Endogenous fibrinolysis inhibitors in acute coronary syndrome

Abstract

Patients with acute coronary syndrome have a high residual risk of ischemic events despite current treatment methods, both invasive and antithrombotic strategies. The strategy of very early revascularization although has been suggested to improve patient outcome, remains associated with a high residual risk of adverse events. Stenting of nonflow-limiting vulnerable plaques in addition to stenting of hemodynamically significant lesions in patients with acute coronary syndrome, has not shown a beneficial effect on major adverse cardiovascular events in early studies. Current antithrombotic therapy in acute coronary syndrome is focused mainly on antiplatelet agents, and to a lesser extent on oral anticoagulants. Besides thrombotic atherosclerotic plaque rupture and activated platelets, impaired fibrinolysis has gained attention as a strong independent risk factor for cardiovascular mortality and adverse outcome in patients with acute coronary syndrome. Various endogenous fibrinolysis inhibitors that act at different levels of the hemostatic process have been associated with the impaired fibrinolysis. This review presents available data for association of impaired fibrinolysis with major adverse cardiovascular outcome in acute coronary syndrome, and the potential role of endogenous fibrinolysis inhibitors in acute coronary syndrome. In addition, experimental evidence for modulation of impaired fibrinolytic state with profibrinolytic agents that target endogenous fibrinolysis inhibitors is summarized.

Highlights

• •Patients with acute coronary syndrome have a high residual risk of ischemic events.
• •Impaired fibrinolysis is an independent risk factor for major adverse cardiovascular outcome.
• •Elevated levels of endogenous fibrinolysis inhibitors have been demonstrated in patients acute coronary syndrome.
• •Modulation of the impaired fibrinolysis in acute coronary syndrome is a yet untapped treatment option.

1. Introduction

Patients with acute coronary syndrome (ACS) have a high residual risk of ischemic events, in spite of current treatment strategies. In a large retrospective study of survivors of myocardial infarction (n = 97,254), major adverse cardiovascular events (MACE) of 18.3% was observed at 1-year, patients who were event-free at 1-year had a risk of 20% in the subsequent 3 years [1]. A multicenter prospective registry reported an event rate of up to 40% at 3 years in patients with ACS [2]. In randomized control trials analyzing dual anti-platelet therapy (DAPT) in ACS, a residual risk of ischemic events of 10% at 12 to 15 months has been reported, irrespective of the type of antiplatelet therapy [3], [4], [5]. Although very early revascularization has been suggested to improve patient outcome in ACS, the high residual risk of ischemic events appears to persist despite this strategy. A 20.4% incidence of MACE at 1-year has been reported in an observational study of patients undergoing very early revascularization [6]. In the VERDICT randomized trial, patients undergoing very early revascularization had a of MACE of 27.5% at a median of 4.3 years [7]. Lipid-rich nonobstructive vulnerable plaques in non-culprit segments have been demonstrated to be associated with a higher incidence of long-term MACE, after percutaneous coronary intervention (PCI) of culprit lesions [8]. However, PCI of nonflow-limiting vulnerable plaques in ACS with bioresorbable vascular scaffold has not shown to significantly reduce cardiovascular death, myocardial infarction, or target-lesion revascularization at a median follow-up of 4.1 years [9], [10]. In the PROSPECT-ABSORB study (non-powered), a favorable long-term clinical outcome (not statistically significant) with stenting was primarily due to a lower incidence of new-onset angina [9].

Besides thrombotic atherosclerotic plaque rupture and activated platelets, an impaired fibrinolytic state has been demonstrated in patients with ACS. In this review, impaired fibrinolysis as a risk factor for clinical outcome, potential endogenous fibrinolysis inhibitors mediating the impaired fibrinolytic state, and possible emerging experimental therapeutic options with profibrinolytic agents are discussed.

2. Current long-term antithrombotic therapy

Current long-term antithrombotic therapy in ACS is focused mainly on antiplatelet agents and to a lesser extent on direct oral anticoagulants (DOACs), the potential of profibrinolytic agents is yet to be defined (Fig. 1).

Fig. 1.

Long-term antithrombotic therapy in ACS.

Legend: Various antiplatelet agents and anticoagulants have been evaluated in ACS, while the potential of profibrinolytic agents is yet to be defined.

ACS = acute coronary syndrome.

