Recent Pathophysiological Insights Are Advancing the Treatment of Venous Thromboembolism

Central Illustration

Highlights

• •Therapeutic treatment for VTE with currently available anticoagulants and fibrinolytic agents has limitations such as incomplete resolution, disease recurrence, and bleeding.
• •We discuss the mechanisms of acute-to-chronic transitioning of venous thrombosis, their translational insights, and the latest clinical developments in pharmacotherapeutic approaches.
• •No effective therapeutics are available to resolve chronic thrombi; clinical evaluation of anti-inflammatory agents and vasodilators continues.
• •Future research should focus on venous thrombus remodeling for novel drug discoveries that support thrombus age and composition-based treatment approaches.

Summary

Venous thromboembolism, defined as deep vein thrombosis and pulmonary embolism, is the third leading cause of cardiovascular deaths globally. Long-term complications of unresolved venous thrombi include post-thrombotic syndrome in the legs and chronic thromboembolic pulmonary hypertension. As the venous thrombus ages, the acute, fibrin, and red blood cell-rich composition changes to a chronic cellular, fibrotic mass that does not respond to presently available therapeutic approaches. Standard anticoagulation treatment does not fully prevent recurrent thrombosis and may cause serious bleeding. Thrombolytic therapy may resolve thrombi but it has unacceptable bleeding risks. Recent drug discovery for acute venous thromboembolism has focused on novel targets that may provide enhanced safety and efficacy. Additional therapeutic strategies have focused on the transition phase of acute-to-chronic venous thromboembolism with anti-inflammatory agents, statins, and vasodilator drugs. In this review, we discuss the mechanisms of venous thrombus aging, its clinical implications, and the latest developments in pharmacotherapeutic approaches for venous thromboembolism.

Venous thromboembolism (VTE) collectively refers to deep vein thrombosis (DVT) and pulmonary embolism (PE), and their respective chronic complications including post-thrombotic syndrome (PTS) and chronic thromboembolic pulmonary hypertension (CTEPH).1, 2, 3, 4 DVT occurs when the blood clots are formed in the deep veins of the legs.² Acute DVT is characterized by pain, swelling, warmth, and tenderness usually in one leg, or may be asymptomatic until it becomes chronic. Rarely, the formation of large venous blood clots may lead to “phlegmasia cerula dolens” with marked pain, swelling, and cyanosis developing into gangrene with potential limb amputation and life-threatening complications.⁵ If these clots detach from the vascular wall and travel to the lungs, they may cause life-threatening complications of PE such as right ventricular dysfunction, cardiogenic shock, and death. Undissolved chronic thrombi in the lungs may lead to vascular scarring, persistent pulmonary hypertension, right ventricular failure, and CTEPH or postpulmonary embolism impairment.^1,2,4 In the legs, undissolved venous thrombi undergo inflammatory and remodeling processes over time that cause permanent vascular changes, with vein thickening and fibrosis.³ The persistence of venous thrombi (incomplete resolution) and/or valvular reflux caused by inflammation-driven vein wall injury may lead to PTS, a debilitating chronic health condition characterized by discomfort, swelling, ulcers, and redness.³

VTE continues to affect 1 to 2 individuals per thousand person-years globally and is the third leading cause of cardiovascular deaths.⁶ Acute venous thrombi, also known as “red thrombi,” are rich in fibrin and red blood cells (RBCs). The initiation and growth phase of thrombi involves RBCs, fibrin, platelets, and neutrophils/monocytes; this is succeeded by matrix-producing and inflammatory cells such as fibroblasts, smooth muscle cells, macrophages, and endothelial cells.⁷ Fibrin is an important component of acute venous thrombi and thus is the primary choice for drug targeting and treatment approaches in VTE. Anticoagulation prevents the formation of fibrin, and plasminogen activator-based thrombolytic therapy is designed for fibrin degradation and clot dissolution through plasmin. Despite their continued and significant contributions to patient care over the years, these approaches have inherent limitations. Current standard-of-care treatment with anticoagulants, including direct oral anticoagulants (DOACs), carries the risk of bleeding, VTE reoccurrence, and chronic complications.⁸ Thrombolytic therapy is restricted to a subset of carefully selected VTE patients because of unacceptable bleeding risks.^9,10

Significant achievements have been made recently in VTE research and drug discovery and development. The most notable advancements in therapeutic approaches include new anticoagulants targeting the intrinsic pathway of coagulation and thrombolytic agents targeting fibrinolytic inhibitors. The primary goals of these approaches are to safely relieve the immediate life-threatening risk of acute VTE and to prevent the longer-term complications of chronic VTE. Although no effective therapeutics are available for chronic complications of VTE such as PTS and CTPEH, anti-inflammatory agents, statins, and vasodilator drugs (for pulmonary hypertension) are being evaluated in clinical trials. In this paper, we discuss developing insights into the molecular composition and mechanisms of acute-to-chronic VTE progression; innovative molecular therapeutic approaches that are expected to transform the care of acute VTE; and novel anti-inflammatory and vasodilation-based approaches to address the chronic complications of VTE.

