary artery disease [54, 55]. The effect of colchicine on venous thrombosis, however, is inconclusive. In preclinical studies, colchicine administration in mice models of venous thrombosis decreased thrombus size [56]. A clinical case report found that colchicine was effective in treating inflammation-induced thrombosis in a patient with Behçet disease [57]. The CANTOS and COLCOT clinical trials have shown an anti-ischemic effect of colchicine, which could be partly explained by reduced thrombotic events [54, 55]. Conversely, a systematic review and meta-analysis concluded that colchicine inhibits endothelial adhesion and platelet aggregation without inhibiting DVT [58]. Currently, the Conquer-DVT phase 3 trial (NCT06440694) is investigating whether colchicine reduces the risk of post-thrombotic syndrome after DVT.

Table 2:

Inflammasome pathway-targeted therapies as emerging treatment strategies for venous thrombosis

Agent	Target	Effect on Pathway	Effect on Thrombosis	Ref
Colchicine (nonselective)	NLRP3	Inhibits NLRP3 inflammasome assembly	Reduces thrombogenesis and inflammation	[56]
PAD4 inhibitor (nonselective)	NLRP3	Inhibits NLRP3 inflammasome assembly	Reduces NET formation and thrombogenesis	[34]
DNAse I (nonselective)	NLRP3	Inhibits NLRP3 activation	Reduces thrombin generation and venous thrombosis	[100]
MCC950 (selective)	NLRP3	Decreases the expression of TXNIP, NLRP3, and IL-1β	Reduces thrombosis, NET formation, and inflammation	[33]
Canakinumab, Anakinra (selective)	IL-1β	Neutralizes IL-1β or blocks its receptor	Reduces thrombosis and inflammation and improves outcomes in COVID-19 and cancer models	[61]
Disulfiram (selective)	GSDMD	Inhibits GSDMD pore formation	Potential for reducing thrombotic microangiopathy	[82]
4-Sulfonic calixarenes (selective)	AIM2	Inhibits AIM2 assembly	No direct effects reported	[87]
ac-YVAD-cmk (selective)	Caspase-1	Inhibits inflammasome complex activation, IL-1β secretion, and expression of caspase-1 and NETs	Reduces thrombosis	[41]
vx-765 (selective)	Caspase-1	Decreases expression of caspase-1 and production of cleaved GSDMD and IL-1β and IL-18	Reduces thrombosis	[84]

Figure 3: Therapeutic role of inflammasome inhibitors in DVT.

Upon inflammasome activation, NETs are released by neutrophils, promoting platelet aggregation and thrombus formation. A variety of pharmacological agents reduce thrombosis by modulating different components of the inflammasome pathway. Decreased NLRP3 assembly was observed via treatment with Colchicine, CL-amidine and MCC950. Disulfiram was shown to inhibit GSDMD pore formation and prevent the release of pro-inflammatory cytokines. Canakinumab and Anakinra were observed to inhibit IL-1β. Decreased NET formation, platelet activation, and thrombosis have been achieved through treatment with the inflammasome complex inhibitors, MCC950, AC-YVAD-CMK, and Vx765. Thrombosis is also reduced in mutant mice deficient in the inflammasome-related genes, caspase-1 and NLRP3. For abbreviations see text.

Cl-Amidine inhibits PAD4 activity and reduces inflammasome assembly, highlighting the potential therapeutic relevance of PAD4 inhibitors, especially in neutrophils [34]. As mentioned above, NETs can activate NLRP3. Therefore, pharmacological tools to reduce IL-6-dependent NETosis independently of NLRP3 could provide therapeutic value. IL-6/IL-6R inhibitors are also under investigation in clinical trials, including Siltuximab, Ziltivekimab, Tocilizumab, and Sarilumab . Interestingly, Tocilizumab treatment reduces the circulating levels of NET markers [59] and NETosis [60].

Canakinumab, Anakinra, and Rilonacept are selective IL-1β inhibitors. Although IL-1 blockade can reduce thrombus weight in animal models [61], and Anakinra reduces inflammation in Kawasaki disease and COVID-19 [62], the impact of selective IL-1β inhibitors on clinical thrombosis is unclear. The impact of NLRP3 and IL-1β inhibitors on venous thrombosis should be studied in large scale clinical trials.