2.1. Antiplatelet therapy

Activated platelets play a central role as mediators of thrombogenesis in patients presenting with ACS [11], and DAPT is a mainstay in the treatment [12]. The CURE trial reported a 28% relative reduction in primary outcome with DAPT using clopidogrel compared to aspirin [3]. The TRITON-TIMI 38 and PLATO trials reported a 19% and 16% relative reduction in primary outcome with prasugrel and ticagrelor respectively compared to clopidogrel [4], [5]. In patients with ACS treated conservatively, no difference in primary efficacy between prasugrel and clopidogrel was observed [13]. In a study comparing DAPT with ticagrelor or prasugrel, a higher incidence of primary outcome was seen with ticagrelor (9.3% versus 6.9%, hazard-ratio [HR] 1.36, 95% confidence interval [CI] 1.09-1.7, p = 0.006) [14]. The authors of the study, however, have remarked that this observation could not be clearly explained as the predicted primary outcome assumed during the study design for prasugrel and ticagrelor were 12.9% and 10.0% respectively. The addition of oral protease-activated–receptor 1 antagonist vorapaxar, that competitively inhibits thrombin-induced platelet aggregation to standard DAPT had no effect on the primary outcome, but was associated with major bleeding [15].

As summarized in Table 1, a residual risk of cardiovascular events of 10% or higher has been observed in most patients irrespective of the type of antiplatelet therapy.

Table 1.

Trials of DAPT in ACS and residual risk of ischemic events.

Study (ref #)	Study population	Drug	Primary end-point (%)
CURE (ref #3)	NSTEMI	Clopidogrel vs placebo	9.3 vs 11.4 (RR 0.80, 95% CI 0.72-0.90, p˂0.001)
TRITON-TIMI 38 (ref #4)	NSTEMI/ Unstable angina (74%) STEMI (26%)	Prasugrel vs Clopidogrel	9.9 vs 12.1 (HR 0.81, 5% CI 0.73-0.90, p˂0.001)
PLATO (ref #5)	NSTEMI/ Unstable angina (59%) STEMI (37.5%)	Ticagrelor vs Clopidogrel	9.8 vs 11.7 (HR 0.84, 95% CI 0.77-0.92, p˂0.001)
TRILOGY ACS (ref #13)	NSTEMI/ Unstable angina	Prasugrel vs Clopidogrel	13.9 vs 16.0 (HR 0.91, 95% CI 0.79-1.05, p = 0.21)
ISAR-REACT 5 (ref #14)	NSTEMI/ Unstable angina (58.6%) STEMI (41.4%)	Ticagrelor vs Prasugrel	9.3 vs 6.9 (HR 1.36, 95% CI 1.09-1.70, p = 0.006)a
TRACER (ref #15)	NSTEMI	Vorapaxar + DAPT vs placebo + DAPT	18.5 vs 19.9 (HR 0.92, 95% CI 0.85-1.01, p = 0.07)

ACS = acute coronary syndrome, CI = confidence interval, DAPT = dual antiplatelet therapy, GP IIb/IIIa inhibitor = glycoprotein IIb/IIIa inhibitor, HR = hazards ratio, NSTEMI = non-ST–segment elevation myocardial infarction, OR = odds ratio, RR = relative risk, STEMI = ST–segment elevation myocardial infarction.

^aLow primary endpoint with prasugrel was unexplained in the study (please refer text).

2.2. Anticoagulation with DOACs

Patients with ACS demonstrate a prothrombotic state that is most prominent in ST–segment elevation myocardial infarction (STEMI), and peak thrombin generation is significantly associated with MACE [16]. This has prompted interest in the evaluation of DOACs in patients with ACS (Table 2). The DOACs apixaban and dabigatran did not demonstrate a significant effect on MACE, and were associated with a higher incidence of major bleeding [17], [18], [19]. A phase 2 trial with rivaroxaban showed no benefit on MACE [20], but in the larger ATLAS ACS 2-TIMI 51 trial a 16% relative reduction in MACE was observed, with more frequent major bleeding [21]. Recently published guidelines have recommended long-term low-dose rivaroxaban use in selected patients with ACS in addition to aspirin and clopidogrel as a class IIb indication [22]. It should be emphasized that there is lack of data on combining DOACs with the more potent P2Y12 inhibitors prasugrel and ticagrelor. Additionally, among patients with ACS, a differential treatment effect of DOACs in non-ST–segment elevation myocardial infarction (NSTEMI) and STEMI present. Chiarito et al. have reported in their meta-analysis that the addition of DOACs to DAPT was not associated with a benefit in NSTEMI, whereas, a significantly lower incidence of MACE was seen in STEMI [23].

Table 2.

DOAC + DAPT in ACS.