Natural History of VTE

Clinical histological evidence of acute-to-chronic VTE

Pathological studies of the thrombi isolated from VTE patients provide useful information about the time-dependent changes in thrombus composition and their relationship to disease symptoms, outcomes, and therapies. Scanning electron microscopy of thrombi from VTE patients shows that acute DVT thrombi are rich in fibrin (28%-35%) and RBCs (35%-63%) arranged in heterogeneous layers and irregular shapes, respectively.^11,12 Fibrin is present in the form of thick bundles and thin fibers, and RBCs are in the form of polyhedrocytes or some intermediate shapes, suggesting clot retraction and compression. The composition of pulmonary emboli is similar to venous thrombi with similar fibrin (21%-41%) and RBC (47%-49%) content.^11,12 Still, the RBC vs fibrin content ratio of these thrombi may vary widely with early fresh thrombi primarily consisting of RBCs with low fibrin (80:20 ratio) or more developed thrombi with higher fibrin content (30:70 ratio).¹³ These observations agree with the previous histological observations suggesting that a fresh (acute) pulmonary embolus consists of structured layers, called lines of Zahn, in which fibrin and platelets alternate with RBCs.¹⁴ Over time, the thrombus organizes into a vascularized connective tissue caused by the invasion of fibroblasts, smooth muscle cells, and capillary buds, which appear in the form of bands and webs to restore recanalization.¹⁴ In the organizing (subacute) phase of thrombi in VTE patients, neutrophils disintegrate to form neutrophils extracellular traps (NETs), a fibrous network of DNA and histone proteins that are typically absent in chronic or fully organized areas of thrombus.¹⁵ A histological examination of 23 venous thrombi removed by mechanical thrombectomy in patients with extensive iliofemoral DVT, within 14 days of symptoms onset, showed signs of early thrombus remodeling involving low fibrosis and endothelial cell infiltration in ∼50% of cases.¹⁶ The histopathological analysis of CTEPH thrombi (removed by endarterectomy) showed an increased thrombus remodeling involving more collagenous fibrosis, loose fibroblast proliferation, smooth muscle cells, and endothelial cell infiltration.¹⁷ These observations are in agreement with time-dependent histopathological changes observed in postmortem pulmonary emboli previously used to assess the chronological transformation of the thrombus.¹⁸ The acute DVT or PE thrombi in phase 1 (∼1 week) are rich in fibrin and RBCs and contain platelets and inflammatory cells, whereas the thrombus remodeling in the subacute or early chronic phase 2 (∼2-8 weeks) is characterized by early remodeling with collagenous fibrosis, infiltration of smooth muscle cells, fibroblasts, and endothelial cells. In the later chronic phase 3 (>8 weeks), there is the formation of neovascular channels (bands and webs) for recanalization^18,19 (Figure 1). In VTE patients, based on the appearance and severity of the symptoms, the disease progression may be classified as acute, subacute, or chronic, but requires a careful diagnosis to accurately determine the thrombus age that might critically decide the choice of treatment.

Figure 1.

Natural History of Venous Thromboembolism

The diagram represents the current understanding of the mechanisms of the acute-to-chronic transition of venous thrombi. (A) Acute fibrin and red blood cell (RBC)–rich thrombi (B) propagate and (C) remodel to collagen-rich fibrotic thrombi with cellular infiltration (smooth muscle cells, fibroblasts, endothelial cells, and macrophages) that are (D) increasingly fibrotic with the formation of neovascular channels. Accumulation of cellular and fibrous tissue in the venous wall and within the thrombus leads to venous wall stiffness, reduced blood flow, and hypertension. Anticoagulation therapy prevents thrombus propagation (B), but it is unclear if affects remodeling (C) or fibrosis (D). Thrombolytic therapy (E) prevents thrombus propagation and (F) dissolves existing thrombi leading to resolution which appears to reduce remodeling (C) and fibrosis (D). Still, limited clinical data is available for the effectiveness of these approaches for chronic complications of VTE. Parts of the figure were drawn by using pictures from Servier Medical Art (https://creativecommons.org/Licenses/by/3.0/).

Mechanistic Insights of Acute-to-Chronic VTE From Animal Models

Fibrin and RBC-rich acute venous thrombi

To understand the basic mechanisms of DVT, different models of venous thrombosis have been developed in rodents such as inferior vena cava (IVC) ligation-induced venous thrombosis, FeCl₃ injury, and electrolytic injury of the IVC.²⁰ IVC ligation models are thought to best recapitulate human DVT.^21,22 Complete stasis in the IVC ligation model initiates reproducible thrombus formation within hours; the thrombus becomes stable by 24 hours and reaches its maximal growth in 2 to 3 days^21,23 (Figure 1). Mechanistically, according to the classical “Virchow triad,” stasis, hypercoagulation, and endothelial injury are the initial triggers for DVT.²⁴ The acute thrombus is fibrin- and RBC-rich (similar to thrombi in DVT patients), along with platelets, neutrophils, and monocytes, which play important roles in thrombus initiation and its development^21,25 (Figure 1, Table 1). The roles of different blood cells, inflammatory cells, coagulation proteins, fibrinolysis, and vascular endothelium in thrombus initiation are reviewed elsewhere.^2,7

Table 1.

Thrombus Composition During the Acute-to-Chronic Transition- Preclinical (IVC Ligation) Evidence

Cellular/Molecular Contributors	Acute-to Chronic Venous Thrombosis
	Acute (1-2 d)	Subacute (7-10 d)	Chronic (>14 d)
Thrombus weight23,92	⇧⇧⇧	⇧⇧	⇧
RBC11,12,35,37	⇧⇧⇧	⇧⇧	⇧
Fibrin11,12,35,36	⇧⇧⇧	⇧⇧⇧	⇧
Platelets64	⇧⇧	⇧⇧	⇧
Neutrophils7,40,93	⇧⇧⇧	⇧⇧	⇧
Macrophages/monocytes7,40,93	⇔	⇧⇧	⇧⇧⇧
Fibroblasts, smooth muscle cells, endothelial cells39,64	⇔	⇧⇧	⇧⇧⇧
Collagen7,39,64	⇔	⇧⇧	⇧⇧⇧

The table shows the cellular and molecular transition of acute thrombi to subacute and chronic stages. The symbols indicate basal level (⇔) or increased levels (⇧).

RBC = red blood cell.

Acute experimental IVC stasis or stenosis thrombi recapitulate compositional (RBC and fibrin-rich) and organizational (layered concentric) architecture observed in the patients’ thrombi. Acutely, fibrin-fibrin crosslinking, crosslinking of fibrinolysis inhibitors to fibrin, and clot retraction make the thrombus more resistant to lysis.²⁶ In a series of studies in genetically modified mice, hypofibrinogenemia (sequence variation in Aα chain for reduced fibrinogen production) and dysfibrinogenemia (nonpolymerizable fibrinogen production) protected against venous thrombosis induced by IVC ligation (stasis).^27,28 Reduced fibrinogen levels in mice (∼10% of the normal blood levels) were sufficient to maintain hemostasis but drastically reduced the capability for venous thrombosis. Although previously debated,^29,30 recent preclinical studies using genetically modified mice with defective fibrin crosslinking ability or factor XIIIa inhibition have provided strong evidence that factor XIIIa-mediated fibrin-fibrin crosslinking is an important determinant of strength and stability of in vivo venous thrombi.³¹ Notably, the role of factor XIII in thrombus resistance with potential therapeutic opportunity has been previously validated in animal models.³⁰ Recent in vivo studies showed that factor XIIIa-mediated fibrin-fibrin crosslinking is responsible for the dynamic retention of RBCs in venous thrombi, which significantly alters thrombus composition and weight.³² Fibrinogen-targeting strategies such as fibrinogen-depleting agents and factor XIIIa inhibitors are currently being investigated preclinically as potential drug targets (reviewed elsewhere³³). These new findings have highlighted the organizational and compositional complexity of venous thrombi and the hope for innovations beyond conventional anticoagulation. The recent translational efforts in the anticoagulation, e.g., factor X and XI inhibitors, have also helped us to understand the mechanistic complexity of thrombosis. Platelets provide a surface for the binding of factor X and XI for thrombin activation. Beyond its role in the coagulation cascade, inhibition of factor XI by domain-specific monoclonal antibodies demonstrate its capability to promote the activation of platelets, neutrophils, and inflammation.³⁴

