The susceptibility of the fibrin network to lysis is dictated by its structure (21). Several post-translational modification processes were found responsible for fibrin network maturation and fibrinolysis resistance. Post-translational modifications are (bio)chemical reactions in which amino-acid residues of proteins or peptides are covalently modified (22). This process significantly increases the structural and functional diversity of proteins, thus introducing a complexity to the proteome (23). Concomitant with converting fibrinogen to fibrin, thrombin activates transglutaminase factor XIII. Activated factor XIII (factor XIIIa) contributes to the conversion of the initially loose fibrin network into the firm insoluble polymer capable of providing biophysical and biochemical support to the thrombus (24). Factor XIIIa also inhibits fibrinolysis by forming fibrin–α2 anti-plasmin cross-links that prevent this plasmin inhibitor from being fully expelled from the thrombus during compaction/retraction (25). Like α2 anti-plasmin, PAI-2, which is derived from macrophages, can also be cross-linked to fibrin by factor XIIIa (26). An in vitro study found that under experimental conditions that increased the rate of non-enzymatic protein glycosylation, fibrin networks were associated with correspondingly greater degrees of resistance to degradation by plasmin (27). As noted above, the fibrin network found at the surface of AIS thrombi showed an altered morphology. Fibrin in the shell of AIS thrombi indeed displays a sealed aspect that strikingly resembles that of fibrinolysis-resistant, dense, matted fibrin deposits produced by various modifications of fibrin, such as oxidation (28), carbamylation (29), and exposure to platelet factor 4 (30) or air (31). Together, these findings indicate that in addition to factor XIIIa-mediated cross-linking of the fibrin network, other chemical modifications likely help to increase the resistance of AIS thrombi to fibrinolysis.

In addition to chemical modifications, mechanical compaction and/or deformation also affect the susceptibility of the fibrin network to tPA-mediated fibrinolysis. Thrombus contraction indeed impairs external fibrinolysis by limiting thrombus permeability and thus the accessibility of fibrin fibers to fibrinolytic agents (25). Of note, platelets, which are essential for thrombus retraction, were recently found required for AIS thrombus shell formation (14). Thus, the compaction of fibrin fibers with other thrombus components in the shell of AIS thrombi might result from a platelet-mediated mechanical effect.

Platelets are crucial to the fibrinolysis response (32). Platelet aggregation plays a central role in thrombus formation, as an anchor for the initiation and growth of the thrombus scaffold. Interaction of platelets with the exposed components of the injured or activated vessel wall generates the initial plug. When platelets bind matrix proteins or activated endothelial cells, they undergo rapid activation to release coagulation factors into the surrounding milieu. Upon activation, platelets release more than 300 proteins, most arising from their secretory α-granules. Several fibrinolytic inhibitors exist within platelet α-granules and as a result, platelet-rich thrombi are reportedly more resistant to tPA-mediated lysis than are RBC-rich thrombi (33). PAI-1 is abundant in α-granules and is released upon platelet activation, accounting for most of the circulating PAI-1 (34). In addition, platelets change both the shape and composition of their membrane, which allows for direct interaction with coagulation proteins, circulating fibrinogen and neutrophils. Finally, platelets undergo a reorganization of their cytoskeleton, mainly because of activation of the fibrin–αIIbβ3–myosin axis allowing them to exert a contractile force on extracellular fibrin fibers responsible for thrombus retraction (35). As compared with non-retracted thrombi, retracted thrombi are more resistant to fibrinolytic degradation (36) and lyse more slowly (37). In an experimental model of embolic stroke in rats, platelet-rich thrombi resulted in high rates of middle cerebral artery occlusion, low rates of spontaneous thrombolysis, and large infarction. These platelet-rich thrombi were highly resistant to tPA (38). A clinical study found that patients with platelet-rich thrombi had poorer revascularization outcomes than those with RBC-rich thrombi. Platelet and vWF levels in AIS thrombi were correlated with each other and both were inversely related to the amount of RBCs (39).