Study (ref #)	Drug	MACE (%)	Comments
APPRAISE (ref #17)	Apixaban vs placebo	7.6 vs 8.7, HR 0.73, 95% CI 0.44-1.19, p = 0.21 (2.5 mg bid dose) 6.0 vs 8.7, HR 0.61, 95% CI 0.35-1.04, p = 0.07 (10 mg od dose)	Dose-dependent increase in major bleeding, with no significant effect on MACE
APPRAISE-2 (ref #18)	Apixaban vs placebo	7.5 vs 7.9 (HR 0.95, 95% CI 0.80-1.11, p = 0.51)	Terminated prematurely due to safety concerns
RE-DEEM (ref #19)	Dabigatran vs placebo	4.6 vs 3.8 (50 mg dose) 4.9 vs 3.8 (75 mg dose) 3.0 vs 3.8 (110 mg dose) 3.5 vs 3.8 (150 mg dose)	Dose-dependent increase in major bleeding, with no significant effect on MACE
ATLAS ACS-TIMI 46 (ref #20)	Rivaroxaban vs placebo	5.6 vs 7.0, (HR 0.79, 95% CI 0.60-1.05, p = 0.10)	Dose-dependent increase in major bleeding, with no significant effect on MACE
ATLAS ACS 2-TIMI 51 (ref #21)	Rivaroxaban vs placebo	8.9 vs 10.7 (HR 0.84, 95% CI 0.74-0.96, p = 0.008)	Lower MACE, with increased risk of major bleeding

ACS = acute coronary syndrome, CI = confidence interval, DAPT = dual antiplatelet therapy, DOAC = direct oral anticoagulant, HR = hazards ratio, MACE = major adverse cardiovascular events.

3. Impaired fibrinolysis in ACS

The hypercoagulable state in ACS activates endogenous fibrinolysis as a counter regulatory mechanism, an impairment in the fibrinolytic mechanism would result in persistence of thrombotic risk [24]. An impaired fibrinolytic state that is significantly associated with cardiovascular mortality and MACE has been reported in patients presenting with ACS [25], [26], [27], [28], [29], [30].

In the study by Saraf et al., patients with ACS and established on DAPT (n = 300) had significantly impaired fibrinolysis evidenced by prolonged thrombus lysis-time compared to controls [25]. A cutoff thrombus lysis-time ≥ 3000 s that predicted MACE was seen in 23% of ACS and in none of the controls. On multivariate analysis, prolonged thrombus lysis-time ≥ 3000 s was an independent predictor of 1-year cardiovascular mortality (HR 4.2, 95% CI 1.3-15.62, p = 0.033) and MACE (HR 2.52, 95% CI 1.34-4.71, p = 0.004). In a PLATO substudy (n = 4354), impaired fibrinolysis in the highest quartile was as an independent predictor of cardiovascular death (HR 1.92, 95% CI 1.19-3.10, p˂0.001) and MACE (HR 1.48, 95% CI 1.06-2.06, p = 0.027) at 1-year, irrespective of the DAPT used [26]. Furthermore, each 50% increase in clot lysis-time was associated with increased risk of cardiovascular death (HR 1.36, 95% CI 1.17-1.59, p˂0.001) and MACE (HR 1.17, 95% CI 1.05-1.31, p = 0.006). Recently, similar results were reported in diabetic population of the PLATO study (n = 974), wherein after adjustment for clinical risk factors and known prognostic biomarkers, impaired fibrinolysis remained an independent risk factor for MACE at 1-year (HR 1.23, 95% CI 1.02-1.49, p = 0.034) [27].

In the RISK-PPCI study of patients with STEMI undergoing primary PCI (n = 496), impaired fibrinolysis (prolonged lysis-time ≥ 2500 s) was seen in 14% of patients, and persisted unchanged at 30 days [28]. The baseline impaired fibrinolysis was strongly associated with MACE at 1-year (HR 8.03, 95% CI 4.28-15.03, p˂0.001). Furthermore in the study, patients with impaired fibrinolysis had significantly higher incidence of cardiogenic shock (8.6% vs 2.8%, p˂0.05) and acute stent thrombosis (4.3% vs 0.7%, HR 5.38, 95% CI 1.13-27.63, p˂0.05), than those in whom lysis-time was ˂2500 s. In a study of 47 patients with definite stent thrombosis (90% of whom had the index PCI for ACS), impaired fibrinolysis as measured by rate of increase in D-dimer levels (marker of fibrin degradation) was the strongest independent predictor of stent thrombosis along with clot permeability and stent length, while vessel size and markers of platelet activation/platelet count did not achieve significance [29]. In patients with STEMI, Spinthakis et al. have shown that fibrin fiber thickness correlated inversely with lysis-time (r = -0.89, p = 0.001), and that impaired fibrinolysis was strongly associated with abnormal fibrin architecture [30]. Using scanning electron microscopy, they were able to demonstrate that with increasing quartiles of lysis-time, fibrin architecture became more compact with denser and thinner fibers.