Chronic collagenous venous thrombi

Collagen is absent in the acute thrombus and vein wall collagen content is minimal (Figure 2).²¹ The thrombus slowly begins to decline in mass and fibrin content caused by endogenous fibrinolysis during the subacute phase (after 1 week) (Figures 1 and 2, Table 1) but fibrinolysis also slows as evidenced by declining D-dimer levels in the chronic phase.35, 36, 37 The components of the fibrinolytic system are present in acute as well as chronic thrombi including plasminogen, plasminogen activators, and the fibrinolysis inhibitors such as plasminogen activator inhibitor-1 (PAI-1) and alpha2-antiplasmin (α2AP).^23,38 RBCs decrease in number and potentially disintegrate as evidenced by time-dependent decreased staining and increased iron (Fe²⁺ to Fe³⁺) oxidation during the acute to subacute and chronic phase.³⁷ The process of the organization begins with the appearance of fibroblasts, smooth muscle cells, and the development of collagen meshwork in the thrombus and the vein wall, which progressively becomes fibrotic and thickened (Figure 1).³⁹

Figure 2.

Clinical and Preclinical Evidence of Acute and Chronic Venous Thromboembolism

The figure shows clinical and preclinical evidence of acute-to-chronic transition of venous thrombi. (A and B) Stained sections of lungs after 4 hours of experimentally induced pulmonary embolism in mice. (A) FITC-fibrin labeled (green) plasma clots in lungs (with DAPI stained nuclei) and its magnified image. (B) Martius Scarlet Blue stained fibrin (red) clot in the lungs. (C and D) Acute-to-chronic IVC thrombi in mice. (C) The thrombi harvested after IVC ligation in mice at different time points, i.e., 1 day (acute), 7 days (subacute or early chronic), and 14 days (chronic), which turn from red to white over time. (D) Immunostained (type IV collagen, red color) sections of IVC thrombi represent fibrosis from acute (minimum collagen) to subacute and chronic stages (increasing collagen), (Guy Reed’s laboratory, unpublished data, December 15, 2017). (E) Red clots removed from the pulmonary artery of a patient by thrombectomy, an example of an acutely formed extensive venous thrombus.⁹⁰ The image is reused with permission under a Creative Commons license (https://creativecommons.org/Licenses/by/4.0/) and public domain dedication waiver ((http://creativecommons.org/publicdomain/zero/1.0/). (F) The image shows chronic fibrous thrombi from a patient removed by pulmonary endarterectomy; reused in modified form (cropped) from a case study⁹¹ under a Creative Commons license (https://creativecommons.org/Licenses/by/4.0/).

Inflammation is driven primarily by neutrophils in acute thrombi, whereas macrophages are abundantly present in chronic thrombi.⁴⁰ In the subacute phase, the neutrophils strengthen the clot structure through the formation of NETs to further enhance thrombosis.¹⁵ NETs may also promote monocyte differentiation into fibroblasts for the production of more collagenous matrix.⁴¹ Neutrophils and monocytes/macrophages are actively involved in the resolution of the thrombus also, because of the expression of key fibrinolytic components such as urokinase plasminogen activator, urokinase receptor, as well as matrix-degrading enzymes, i.e., matrix-metalloproteinases-2 and -9.⁷ The exacerbating effects of NETs-associated thrombo-inflammation have been observed in COVID-19 patients, with increased levels of NETs markers (cell-free DNA, histone H3) and neutrophils-platelets aggregates in plasma. The widespread NETs-rich microvascular thrombi in different organs were observed in patients with severe COVID-19 including lungs showing the pathology of acute respiratory distress syndrome.⁴²

In the second week, the resolving thrombus becomes increasingly cellular, with further collagen deposition and thickening evident in the vessel wall. In addition to fibrinolysis, chronic thrombus resolution occurs in part through neovascularization with the formation of vascular channels.⁴³ The external margin of the thrombus becomes encircled by endothelial cells, and neovascular channels form in about 10 days.^39,43 In the chronic phase (2-4 weeks) the thrombus has become a condensed, fibrotic (collagen-rich) mass contained within a thickened fibrotic vessel. The actual initial thrombus decreases in size, the remodeled structure at the thrombus site becomes thickened and part of the vascular wall, resembling a healing wound,⁴⁴ which restricts the vascular lumen. These preclinical insights suggest that progressive compositional changes in venous thrombi are crucial determinants of the time-related susceptibility of thrombi to different treatment modalities. They also represent potential avenues for novel therapeutic discovery.

Emerging Therapeutic Approaches

“Anticoagulation”—the first line of treatment

The standard of care for VTE is the prevention of thrombus expansion or recurrence with anticoagulants. Recent guidelines of major international societies, e.g., CHEST, European Society of Cardiology, and American Society of Hematology, recommend DOACs over conventional anticoagulants (heparins and vitamin antagonists) for acute VTE.^9,45,46 DOACs cause less bleeding, are easy to use because of their better pharmacokinetic and pharmacodynamic profile, and were recently approved by the U.S. Food and Drug Administration (FDA) for clinical use in VTE patients (Figure 3). The risk of reoccurrence in patients who completed 3 months of anticoagulation after the first episode of unprovoked VTE is as high as 36% at 10 years if anticoagulation is discontinued.⁴⁷ These anticoagulants have been beneficial for VTE patients to a large extent in preventing the propagation of acute thrombosis and VTE reoccurrence, but bleeding remains a serious concern.⁸

Figure 3.