Neutrophil extracellular traps are fibrous networks of extracellular DNA released by neutrophils in response to stimuli; they are in the form of decondensed chromatin associated with citrullinated histones and neutrophil granule proteins (40). In recent years, the important contribution of neutrophils, particularly via their NETs, to thrombus pathogenesis has been increasingly recognized (41, 42). NETs provide a physical scaffold for thrombus growth by binding platelets and RBCs (43). Recent immunostaining analyses found that every AIS thrombus contained NETs. A study found a significantly higher amount of NETs in AIS thrombi from a cardiac than non-cardiac origin (8). NETs were predominantly located in platelet-rich areas that include the outer layer of AIS thrombi (7, 15). In ex vivo thrombolysis assays, tPA and deoxyribonuclease 1 (DNAse 1) incubation accelerated thrombolysis as compared with tPA or DNAse 1 alone (7, 8). More recently, we confirmed in a new ex vivo study that the ability of DNAse 1 to improve tPA-mediated thrombolysis was linked to a potentiation of fibrinolysis, as indicated by increased release of plasmin-generated D-dimers in the presence of DNAse 1 (44). These data confirm that NETs contribute to thrombus composition regardless of their origin and participate in fibrinolysis resistance.

Regarding resistant thrombi to EVT, those are rare, both in our experience and in large recent series of EVT-treated patients, with proximal recanalization rates now nearing 90% (45). Data on the characteristics of those remaining 10% proximal thrombi that are resistant to both thrombolysis and EVT are scarce. Nonetheless, information on this thrombus category may be extrapolated from analyses of thrombi that were particularly difficult to retrieve mechanically. NET thrombus content was found to be positively correlated with endovascular procedure length and device number of passes (7). In a recent case report by Staessens et al., the authors described the histological features of a thrombus that had required 11 attempts to be retrieved mechanically. This nearly EVT-resistant thrombus was characterized by a core that was particularly rich in extracellular DNA, vWF, and microcalcifications (46). With the exception of the presence of microcalcifications, comparable observations were made by Abbasi et al. who reported increased levels of NETs and vWF in thrombi with delayed time to recanalization, as compared to thrombi with early recanalization time (47). Considering that longer recanalization delay is associated with a reduced recanalization success rate, the results from these recent studies converge to support the hypothesis that NETs, vWF, and extracellular DNA with microcalcifications favor EVT resistance.

von Willebrand factor is a large multimeric plasma glycoprotein and plays a crucial role in arterial thrombus formation. Particularly, vWF recruits platelets at sites of vascular injury by acting as a molecular bridge between circulating platelets and exposed components of the subendothelium (48). Together with fibrin(ogen), vWF links platelets together, which further stabilizes platelet aggregates. High vWF level and low level of a disintegrin-like, metalloprotease thrombospondin type 1 motif, and member 13 (ADAMTS13) in plasma were identified as important risk factors for AIS (49, 50). After immunostaining analysis of AIS thrombi, one study found 20% vWF content, which was inversely correlated with thrombus RBC content (12). In addition, reduced plasma ADAMTS13 activity was associated with poor response to recanalization therapies (51).

Tenecteplase is a genetically modified form of human tPA. These mutations confer 15-fold higher fibrin specificity than the recombinant human form of tPA (alteplase) and reduced binding to PAI-1, which leads to greater resistance to its inactivation (52). In patients with AIS, tenecteplase consumed less plasminogen and fibrinogen than did alteplase (53). Tenecteplase is also associated with an ~10-fold prolonged plasma half-life when compared with alteplase (52). This new pharmacokinetic property allows for a unique IV bolus injection when compared with the continuous 1-h infusion required with alteplase therapy. Of note, AIS trials suggest that tenecteplase may be associated with lower hemorrhagic transformation (HT) risk than alteplase but with a similar or superior recanalization rate (54). A randomized trial revealed that IV tenecteplase before EVT was associated with a significantly higher incidence of reperfusion and better functional outcome than with IV alteplase (55). Several international randomized clinical trials are still ongoing to confirm this superiority of tenecteplase over alteplase. The last US and EU guidelines stipulate that tenecteplase might be considered an alternative to alteplase before EVT (56, 57).

To date, only a few preclinical studies have explored this therapy. Nanomedical approaches for local or targeted delivery of thrombolytic drugs have aroused growing interest (58). However, more in vivo studies are needed to consider this strategy in the future.

Targeting anti-fibrinolytic proteins to boost endogenous fibrinolysis, raise the success of plasminogen activators, and lower the required therapeutic dose constitutes a promising approach for improving AIS treatment (59).

In the past three decades, many research groups and pharmaceutical companies have invested in the development of fibrinolysis inhibitors, such as mainly thrombin-activated fibrinolysis inhibitor (TAFI) and/or PAI-1 inhibitors. However, the question is still whether fibrinolysis inhibitors could be considered a new treatment strategy for improving fibrinolysis.