An interesting observation by Sumaya et al. is the association between impaired fibrinolysis and B-type natriuretic peptide levels in patients with ACS [26]. Levels of B-type natriuretic peptide increased significantly with increasing quartiles of clot lysis-time. It has been shown that a single measurement of B-type natriuretic peptide obtained in the first few days of ACS is a strong predictor of long-term mortality, independent of the extent of myocardial necrosis [31]. The mechanism underlying the association between elevated B-type natriuretic peptide levels and impaired fibrinolysis in ACS is not exactly determined, but it is of note that both states are associated with increased neuro-hormonal activation, a well-documented phenomenon in ACS patients.

4. Role of endogenous fibrinolysis inhibitors

Endogenous fibrinolysis inhibitors that underlie the impaired fibrinolysis include thrombin-activatable fibrinolysis inhibitor (TAFI), microparticles, complement C3, plasminogen activator inhibitor-1 (PAI-1), lipoprotein (a), and alpha2-antiplasmin (Fig. 2). Inhibition of fibrinolysis by endogenous inhibitors can occur at various levels [32] in the hemostatic process (Fig. 3). It has not been determined whether any single endogenous fibrinolysis inhibitor is responsible for the impaired fibrinolysis in ACS, it may well be possible that multiple agents could be contributive (Table 3).

Fig. 2.

Endogenous fibrinolysis inhibitors.

Legend: Schematic representation of endogenous fibrinolysis inhibitors. Thrombin generated from the coagulation cascade activates TAFI (particularly in the presence of TM), which cleaves the lysine residues on fibrin. Lysine residues are the binding sites for PLA – tPA complex. MP and C3 bind directly to fibrin, increasing resistance to clot lysis. Lp (a) competes with PLA for binding with lysine residues. PAI-1 and α2AP form inert complexes with t-PA and plasmin, respectively. The breakdown of fibrin to FDP is thus impaired, and the resultant impaired fibrinolytic state is a strong independent risk factor for MACE in ACS.

ACS = acute coronary syndrome, α2AP = alpha2-antiplasmin, C3 = complement C3, FDP = fibrin-degradation product, Lp (a) = lipoprotein (a), Ly = carboxy-terminal lysine residues on fibrin monomers, MACE = major adverse cardiovascular events, MP = microparticles, PLA = plasminogen, PAI-1 = plasminogen activator inhibitor-1, TAFI = thrombin-activatable fibrinolysis inhibitor, TAFIa = thrombin-activatable fibrinolysis inhibitor active form, TM = thrombomodulin, tPA = tissue plasminogen activator.

Fig. 3.

Levels of action of endogenous fibrinolysis inhibitors.

Legend: Inhibition of fibrinolysis by endogenous inhibitors can occur at three levels in the hemostatic process: - at level of fibrin architecture alteration, at level of plasminogen activation, and at level of plasmin action.

TAFI = thrombin-activatable fibrinolysis inhibitor, PAI-1 = plasminogen activator inhibitor-1, α2AP = alpha2-antiplasmin.

Table 3.

Endogenous fibrinolysis inhibitors in ACS.

Fibrinolysis inhibitor	Mechanism of action	Association with ACS and long-term MACE (ref#)
TAFI	Cleavage of carboxy-terminal lysine residues on fibrin monomers	▪TAFI antigen/activity: - varying results with history of past MI (#32-34)a ▪TAFIa: - Higher levels with ACS and MACE (#39,40-42)
Microparticles	Increased fibrin density and resistance to lysis	•Higher levels with ACS (#49,50) •Lower levels with MACE (#51)b
Complement C3	High-affinity complexes with fibrin	Higher levels with ACS (#60,63)
Plasminogen activator inhibitor-1	Inert complexes with tissue plasminogen activator	Higher levels with ACS and MACE (#65-67)
Lipoprotein (a)	Competition with plasminogen for binding with lysine residues on fibrin	▪No association with ACS/MACE (#66,70-72) ▪Higher levels with MACE (#73)
Alpha2-antiplasmin	High molecular-weight complexes with plasmin	•Lower levels with MACE (#78) •Higher levels with MACE (#79)

ACS = acute coronary syndrome, MACE = major adverse cardiovascular events, MI = myocardial infarction, TAFI = thrombin-activatable fibrinolysis inhibitor, TAFIa = activated thrombin-activatable fibrinolysis inhibitor.

^aVarying result are likely due to different assays used in TAFI measurement in these studies (please refer text).

^bLow peripheral levels suggested to be due to increased local capture at site of atherothrombosis.

4.1. Thrombin-activatable fibrinolysis inhibitor

TAFI, a procarboxypeptidase synthesized by the liver is converted to its active form (TAFIa) by thrombin, particularly in the presence of thrombomodulin [33]. TAFIa inhibits fibrinolysis by cleavage of carboxy-terminal lysine residues on fibrin monomers which are the binding sites for plasminogen – tissue plasminogen activator complex, thereby limiting plasmin generation.