Timeline for the U.S. FDA-Approved Anticoagulants and Thrombolytics

(A) The light green boxes represent the timeline for U.S. Food and Drug Administration (FDA) approval of direct oral anticoagulants (DOACs), and the red box (within the light green box) represents the timeline for the promising agents in clinical trials internationally specifically for venous thromboembolism. The DOACs timeline represents the approval year for specific brand names such as dabigatran (a thrombin inhibitor, Pradaxa, Boehringer Ingelheim Pharmaceuticals), and the inhibitors of factor X including Rivaroxaban (Xarelto, Janssen Pharmaceuticals, Inc), Apixaban (Eliquis, Bristol-Myers Squibb and Pfizer, Inc) and Edoxaban (Savaysa, Daiichi Sankyo Inc) specifically for VTE (e.g., based on the results of clinical trials in hip or knee arthroplasty patients or acutely ill medical patients). Factor XI inhibition is a novel anticoagulation strategy in advanced clinical trials (B) Thrombolytic agents including streptokinase, urokinase, and r-tPA were first approved by the FDA several decades ago. r-tPA remains the preferred thrombolytic agent for clinical use in VTE management. The limited use of streptokinase, a thrombolytic agent of bacterial origin, is still reported in developing countries. Monoclonal antibodies inhibiting the antifibrinolytic function of alpha2-antiplasmin (α2AP) are currently in phase 2 trials for submassive acute pulmonary embolism and proximal deep vein thrombosis patients.

Considering these limitations, strategies for developing novel anticoagulants are in progress. The latest entry to the list of anticoagulants is the inhibition of factor XI(a), which uniquely targets the intrinsic coagulation cascade instead of conventional anticoagulation strategies that directly target factor Xa or thrombin. In recently concluded phase II trials for factor XI(a)-inhibiting strategies, abelacimab (a monoclonal humanized antibody), milvexian (an oral inhibitor), and IONIS-FXI_Rx (antisense oligonucleotide) treatments reduced the risk of VTE in patients undergoing knee arthroplasty to a greater extent than enoxaparin and were associated with a reduced risk of bleeding48, 49, 50 (Figure 3, Central Illustration, Table 2). More factor XI(a) inhibiting antibodies are currently being evaluated in phase 2 trials for the prevention of VTE in patients undergoing unilateral total knee arthroplasty (TKA), including KN060 (NCT06180889), SHR2285 (NCT05203705) and 2 monoclonal antibodies from Regeneron Pharmaceuticals: REGN9933 (NCT05618808) and REGN7508 (NCT06454630) (Table 2). Abelacimab is currently being evaluated in phase 3 (ASTER [A Study Comparing Abelacimab to Apixaban in the Treatment of Cancer-associated VTE] and MAGNOLIA [A Study Comparing Abelacimab to Dalteparin in the Treatment of Gastrointestinal/Genitourinary Cancer and Associated VTE]) trials for the treatment of cancer-associated VTE (Table 2). However, the recent failure of asundexian, a small molecule inhibitor of factor XI for preventing stroke in high-risk patients with atrial fibrillation, raises the question of whether the potential benefits of this therapy can be extended to stroke prevention.⁵¹ Overall, factor XI inhibition has shown great promise in clinical trials for the prevention or treatment of VTE (Table 2). In addition to a unique target selection, the simultaneous therapeutic development of different classes of inhibitors (monoclonal antibodies, oral small molecule inhibitors, and antisense oligos) presents a multipronged approach to address the patient-management needs such as parenteral vs oral administration or immediate vs extended inhibition in different subsets of VTE patients including total hip arthroplasty (THA) or TKA, end-stage renal disease, and cancer-associated VTE.⁵²

Central Illustration.

Emerging Therapeutic Approaches for Venous Thromboembolism

Emerging therapeutic approaches (white boxes) in anticoagulation and thrombolytics are inhibitors of factor XI and antagonists of fibrinolysis inhibitors respectively. Additional therapeutic approaches include statins, micronized purified flavonoid fraction (MPFF), and the use of pulmonary artery hypertension drugs for chronic thromboembolic pulmonary hypertension (CTEPH). The list contains the promising approaches in clinical trials for different conditions of venous thromboembolism (VTE). DVT = deep vein thrombosis; PAH = pulmonary artery hypertension; PE = pulmonary embolism; PTS = post-thrombotic syndrome.

Table 2.

Therapeutic Advancements in Anticoagulation: Factor XI Inhibitors

Agent Name	Agent Class/Route of Administration	Sponsor/ Responsible Party	Clinical Trial/Year of Publication or Trial	Results/Comments
Abelacimab (MAA868)	Antibody (IV)	Anthos Therapeutics, Inc	Phase 2- ANT005, TKA; EduraCT Number 2019-003756-3748 (2021)	Phase 2- Postoperative dose (30-150 mg) related superiority over 40 mg (once daily) enoxaparin (i.e., 13%-4% vs 22%). Bleeding 0%-2% vs none in enoxaparin48
			Phase 3- NCT05171049 (ASTER) (2023)	Recruiting patients; vs apixaban; Cancer-associated VTE
			Phase 3- NCT05171075 (MAGNOLIA) (2023)	Recruiting patients; vs dalteparin; Gastrointestinal/genitourinary cancer and associated VTE
Osocimab (BAY1213790)	Antibody (IV)	Bristol Myers Squibb	Phase 2- NCT03276143 (FOXTROT) (2020)94	Non-inferiority vs enoxaparin (40 mg) or apixaban (2.5 mg) at postoperative doses (0.6-1.8 mg/kg). A preoperative high dose (1.8 mg/kg) was superior to enoxaparin. Bleeding 4.7% vs 5.9%94
Milvexian (BMS-986177; JNJ-70033093	Small molecule (oral)	Bristol Myers Squibb; Janssen Research and Development/Johnson and Johnson	Phase 2-NCT03891524 (AXIOMATIC-TKR) (2021)49	Postoperative dose (25-200 mg) related superiority over 40 mg enoxaparin (i.e., 25%-7% vs 21%). Equivalent bleeding in both groups (4%).49
IONIS-FXIRx; (ISIS416858 or ISIS-FXIRx)	Anitsense oligo (Subcutaneous)	Ionis Pharmaceuticals Inc	Phase 2- NCT01713361, TKA (2015)50	Preoperative and postoperative doses; a noninferior dose of 200 mg (27% vs 30%), and a superior dose of 300 mg vs 40 mg (once daily) enoxaparin (4% vs 30%). Reduced bleeding vs enoxaparin (3% vs 8%)50
KN060	Antibody (IV)	Suzhou Alphamab Co, Ltd	Phase 2- NCT06180889	Recruiting patients undergoing TKA for the prevention of VTE; vs enoxaparin
REGN9933	Antibody (IV)	Regeneron Pharmaceuticals	Phase 2- NCT05618808	Prevention of VTE in patients undergoing unilateral TKA; vs enoxaparin, apixaban; completed, no results posted
REGN7508	Antibody (IV)	Regeneron Pharmaceuticals	Phase 2- NCT06454630 ROXI-VTE II	Prevention of VTE in patients undergoing TKA vs enoxaparin; active, not yet recruiting
SHR2285	Small molecule (oral)	Shanghai Hengrui Pharmaceutical Co, Ltd	Phase 2- NCT05203705	Prevention of postoperative VTE in patients undergoing TKA.