Disulfiram, an inhibitor of aldehyde dehydrogenase, is a drug to treat chronic alcohol addiction that was identified as an inhibitor of GSDMD pore formation, preventing the release of pro-inflammatory cytokines in canonical and noncanonical pyroptosis [63]. One study identified GSDMD as a critical mediator of thrombotic microangiopathy via neutrophil pyroptosis and promotion of immune-thrombosis in a mouse model of focal crystalline thrombotic microangiopathy; Disulfiram treatment inhibited clot formation [64], highlighting its promise as a therapeutic agent against inflammatory thrombotic disorders.

Several other small molecules, including MCC950, Ac-YVAD-CMK, and VX-765, target NLRP3 signaling and alleviate thrombotic disorders. MCC950 inhibits NLRP3 activation, while Ac-YVAD-CMK and VX-765 block caspase-1 activity, thereby reducing downstream inflammatory responses that contribute to thrombosis. Treatment with si-TXNIP or MCC950 inhibits thrombosis [33]. Treatment of neutrophils with MCC950 reduces the release of the DAMP molecule, HMGB1, while upregulation of HMGB1 is associated with increased pyroptosis and elevated levels of IL-1β [65]. Treatment with the caspase-1 inhibitor, ac-YVAD-cmk, suppresses thrombus size and platelet activation in a mouse model of DVT [31]. Similarly, the caspase-1 inhibitor, vx-765, reduces thrombus burden, expression of IL-1β and IL-18, and pyroptosis [66].

NLRP3/IL-1-targeted agents are in clinical development. A phase 1b study reported the safety of Selnoflast in inhibiting NLRP3 in moderate to severe ulcerative colitis [67]. DFV890 is an NLRP3 inhibitor that prevents caspase-1 and subsequent activation of IL-1β [68]. Rilonacept and Goflikicept inhibit IL-1α and IL-1β and have been studied for the treatment of pericarditis [69, 70]. Given the involvement of the NLRP3/IL-1 axis in venous thrombosis, drugs that inhibit this pathway represent promising therapeutic strategies for the treatment of DVT.

As mentioned above, AIM2 activation can contribute to DVT, but there is a lack of pharmacological tools to target the AIM2 inflammasome. One study recently discovered 4-sulfonic calixarenes as potent AIM2 inhibitors that prevent caspase-1, IL-1β, and GSDMD activation [71]. They also observed that Suramin, a drug used for the treatment of trypanosomiasis, is an AIM2 inhibitor [71]. Suramin reduces the prothrombotic activity of colorectal cancer cells by decreasing thrombin generation [72]. These data highlight the importance of studying AIM2-mediated mechanisms of venous thrombosis.

6. Future Perspectives on Inflammasome-Targeted Therapies in DVT

Preclinical studies have established a role for NLRP3 in venous thrombosis and the prognostic potential of inflammatory biomarkers, including the NLRP3 inflammasome and inflammasome-derived cytokines, in thrombotic cardiovascular diseases [73, 74]. Given that NLRP3 promotes venous thrombosis, future studies could aim to target this inflammasome for the inhibition or treatment of DVT. Studies should aim towards selective and repurposed therapies that inhibit inflammasome-driven thrombosis, including but not limited to NLRP3/IL-1 inhibitors [75]. It is foreseeable that any inflammasome-targeted therapies that inhibit venous thromboembolism would also reduce the incidence or severity of post thrombotic complications including chronic thromboembolic pulmonary hypertension and post-thrombotic syndrome, although this remains to be proven. It will also be important to consider that NLRP3 can be activated in a canonical and non-canonical manner. In other words, agents that inhibit caspase-1-mediated activation of NLRP3 may not interfere with non-canonical NLRP3 activation.

While the role of NLRP3 in venous thrombosis has been investigated, research on the other types of inflammasome in venous thrombosis is limited. Thus, it remains unclear which other inflammasomes regulate venous thrombosis, and to what extent, as well as the extent to which canonical versus non-canonical NLRP3 signaling controls DVT. Activation of NLRP3 promotes thrombus formation via platelet recruitment and aggregation, fibrin deposition, and NET formation [31]. However, the contributions of NLRP3 to thrombus propagation and resolution are also important considerations, given that NLRP3 amplifies leukocyte-endothelial activation and inhibits fibrinolysis via plasminogen activator inhibitor-1 (PAI-1) [76]. Future studies should aim to investigate the contributions of each inflammasome to thrombus formation, propagation, and resolution. These studies could provide critical insight into the potential therapeutic windows of opportunity for inflammasome-targeted therapies. Studies are also needed to identify whether and how any of the inflammasome complexes inhibit venous thrombosis.