Levels of TAFI antigen or TAFI activity have been reported to be lower in some studies [34], [35], but higher in other studies [36] of patients with a past history of myocardial infarction. These varying results are likely a result of different assays used in TAFI measurement [37], [38]. TAFIa has a very short half-life (around 10 min) at body temperature and is rapidly converted to the isoform TAFIai [33]. Unlike measurement of TAFI antigen, measurement of TAFIa/TAFIai isoforms is valuable in monitoring the anti-fibrinolytic effect [39], [40]. In the AtheroGene study (n = 1668, 31% presented with ACS), patients with ACS had significantly increased levels of TAFIa/TAFIai than those with stable angina [41]. Only TAFIa/TAFIai levels, but not TAFI antigen was associated with long-term cardiovascular mortality. After adjusting for conventional risk factors and inflammation markers, the risk of cardiovascular death increased with each tertile increase in TAFIa/TAFIai (HR 1.69, 95% CI 1.07-2.67).

Several other studies have documented significantly higher levels of TAFIa in ACS compared to controls [42], [43], [44]. Leenaerts et al. also reported that TAFIa levels in intracoronary blood samples obtained from patients with STEMI during primary PCI were significantly higher (up to 8-fold) than that in peripheral blood from the same patients [43], suggesting increased local generation or capture at the site of culprit lesion. Shantsila et al. demonstrated that in ACS, TAFIa levels peaked on day of presentation and were significantly higher in NSTEMI than in STEMI or controls [44]. Markedly high tissue factor-expression in monocytes with increased thrombin generation has been demonstrated at presentation in ACS [45]. This observation assumes significance as tissue factor-expressing monocytes by promoting thrombin formation and increased generation of TAFIa not only impair fibrinolysis, but also make clots more resistant to heparin and enoxaparin [46]. In their study, Semeraro et al. demonstrated that a concentration of tissue factor-expressing monocytes as low as 3% significantly impaired fibrinolysis. This is comparable to the levels seen in patients with ACS, wherein tissue factor-expressing monocyte concentrations of 4.85% have been demonstrated [47]. Brambilla et al. have reported that the concentration of tissue factor-expressing monocyte in ACS is 3-fold that seen in patients with stable angina, and 5-fold that seen in controls.

4.2. Microparticles

Microparticles are vesicles varying in size from 50 nm to 1000 nm that are shed from platelets, endothelial cells or monocytes in response to activation by inflammatory stimuli or apoptosis [48]. Microparticles capable of increased thrombin generation were significantly higher in patients with a history of arterial thrombosis as compared to healthy controls [49]. Aleman et al. have shown that tissue factor-expressing microparticles promoted thrombin generation and accelerated the rate of fibrin formation [50]. The fibrin thus formed had greater density and was more resistant to lysis. Using scanning electron microscopy, Zubairova et al. have shown that microparticles can bind directly to fibrin, thereby getting incorporated into the fibrin network and increasing resistance to lysis [51].

Microparticles have been identified as the main carriers of circulating tissue factor [52]. Giesen et al. have suggested that coronary artery thrombosis maybe initiated by tissue factor released from atherosclerotic plaques, but propagation of thrombus results from the local capture of tissue factor-expressing microparticles. Significantly higher levels of microparticles are found in patients with coronary artery disease than in controls; and among patients with coronary artery disease, levels are higher in ACS than stable angina [53]. Mavroudis et al. analyzed blood samples from right atrium and from culprit artery in patients undergoing PCI for ACS or stable angina [54]. They reported that levels of microparticles were higher in patients with ACS than with stable angina; ACS patients also showed greater levels of microparticles in culprit coronary artery than in right atrium. In patients undergoing primary PCI for STEMI, Morel et al. documented significantly higher concentrations of microparticles within the occluded arteries compared to peripheral levels [55]. In another study, lower peripheral levels of microparticles were significantly associated with increased incidence of recurrent cardiovascular events in patients undergoing PCI for ACS [56]. Faille et al. have suggested that the low peripheral levels of microparticles observed in their study (intracoronary blood samples were not obtained) could be due to increased capture at site of coronary atherothrombosis [56]; an effect that has shown to be mediated by the binding of P-selectin glycoprotein ligand 1 on microparticles to P-selectin expressed in platelet-thrombus [57]. Microparticles isolated from patients with ACS promote thrombogenicity by upregulation of tissue factor and downregulation of endothelial nitric oxide synthase, among other mechanisms [58]. This effect was regulated through angiotensin II induced upregulation of angiotensin II receptor type-1 with resultant activation of mitogen-activated protein kinases and phosphoinositide 3-kinase/Akt.