The list contains selected promising compounds that have progressed to advanced stages of clinical trials (Phase 2) specifically for prevention or treatment of venous thromboembolism (VTE). The primary efficacy endpoint was the incidence of VTE confirmed by venography in patients undergoing total knee arthroplasty (TKA) or TKR. The trials for “end-stage renal disease” and others demonstrating the better safety or efficacy profile of these inhibitors including arterial thrombotic diseases such as ischemic stroke, atrial fibrillation, and acute coronary syndrome are not included.

ANT005, TKA = ANT-005 total knee arthroplasty; ASTER = A Study Comparing Abelacimab to Apixaban in the Treatment of Cancer-associated VTE; AXIOMATIC TKR = Antithrombotic Treatment With Factor XIa Inhibition to Optimize Management of Acute Thromboembolic Events in Total Knee Replacement; FOXTROT = FactOr XIa inhibiTion for the pRevention of venOus Thromboembolism in Patients Undergoing Total Knee Arthroplasty; IV = intravenous; MAGNOLIA = A Study Comparing Abelacimab to Dalteparin in the Treatment of Gastrointestinal/Genitourinary Cancer and Associated VTE; ROXI-VTE = Randomized, Open-Label, Active Control Study of REGN7508, a Factor XI Monoclonal Antibody, for Prevention of Venous Thromboembolism.

Thrombolytic therapy

Plasminogen activator-based thrombolytic therapies including recombinant tissue plasminogen activator (r-tPA/Alteplase), urokinase, and streptokinase, were approved for VTE by the FDA nearly 5 decades ago (Figure 3). Traditional thrombolytic therapy is based on the concept of increasing the conversion of plasminogen to plasmin for fibrin degradation and thrombus dissolution (Figure 3, Table 3). However, because of its unacceptable bleeding risks,¹⁰ thrombolysis by plasminogen activators is selectively used in VTE cases such as extensive DVT (e.g., iliofemoral) and massive or submassive PE.⁹ Efforts to extend thrombolytic treatment to lower-risk patients with submassive PE have been unsuccessful caused by serious and fatal bleeding complications, which exceed the net clinical benefit of therapy (PEITHO [Pulmonary Embolism International THrOmbolysis] trial).⁵³ To reduce bleeding complications, systemic low-dose t-PA has been advocated since the 1990s and investigated in multiple clinical trials⁵⁴ (Table 3). PEITHO-3 (NCT04430569) is an ongoing phase 3, multicenter, multinational double-blind randomized controlled trial (RCT) that will examine the safety and efficacy outcomes of low dose r-tPA (0.6 mg/kg ≤50 mg) in patients with intermediate-high-risk PE at 30 days along with long-term follow-up⁵⁵ (Table 3).

Table 3.

Therapeutic Advancements in Thrombolytic Approach in VTE

Agent Name	Target	Agent Class/Route of Administration	Sponsor/Responsible Party	Clinical Trial/Year of Publication or Trial	Results/Comments
r-tPA (low-dose)	Plasminogen activation	Protein (IV)	Beijing Chao Yang Hospital, China	Phase 3-NCT00781378 (2010)95	Similar efficacy and reduced bleeding by low dose vs standard dose r-tPA in acute PE patients.95
			MOPETT investigators, Arizona Cardiovascular Consultants, USA.	MOPETT trial (2013)96	Decreased pulmonary hypertension by low-dose r-tPA with anticoagulation (16%) vs anticoagulation alone (57%).96
			Assistance Publique - Hôpitaux de Paris	Phase 3- NCT04430569, PEITHO-3 (2022)55	Recruiting intermediate- high-risk PE patients; low-dose r-tPA (0.6 mg/kg ≤50 mg).55
DS1040	TAFI	Small molecule (oral)	Daiichi Sankyo	Phase 2- NCT02923115; JapicCTI-163164 (2022)	Safe and well tolerated but no significant benefit in reducing thrombus burden in acute submassive PE.56
TS23	α2AP	Antibody (IV)	Translational Sciences	Phase 2-NCT05408546, NAIL-IT (2022)	Recruiting intermediate-risk (submassive) PE patients.
BAY3018250	α2AP	Antibody (IV)	Bayer	Phase 2-NCT06149520, SIRIUS (2024)	Recruiting proximal DVT patients.

The table includes the clinical trials for low-dose recombinant tissue plasminogen activator (r-tPA) (systemic) and, the antagonists of fibrinolysis inhibitors specifically the monoclonal antibodies against alpha2-antiplasmin (α2AP) (TS23 and BAY3018250).

MOPETT = moderate pulmonary embolism treated with thrombolysis; NAIL-IT = Novel α2-Antiplasmin Inactivation for Lysis of Intravascular Thrombi Trial; PE = pulmonary embolism; PEITHO = Pulmonary Embolism International Thrombolysis; SIRIUS = A Study to Learn More About How Well BAY3018250 Works and How Safe it is for People With Proximal Deep Vein Thrombosis; other abbreviations as in Table 2.