Future studies could aim to assess the efficacy of NLRP3 inhibitors in reducing DVT; identify new therapeutic targets involved in NLRP3-dependent DVT; and characterize any inhibitory impact of anti-coagulants on inflammasome signaling. For example, heparin and rivaroxaban could dampen inflammasome signaling. Preclinical studies showed that rivaroxaban reduces NLRP3–caspase-1 signaling and IL-1β in diabetic mice, which suppressed pyroptosis; meanwhile, heparin prevented caspase-11-dependent immune response in a sepsis model, suggesting that anti-coagulants impact upon inflammasome-related signaling [77, 78].

As described above, several FDA-approved drugs inhibit the NLRP3 pathway. Unfortunately, owing to the ubiquity of inflammasomes and their role in homeostasis, even highly specific inflammasome inhibitors could be limited by off-target effects. To address this issue, the field has focused on multifunctional nanoparticles as drug carriers that can target diseased tissue with inflammasome activity and then release a therapeutic payload. An important consideration in the development of these nanomedicines for inflammasome modulation is that nanoparticle uptake can result in inflammasome activation [79]. However, drug delivery without aberrant inflammasome activation is possible and nanoparticles have been used to deliver MCC 950, disulfiram, and NLRP3/AIM2-IN-3 in sepsis and psoriasis models [80–83]. To our knowledge, nanoparticle delivery of inflammasome inhibitors for the treatment of DVT has not been reported. On the other hand, thrombus-targeted nanoparticles for the delivery of thrombolytics is an area of high activity [84, 85]. Thrombus-targeting nanoparticles can decrease the dose of thrombolytics needed by increasing drug delivery to the thrombus thereby decreasing the incidence of side effects such as uncontrolled bleeding. Delivery of inflammasome inhibitors in thrombus-targeted nanoparticles may represent a safe and effective therapeutic strategy for DVT.

Recent studies indicate that TLR9 participates in inflammasome pathway activation by promoting NLRP3 assembly and caspase-1 activation, resulting in the maturation of IL-1β and IL-18 [86, 87]. TLR9 antagonists such as ODN 2088 have been used to block TLR9 activity in animal models and human cells, but there’s a lack of studies investigating the role and targeting of the TLR9-NLRP3 pathway in venous thrombosis.

In platelets, the cyclic GMP/AMP synthase-stimulator of interferon genes (cGAS-STING) pathway promotes platelet activation and NETosis, therefore exacerbating sepsis-induced thrombosis [88]. The cGAS-STING pathway has also been shown to interact with AIM2 and NLRP3, leading to canonical and non-canonical inflammasome activation [89]. Future studies could aim to test inhibitors of the cGAS-STING pathway for the treatment of DVT.

In contrast to classic forms of cell death, PANoptosis is coordinated by protein complexes called PANoptosomes . These complexes activate pyroptosis, apoptosis, necroptosis, and inflammation, via molecules such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and caspase-8 [90]. The NLRP3 inflammasome has also been identified as an integral component of multiple PANoptosomes [91], but there are no studies to date that assess the role of PANoptosis in venous thrombosis. However, RIPK1 inhibition reduces NET-driven thrombosis [92], while RIPK3 accumulates within murine thrombus [93]. Improved understanding of the role of PANoptosis in venous thrombogenesis could lead to the identification of new targets for novel DVT treatments.

Organ-on-chips, also referred as microphysiological systems, are contemporary experimental models that have recently been shown to effectively recapitulate the onset of thrombosis in arteries, veins, and small capillaries [94] . A major advantage of these tools is that they offer an approach to assemble molecules and cells within their physiologically relevant mechanical and biochemical microenvironment. A second advantage is that organ-chip technology enables inclusion of primary human cells and blood obtained from patients. In future studies of the inflammasomes and their targeting in venous thrombosis, human vein-chips may serve as a valuable tool.

7. Conclusions

The inflammasome complexes regulate venous thrombosis, with the pro-thrombotic role of NLRP3 signaling being the most widely studied. The roles and potential therapeutic targeting of non-NLRP3 inflammasomes are under-studied in DVT. While the inflammasome complexes represent putative targets for treatments that aim to inhibit venous thrombosis, further studies are required to identify the contributions of each inflammasome and pathway to venous thrombus formation and propagation. The benefits and potential side effects of inflammasome-targeted treatments that aim to reduce venous thrombosis should be carefully considered.