4.3. Complement C3

Complement C3 is the convergence point of all the three complement pathways (classical, lectin, and alternative pathways). Complement C3 prolongs clot-lysis by forming high-affinity complexes with fibrin, and the antifibrinolytic effect is concentration-dependent [59]. Using proteomic analysis of plasma samples obtained from peripheral blood and from the site of culprit lesion, Distelmaier et al. reported the first direct evidence for localized activation of complement at the site of coronary occlusion in patients with acute myocardial infarction [60]. In a cohort of healthy subjects who were followed for 4 years, complement C3 level was independently associated with MACE, particularly in men [61]. Patients in the highest tertile had a 4-fold increased risk of MACE compared to the other two tertiles (risk ratio 4.2, 95% CI 1.5-11.7). Szeplaki et al. analyzed serum complement C3 levels in patients who underwent coronary artery bypass surgery, with a follow up period of 5 years [62]. They demonstrated that high serum concentrations of complement C3 predicted MACE only in women (odds-ratio [OR] 4.1, 95% CI 1.23-13.61). Complement C3 levels were reported to be significantly higher in survivors of myocardial infarction compared to controls, and was independently associated with the risk of myocardial infarction (OR 1.47, 95% CI 1.16-1.87) [63]. There is lack of data, however, on the association of complement C3 with long-term adverse outcomes in patients with ACS.

4.4. Plasminogen activator inhibitor-1

PAI-1 is the major endogenous inhibitor of tissue plasminogen activator, with which it forms inert complexes with resultant impairment of fibrinolysis [64]. Impaired fibrinolysis due to elevated PAI-1 levels has been reported to predispose to risk of MACE in patients presenting with ACS [65]. Analyzing risk factors for MACE during 3-year follow up in ACS patients, Hamsten et al. observed that elevated level of PAI-1 was independently associated with risk of MACE, along with dyslipidemia, poor left ventricular function, and multi-vessel coronary artery disease [65]. PAI-1 levels measured at the time of admission in patients with ACS undergoing PCI was significantly higher in patients who subsequently developed MACE, and PAI-1 level in the highest quintile was independently associated with risk of MACE compared to the lowest quintile (OR 5.3, 95% CI 1.2-23.8, p˂0.05) [66]. In their study, Marcucci et al. also observed that patients with elevated PAI-1 levels at admission had a significantly lower event-free survival during a mean follow up of 22.2 ± 3.9 months. Among patients presenting with ACS, analysis of samples obtained within six hours of presentation showed that STEMI patients had significantly higher levels of PAI-1 than NSTEMI patients, which is thought to contribute to the higher predilection for occlusive thrombi in STEMI [67].

4.5. Lipoprotein (a)

The structural similarity to low density lipoprotein cholesterol and plasminogen confers lipoprotein (a) with proatherogenic and antifibrinolytic effects, respectively [68]. Lipoprotein (a) particles consist of low density lipoprotein particles to which an apolipoprotein (a) chain is covalently attached. Because low density lipoprotein cholesterol is a risk factor for atherosclerosis, lipoprotein (a) particles are considered proatherogenic. Apolipoprotein (a) has structural similarity to plasminogen and by competing with plasminogen for available binding sites, lipoprotein (a) is considered antifibrinolytic. This competitive binding of lipoprotein (a) and plasminogen for lysine residue sites on fibrin results in decreased plasmin generation [69]. There are conflicting data regarding the role of lipoprotein (a) levels in patients with ACS. In a study of patients undergoing cardiac catheterization for ACS, no association was observed between lipoprotein (a) levels and cardiovascular mortality at 1-year after adjustment for conventional cardiovascular risk factors [70]. Roth et al., in their 5-year median follow up of patients with ACS were unable to demonstrate an association of lipoprotein (a) levels with cardiovascular mortality [71]. The dal-Outcomes trial reported that lipoprotein (a) levels were similar in patients with ACS and in controls and that lipoprotein (a) level was not associated with MACE, raising the question whether targeted therapy to reduce lipoprotein (a) levels would reduce the ischemic risk after ACS [72]. In patients with ACS undergoing PCI, Marcucci et al. reported that lipoprotein (a) levels measured at the time of admission were not elevated in patients who developed MACE, and had no effect on long-term event-free survival [66]. However in the ODYSSEY OUTCOMES trial, baseline lipoprotein (a) levels predicted risk of MACE after ACS, and reduction in lipoprotein (a) levels with proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor was associated with a significant reduction in MACE [73]. While atherosclerotic plaque regression and reduction in platelet reactivity with PCSK9 inhibitors have been documented [74], [75], there is no evidence till date demonstrating enhanced fibrinolytic effect with PCSK9 inhibitors.