A novel therapeutic paradigm is now emerging that venous thrombi can be safely dissolved without significantly increased bleeding by targeting fibrinolysis inhibitors such as α2AP, PAI-1, and thrombin activable fibrinolysis inhibitor (TAFI).^23,38,56 These fibrinolysis inhibitors act as the risk factors for VTE and play an important role in increasing thrombus resistance against endogenous and therapeutic dissolution.^29,57,58 Antagonists of fibrinolytic inhibitors directly target thrombus resistance and accelerate thrombus dissolution, either independently or in combination with several-fold lower doses of r-tPA to increase safety.^23,38,59 Inhibitors directed against α2AP, PAI-1, and TAFI have been preclinically tested in experimental VTE.^57,58,60 An oral inhibitor of TAFI (DS1040) was safe and well-tolerated in humans but did not show beneficial effects in reducing thrombus burden in acute submassive PE patients in phase II trials recently.^56,59 Phase 2 trials for monoclonal antibodies against α2AP, i.e., TS23 (NCT05408546), and BAY3018250 (NCT06149520) are currently ongoing in acute submassive PE and proximal DVT patients respectively (Figure 3, Central Illustration, Table 3). These trials will inform if acutely dissolving the thrombi will alleviate immediate symptoms or their potential utility in providing longer-term benefits of timely removal of thrombi for chronic complications. To support the longer-term benefits of timely resolution of the thrombus, a post hoc analysis of the ATTRACT (Acute Venous Thrombosis: Thrombus Removal With Adjunctive Catheter-Directed Thrombolysis) trial data showed that PTS and quality-of-life scores improved when pharmacomechanical catheter-directed thrombolysis was administered within 4 to 8 days after the onset of DVT symptoms, highlighting the importance of thrombus age in treatment effects.⁶¹ This was further supported by preclinical evidence that the early restoration of blood flow reduces thrombus size and provides benefits for later outcomes including vein wall fibrosis and inflammation in chronic thrombi.⁶¹ These novel therapeutics may fulfill the requirement of safely dissolving the large thrombi in DVT and PE patients who urgently require aggressive approaches for thrombus removal to alleviate immediate limb or life-threatening consequences.

Antiplatelet approach

Other interesting approaches for the prevention of thrombosis include antiplatelet agents such as aspirin, and antagonists of cell-surface receptors of platelets including PAR1, P2Y_12, and αIIbβ3.⁶² In addition to initiating thrombus formation by activating coagulation cascade on injured endothelium,²⁵ platelets dense granules release antifibrinolytic factors such as α2AP, PAI-1, and other components to enhance clot strength.⁶³ Platelets also contribute to an increase in intrathrombus and vein wall fibrosis in experimental chronic thrombi.⁶⁴ Several trials have examined the effect of aspirin on the prevention of recurrence after the term of initial anticoagulation treatment for VTE (Table 4). A combined analysis of the phase 3 ASPIRE (Aspirin to Prevent Recurrent Venous Thromboembolism) and WARFASA (Aspirin for the Prevention of Recurrent Venous Thromboembolism [Warfarin and Aspirin]) trials showed a significant reduction (42%) in VTE recurrence in patients with unprovoked VTE receiving aspirin vs placebo, without increased bleeding in patients.⁶⁵ However, in the EINSTEIN CHOICE (Reduced-dosed Rivaroxaban in the Long-term Prevention of Recurrent Symptomatic VTE) trial, the risk of recurrent VTE was significantly lower with rivaroxaban vs aspirin patients⁶⁶ (Table 4). The therapeutic value of aspirin vs anticoagulation for the prevention of VTE postorthopedic surgery has shown mixed results.^67,68,69 The Phase 3 PEPPER (Pulmonary Embolism Prevention after hiP and kneE Replacement; NCT02810704) trial will compare aspirin vs anticoagulants, rivaroxaban, or warfarin for the prevention of VTE in patients who have elective THA or TKA.⁷⁰ Although not the primary focus of the COMPASS (Cardiovascular Outcomes for People Using Anticoagulation Strategies; NCT01776424) and VOYAGER PAD (Vascular Outcomes Study of Acetyl Salicylic Acid Along With Rivaroxaban in Endovascular or Surgical Limb Revascularization for Peripheral Artery Disease; NCT02504216) trials, in later analysis, it was found that the combination of rivaroxaban and aspirin reduced the risk (39%) of VTE in chronic atherosclerosis patients.⁷¹ The EPCAT-III (VTE Prevention Following Total Hip and Knee Arthroplasty; NCT04075240) trial will examine the effectiveness of aspirin alone or with rivaroxaban (dual pathway inhibition) in the prevention of VTE in patients undergoing THA or TKA.

Table 4.

Clinical Trials of Aspirin, an Antiplatelet Agent in VTE

Sponsor/Responsible Party	Clinical Trial/Year of Publication or Trial	Results/Comments
National Health and Medical Research Council, Australia, and NZ Health Research Committee	Phase 3-ACTRN12605000004662 ASPIRE97 (2012)	No significant reduction in VTE recurrence but a reduction in other cardiovascular events vs placebo.
University of Perugia	Phase 3-NCT00222677 WARFASA98 (2012)	Reduced recurrence in idiopathic VTE patients vs placebo; no increase in bleeding.
David Anderson, Nova Scotia Health Authority	Phase 3-ISRCTN11902170 EPCAT-I67 2013	Aspirin was safe and non-inferior to dalteparin in the prevention of VTE after THA/TKA.67
David Anderson, Nova Scotia Health Authority	Phase 3- NCT01720108, EPCAT-II, (2018)68	No significant difference between aspirin and rivaroxaban in the prevention of VTE after THA/TKA.68
Bayer	Phase 3-NCT02064439, EINSTEIN CHOICE (2017)66	Increased risk of recurrent VTE vs rivaroxaban; no increase in bleeding.66
University of New South Wales, /Medical Research Future Fund, Australia	Phase 4-ACTRN12618001879257 CRISTAL (2022)69	Increased risk of symptomatic VTE as a monotherapy vs enoxaparin after THA/TKA.69
Sudeep Shivakumar, Nova Scotia Health Authority	Phase 3-NCT04075240, EPCAT-III (2024)	Recruiting patients; aspirin with or without rivaroxaban in the prevention of VTE after THA or TKA.
Dartmouth-Hitchcock Medical Center, USA	Phase 4- NCT02810704, PEPPER, (2022)70	Recruiting VTE patients, THA/TKA; Aspirin vs rivaroxaban vs warfarin.70

The clinical trials of aspirin vs placebo or standard direct oral anticoagulants (DOACs) alone or in combination are listed.

ASPIRE = Aspirin to Prevent Recurrent Venous Thromboembolism; ATTRACT = Acute Venous Thrombosis: Thrombus Removal With Adjunctive Catheter-Directed Thrombolysis; COMPASS = Cardiovascular Outcomes for People Using Anticoagulation Strategies; CRISTAL = A cluster-randomised, crossover, non-inferiority trial of aspirin compared to low molecular weight heparin for venous thromboembolism prophylaxis in hip or knee arthroplasty; EINSTEIN CHOICE = Reduced-dosed Rivaroxaban in the Long-term Prevention of Recurrent Symptomatic VT; EPCAT-III = Extended Venous Thromboembolism Prophylaxis Comparing Rivaroxaban to Aspirin Following Total Hip and Knee Arthroplasty; PEPPER = Pulmonary Embolism Prevention after hiP and kneE Replacement; THA = total hip arthoplasty; VOYAGER PAD = Vascular Outcomes Study of Acetyl Salicylic Acid Along With Rivaroxaban in Endovascular or Surgical Limb Revascularization for Peripheral Artery Disease; WARFASA = Aspirin for the Prevention of Recurrent Venous Thromboembolism (Warfarin and Aspirin); other abbreviations as in Table 2.

Platelet P2Y₁₂ receptor antagonists (e.g., clopidogrel, prasugrel, ticagrelor, cangrelor) are FDA-approved for the prevention of thrombosis in patients with coronary/peripheral artery diseases,⁷² but have not been investigated in randomized trials for VTE.

Anti-inflammation approach

Inflammation plays an important role in venous thrombosis, thrombus propagation, and chronic effects on vein wall health.⁷ P- and E-selectin receptors on endothelial cells promote leukocyte binding (through P-selectin glycoprotein ligand-1 of leukocyte) and thrombosis, and serve as potential drug targets for anti-inflammatory therapeutic approaches against VTE.⁷³ In phase 2 trials of SelK-2 (Tetherex Pharmaceuticals Corporation), a promising inhibitory antibody blocking P-selectin glycoprotein ligand-1 and P-selectin interaction did not reduce the risk of DVT after elective knee arthroplasty (NCT03812328) (Table 5). Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors and are used as antilipemic or lipid-lowering agents. During VTE, statins may induce anti-inflammatory, antithrombotic, and profibrinolytic functions; however, the exact mechanisms of statins are not yet clear. Statins have shown beneficial effects in experimental venous thrombosis, thrombus resolution, reduction in inflammation, and vein wall scarring.74, 75, 76 In a pooled analysis of the large JUPITER (Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin) and HOPE-3 (Heart Outcomes Prevention Evaluation -3) trials, rosuvastatin was associated with a significant (47%) reduction in the risk of VTE^77,78 (Table 5). A larger placebo-controlled RCT (NCT04319627) is investigating the effects of rosuvastatin in the prevention of symptomatic major VTE recurrence as a primary outcome.

Table 5.

Anti-Inflammatory and Vasodilator Agents

Agent Name	Target	Agent Class/Route of Administration	Sponsor/ Responsible Party	Clinical Trial/Year of Publication or Trial	Results/Comments
Anti-inflammatory approach for VTE
SelK2	PSGL-1	Antibody (I.V.)	Tetherex Pharmaceuticals Corporation	Phase 2- NCT03812328, COURSE (2021)	No benefit in reducing DVT or bleeding over enoxaparin in total knee replacement patients
Pulmonary artery hypertension drugs for CTEPH
Riociguat (BAY63-2521	Soluble guanylate cyclase	Small molecule (oral)	Bayer	Phase 3, CHEST-1 and CHEST-285	Significant Improvement in exercise capability, i.e., 6-min walking test and reduction in pulmonary vascular resistance.85
Macitentan	Endothelin Receptor	Small molecule (oral)	Actelion Ltd, Janssen	Phase 2- NCT02021292, MERIT-1 (2017)	Significant reduction in pulmonary vascular resistance vs placebo; associated with peripheral edema and anemia87
			Actelion Ltd, Janssen	Phase 3- NCT04271475, MACiTEPH (2023)	Trial stopped caused by futility99
Treprostinil	Prostacyclin and prostaglandin receptors	A prostacyclin analog, small molecule (s.c.), also available oral, IV	SciPharm SaRL	Phase 3- NCT01416636; EudraCT number 2008-006441-10, CTREPH (2018)89	Improvement in exercise capability, i.e., 6-min walking test
Statin therapy for VTE
Rosuvastatin	HMGCR	Statin; antilipemic agent (small molecule; oral)	AstraZeneca	Phase 3- NCT00239681, JUPITER78 (2009)	Significant reduction in symptomatic VTE in apparently healthy people.
			AstraZeneca; Canadian Institutes of Health Research	Phase 4-NCT00468923, HOPE-3100 (2016)	Significant reduction in symptomatic VTE
			Ottawa Hospital Research Institute, Canada	Phase 2- NCT02679664, SAVER101 (2022)	No benefit in PTS, measured by no reduction in Villalta score at 180 days. Warranted longer treatment studies.
				Phase 3- NCT04319627, SAVER (2023)	Recruiting patients; measuring VTE reoccurrence and PTS

Additional promising therapies (other than anticoagulants/antiplatelets and thrombolytics) include anti-inflammatory agents, statins (for deep vein thrombosis [DVT], pulmonary embolism, and post-thrombotic syndrome [PTS]), and pulmonary artery hypertension (PAH) drugs for chronic thromboembolic pulmonary hypertension (CTEPH).