4.6. Alpha2-antiplasmin

Alpha2-antiplasmin inhibits plasmin activity by forming high molecular-weight complexes with plasmin [32]. Alpha2-antiplasmin rapidly inactivates plasmin, and measurement of plasmin–alpha2-antiplasmin complexes is a useful marker for evaluation of fibrinolytic system in-vivo [76]. The evidence for an independent association between elevated plasmin–alpha2-antiplasmin complex levels and MACE is less clearly established. In a nested case-control study of healthy elderly subjects, increasing quartiles of plasmin–alpha2-antiplasmin complex levels were independently associated with increased risk of MACE over a follow-up period of 2.4 years [77]. On the other hand, in their study of patients with ACS, Redondo et al. have reported that in survivors of myocardial infarction, low levels of plasmin–alpha2-antiplasmin complex was independently associated with up to a 5-fold increase in relative risk of MACE over 2 years [78]. In a prospective cohort of patients with coronary artery disease from the AtheroGene study (n = 1057, 30% presented with ACS), although increase in plasmin–alpha2-antiplasmin complex levels was associated with cardiovascular mortality (HR 1.46, 95% CI 1.17-1.82), it did not achieve significance as an independent risk factor for cardiovascular mortality [79].

5. Profibrinolytic therapy

The recognition that impaired fibrinolysis is an independent risk factor for cardiovascular mortality and MACE, offers hopes that therapeutic modulation with profibrinolytic agents may provide an opportunity to improve outcome in ACS. Several pharmacological agents targeting the various fibrinolysis inhibitors have potential to promote fibrinolysis (Table 4), some of which are discussed briefly below.

Table 4.

Modulation with profibrinolytic therapy.

Compound	Target	Effect on fibrinolysis
Heparin	TAFI	Efficacy markedly reduced by platelet-rich clot
Parenteral direct thrombin inhibitors	TAFI	Promote clot lysis
Warfarin	TAFI	Promotes clot lysis
Fondaparinux	TAFI, Microparticles	Promotes clot lysisa
Oral direct thrombin inhibitor	TAFI	Promotes clot lysis
Imidazole derivative DS-1040	TAFI	Promotes clot lysis
Monoclonal antibodies/nanobodies ➢MA-T1C10 and MA-T94H3 ➢Vhh-TAFI-i and Vhh-TAFI-a ➢α2AP-I and 4H9 ➢MEDI-579	TAFI TAFI Alpha2-antiplasmin PAI-1	Promote clot lysis Promote clot lysis Promote clot lysis Effect on clot lysis not known
Oral factor Xa inhibitors	Fab Xaβ	Promote clot lysis
Compstatin	Complement C3	Promotes clot lysis
PAItrap	PAI-1	Promotes clot lysis
Clopidogrel	PAI-1, Microparticles	Effect on clot lysis not known
Rosuvastatin	Microparticles	Effect on clot lysis not known
PCSK9 inhibitors	Lipoprotein (a)	Effect on clot lysis not known
Antisense oligonucleotide IONIS-APO(a)Rx	Lipoprotein (a)	No effect on clot lysis
Aspirin	Not determined	Promotes clot lysis
Cangrelor	Not determined	Promotes clot lysis

PAI-1 = plasminogen activator inhibitor-1, PCSK9 inhibitors = proprotein convertase subtilisin/kexin type 9 inhibitors, TAFI = thrombin-activatable fibrinolysis inhibitor.

^aEffect relating to microparticles inhibition has not been reported.

5.1. Heparin and parenteral DTI

Heparin inhibits activation of TAFI by fluid-phase thrombin, however, it is unable to enhance clot-lysis as it has negligible effect on clot-bound thrombin [80]. This is because of the presence of activated platelets in clots, which markedly reduce the ability of heparin to inhibit TAFIa generation [81]. Clot-bound thrombin plays a major role in the local generation of TAFIa, and is inhibited by the parenteral direct thrombin-inhibitor (DTI) hirudin [80]. A meta-analysis of randomized trials with parenteral DTI in ACS showed a significant reduction in MACE at 30 days compared to heparin, with variable effects on bleeding [82]. The reduction in MACE with intravenous direct DTI compared to heparin was more pronounced in patients undergoing early PCI (9.50% vs 13.86%, OR 0.64, 95% CI 0.47-0.89) [83]. Lepirudin and desirudin are the most potent parenteral DTI, while bivalirudin and argatroban have much lower thrombin-inhibition activity [84]. Marketing of lepirudin was withdrawn in 2012, and desirudin is not commercially available in the United States since 2019. Besides major bleeding, other issues of concern are the rebound activation of coagulation after discontinuation of hirudin, and allergic-reactions including anaphylaxis [85], [86]. To overcome some of the major limitations of recombinant hirudin, recently an alternative cell-free synthesis of hirudin (WT-HV1) has been described, which has several-fold greater potency on thrombin inhibition [87].

5.2. Vitamin K-antagonist

Plasma obtained from patients on treatment with warfarin for various indications promoted fibrinolysis by reducing TAFIa generation in-vitro, however paradoxically, higher circulating levels of TAFIa/TAFIai were observed in patients compared to controls, the significance of which is unclear [88]. Furthermore, prothrombin time-international normalized ratio level is unsuitable to monitor changes in impaired fibrinolysis, as they do not correlate with clot lysis-time [88].

5.3. Direct factor Xa-inhibitors and oral DTI

The parenteral factor Xa-inhibitor fondaparinux inhibited microparticles-induced thrombin generation in in-vitro studies [89]. It has been demonstrated to promote fibrinolysis by modification of clot structure with the formation of loosely-packed fibrin, and to a lesser extent by inhibition of TAFIa [90]. The oral factor Xa-inhibitors rivaroxaban and apixaban exert their profibrinolytic effect primarily by enabling the function of the factor Xa cleavage product FXaβ, a co-factor in tissue plasminogen activator-mediated plasminogen activation [91]. TAFIa, which is elevated in ACS is a potent inhibitor of tissue plasminogen activator – plasminogen complex activity, and may thus offset some of the profibrinolytic effects of oral factor Xa-inhibitors. This may explain to an extent why the oral DTI dabigatran promotes fibrinolysis to a greater degree than the oral factor Xa-inhibitors rivaroxaban or apixaban, as it has a greater effect on TAFIa inhibition [92]. In therapeutic concentrations, dabigatran increased sensitivity to clot-lysis by ˃4-fold and accelerated clot lysis-time by ≥50% [93]. Dabigatran inhibits both free and clot-bound thrombin [94]. In a study of 19 patients with stroke and non-valvular atrial fibrillation who were prescribed dabigatran, blood samples obtained after treatment with dabigatran demonstrated significantly enhanced clot-lysis compared to baseline [95]. Although the sample size was small, the study provides preliminary direct evidence of profibrinolytic effect in clinical setting with dabigatran treatment.

Despite dabigatran being a potent inhibitor of TAFIa with profibrinolytic effects, no benefit on MACE was seen in the RE-DEEM trial [19]. Whether this could be related to the timing of initiating therapy in the trial may need examination, in which treatment was started at a mean of 7.5 days after ACS. However in ACS, TAFIa levels have been demonstrated to peak by day 1 of presentation [44]. Also, tissue-factor expressing monocytes (promote thrombin formation and increased generation of TAFIa) that are known to impair fibrinolysis are detectable in ACS in high concentrations even at presentation [45], [46].

5.4. Other profibrinolytic agents

Other agents that are under evaluation and may have potential profibrinolytic effects include the low-molecular weight imidazole-derivative DS-1040 [96], [97], [98], [99], monoclonal antibodies and nanobodies that are highly-specific against TAFI [100], [101], [102], monoclonal antibodies to alpha2-antiplasmin [103], [104] and PAI-1 [105], the complement C3 inhibitor cyclic peptide compstatin [106], [107], PAI-1 antagonist PAItrap [108] and clopidogrel [109], [110], microparticle release suppression by clopidogrel [111], [112] and rosuvastatin [113], antisense oligonucleotide to lower elevated lipoprotein (a) levels [114], and the antiplatelet agents aspirin and cangrelor [115], [116].

6. Conclusion

Despite treatment with dual antiplatelet agents, oral anticoagulants, and optimal revascularization strategies, patients with acute coronary syndrome have a high risk of residual ischemic events. Thus, factors other than activated platelets and vulnerable atherosclerotic lesions may underlie the pathogenesis of the persistent risk of ischemic events. Impaired fibrinolysis in acute coronary syndrome is a strong independent risk factor for cardiovascular mortality and major adverse cardiovascular events. Several studies demonstrate evidence of significantly elevated levels of endogenous fibrinolysis inhibitors in patients with acute coronary syndrome, notably in intracoronary blood samples from culprit vessels obtained during percutaneous coronary intervention. A substantial number of pharmacological agents targeting endogenous fibrinolysis inhibitors have shown potent profibrinolytic effects in experimental studies, and some in small clinical studies. Current research in antithrombotic therapy for acute coronary syndrome appears to be focused primarily on the development of more potent antiplatelet agents [117]. However, modulation of impaired fibrinolytic state in acute coronary syndrome has received sparse attention.

Given that literature supports the notion that impaired fibrinolysis is a strong independent risk factor for major adverse cardiovascular events, that it is documented that current therapy can have an impact upon fibrinolysis, and that endogenous fibrinolysis inhibitors have been identified, there is an exigent need to transition from experimental studies to clinical investigations to target fibrinolysis enhancement as a therapeutic approach. Profibrinolytic therapy to modulate impaired fibrinolysis has the potential to emerge as an exciting approach in the management of patients with acute coronary syndrome.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.