BENEFiT = Bosentan Effects in iNopErable Forms of chronIc Thromboembolic pulmonary hypertension; CHEST 1 = Chronic Thromboembolic Pulmonary Hypertension Soluble Guanylate Cyclase–Stimulator-1; COURSE = Study to Assess the Safety and Efficacy of SelK2 to Prevent Blood Clots in Patients Undergoing Total Knee Replacement; CTREPH = Subcutaneous treprostinil for the treatment of non-operable chronic thromboembolic pulmonary hypertension; HMGCR = 3-hydroxy-3-methylglutaryl-CoA reductase; HOPE 3 = Heart Outcomes Prevention Evaluation - 3; JUPITER = Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin; MACiTEPH = A Study to Evaluate Efficacy and Safety of Macitentan 75 mg in Inoperable or Persistent/Recurrent Chronic Thromboembolic Pulmonary Hypertension; MERIT-1 = Macitentan for the treatment of inoperable chronic thromboembolic pulmonary hypertension; MUFFIN-PTS = Micronized Purified Flavonoid Fraction for the Treatment of Post-Thrombotic Syndrome; PSGL-1 = P-selectin glycoprotein ligand-1; SAVER = StAtins for Venous Event Reduction in Patients with Venous Thromboembolism; other abbreviations as in Table 2.

Treatment approaches for chronic complications of VTE

The long-term complications of VTE have been attributed to the fact that anticoagulation treatment itself does not dissolve and clear existing venous thrombi. Even after standard anticoagulation of 3 months, the presence of a residual thrombus is detected in nearly 70% of VTE patients.^79,80 This may lead to complications such as postpulmonary embolism impairment, PTS, and CTEPH.

Post-thrombotic syndrome

Up to 30% to 50% of anticoagulation-treated patients develop PTS, of whom 5% to 10% are severely affected.^80,81 PTS reduces the patient’s quality of life and causes a significant socioeconomic burden; unfortunately, no effective therapy is currently available.³ In addition to exercise and lifestyle modifications, elastic compression stockings are recommended for PTS management, but their therapeutic value is also debatable.⁸² A recent analysis of 10 clinical trials involving 2,361 patients suggested only low-quality evidence of a reduction in PTS by compression stockings and they did not appear to reduce severe PTS and PE.⁸² The pharmacological treatments (rutosides, micronized purified flavonoid fraction, statins) have not shown clear benefits in PTS to date,⁸³ although further results are expected from upcoming larger RCT of statins (NCT04319627) and micronized purified flavonoid fractions (NCT03833024/MUFFIN-PTS trial).

Chronic thromboembolic pulmonary hypertension

The undissolved thrombi in the pulmonary arteries may lead to chronic complications of CTEPH characterized by altered vascular remodeling, suppressed fibrinolysis, and vascular dysfunction, which causes pulmonary hypertension, right ventricular dysfunction/failure with symptoms of fatigue, difficulty in breathing, and reduced exercise capability.^1,4,84 The treatment options (other than anticoagulation) include surgical endarterectomy (if surgically accessible) and balloon pulmonary angioplasty, which aim to remove the blockage of pulmonary arteries to relieve hypertension and other symptoms.⁸⁴ Around 36% of CTEPH patients are not eligible for pulmonary endarterectomy and ∼50% of patients will have persistent recurrence after surgery.⁸⁴ Balloon pulmonary angioplasty carries the risk of catheterization-associated complications like wire injury, vessel rupture and dissection, bleeding, reperfusion injury, and pleural effusions.⁸⁴ The symptoms of pulmonary artery hypertension in CTEPH patients are alleviated with vasodilator drugs (targeting endothelial cell function); limited evidence of benefits from selective agents is available from double-blind, placebo-controlled RCTs. Riociguat (a guanylate cyclase stimulator, Adempas, Bayer), an FDA-approved drug for pulmonary artery hypertension, improves exercise capability and pulmonary hemodynamics in patients (CHEST-1 and -2 trials) with inoperable CTEPH and persistent pulmonary hypertension after endarterectomy or balloon angioplasty^85,86 (Table 5). Macitentan (an endothelin receptor antagonist) showed benefits in reduction in pulmonary vascular resistance and hemodynamics in phase 2 trials in inoperable CTEPH patients,⁸⁷ but the planned phase 3 trials were recently stopped caused by anticipated futility (Table 5). Similar to its precursor drug bosentan, macitentan is not clinically approved for CTEPH.⁸⁸ Treprostinil, another medication for pulmonary artery hypertension, has been clinically shown to be beneficial in phase 3 trials (CTREPH trial) for CTEPH patients.⁸⁹ These therapeutic developments and advancements in medical/surgical/catheter interventions provide new hope for CTEPH patient care.

Concluding Remarks and Future Perspectives

Anticoagulation therapy continues to remain the standard of care for the treatment and prevention of thrombosis in VTE patients with the hope of better safety and efficacy through the development of factor XI inhibitors. Emerging thrombolytic approaches, including antagonists of fibrinolysis inhibitors, may offer improved safety and efficacy profiles in the dissolution of acute venous thromboemboli to further reduce long-term complications such as PTS, postpulmonary embolism impairment, and CTEPH, that occur despite anticoagulation. Advancements in preclinical animal models are illuminating basic mechanisms and leading to innovative composition- and time-based treatment approaches for VTE patients. More research insights are needed into the mechanisms responsible for the acute-to-chronic transition of venous thrombi to support treatment innovations that are appropriately targeted to thrombus age.

Funding Support and Author Disclosures

This work was funded in part by the Ramanujan Fellowship (RJF/2020/000070) by SERB-DST, Government of India. Institutional support was provided by CSIR-IHBT Project MLP0204 (Department of Dietetics and Nutrition Technology) to Dr Singh; and National Institutes of Health funding to Dr Reed (HL092750, HL158376) and Dr Jaffer (HL144550, HL150538, HL16543). Dr Jaffer has sponsored research for Canon, Siemens, Shockwave, Teleflex, Mercator, Boston Scientific, HeartFlow, and Neovasc; has received consultant/speaker fees from Boston Scientific, Siemens, Magenta Medical, Philips, Biotronik, Mercator, Terumo, Abiomed, Shockwave, DurVena, Intravascular Imaging Inc, Medtronic, and FastWave; has equity interest in Intravascular Imaging Inc, DurVena, and FastWave; and his institution, Massachusetts General Hospital, has licensing arrangements with Terumo, Canon, and Spectrawave, for which he has the right to receive royalties. Dr Reed is the founder and CSO of Translational Sciences. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.