Several experimental studies showed a clear benefit of TAFI and/or PAI-1 inhibition but mainly in transient occlusion models of AIS (60–62). This impact was induced by both reduced microvascular thromboinflammation and better collateral perfusion. In a thromboembolic model of AIS, a TAFI inhibitor in association with reduced tPA dosage was associated with reduced ischemic lesion growth as compared to full tPA dosage alone. In this study, the TAFI inhibitor alone had no impact (63).

Two ongoing phase 1–2 clinical trials are assessing the safety of a TAFI inhibitor infusion. The first is recruiting non-selected patients with AIS and the second one focuses on AIS treated by EVT. Both have a primary end point of safety (ClinicalTrials.gov: NCT03198715). To date, to our knowledge, no PAI-1 inhibitor has been tested clinically.

Besides PAI-1, PN-1 represents another platelet-derived serpin with the ability to inhibit tPA-mediated fibrinolysis (64). Inhibitory single-domain antibodies to PN-1 were recently developed and might offer an additional therapeutic option to potentiate fibrinolysis (65). Nonetheless, as compared to PAI-1 inhibition, PN-1 inhibition might be associated with an increased risk of adverse effects, because PN-1 is a dual serpin that limits not only fibrinolysis but also thrombosis (66).

As specified previously, factor XIIIa plays a crucial role in thrombus stability and could represent an interesting target to improve the tPA-induced thrombolysis. In this perspective, a recent preclinical study using a murine model where fibrin cross-linking by factor XIIIa was reduced demonstrated that this strategy might be associated with an increased risk of thrombus fragmentation and embolization without affecting thrombus size or its susceptibility to lysis (67).

Emerging experimental and clinical data demonstrate that platelets have a dual role in the setting of AIS. Undeniably, platelets play a key role in thrombus formation and resistance to thrombolysis. Nevertheless, platelets are also essential to maintain vascular integrity and prevent HT after AIS.

In the early 2000s, two clinical trials failed to demonstrate the benefit of αIIbβ3 inhibitors alone (abciximab and tirofiban) in AIS. In contrast, these trials showed that αIIbβ3 inhibitor infusion increased the HT rate, with significantly worse neurological outcomes (68, 69).

A still growing number of studies are highlighting potentially important differences in the pathways mainly involved in platelet aggregation in physiological hemostasis vs. pathological thrombosis. These studies raise the prospect of developing new antiplatelet drugs that specifically target mechanisms in thrombosis with a low risk of bleeding because they are dispensable for hemostasis. Platelet glycoprotein VI is a typical example of a platelet pathway that is dispensable for physiological hemostasis but critical for thrombus formation and growth. A therapeutic antibody to platelet glycoprotein VI, glenzocimab, was recently tested in healthy volunteers, showing a dose-dependent inhibition of collagen-induced platelet aggregation without affecting template bleeding times (70). A phase 2 randomized trial currently assessed the safety of glenzocimab infusion on the top of IV tPA in patients with AIS (ClinicalTrials.gov: NCT03803007).

Another strategy could be to administer a reversible antiplatelet drug with a short plasma half-life associated with early restoration of platelet function to prevent HT occurrence. This is now possible with cangrelor. This new platelet P2Y12 receptor inhibitor has a plasma half-life of about 5 min, and complete restoration of platelet aggregation is obtained approximately 1 h after the end of IV cangrelor infusion (71). A phase 3 randomized trial is currently assessing the benefit of cangrelor infusion in imaging-selected patients with AIS who were eligible for EVT (ClinicalTrials.gov: NCT04667078).

The other strategy to enhance thrombolysis in AIS could be to target non-fibrin extracellular network components. Platelet cross-linking during thrombosis involves vWF multimers. Therefore, proteolysis of vWF multimers appears promising to disaggregate platelet-rich thrombi and restore vessel patency in AIS.

The first study from Denorme et al. (12) found that AIS thrombi contained about 20% of vWF. The authors suggested that targeting vWF with the specific vWF-cleaving metalloprotease ADAMTS13 could have a thrombolytic effect in an experimental thromboembolic model of AIS associated with reduced infarct volume (12).

More recently, a study assessed a potent thrombolytic effect of N-acetylcysteine (NAC, a clinically approved mucolytic drug). NAC has the ability to break up vWF multimers by reducing intrachain disulfide bonds in large polymeric proteins. This study found an increased recanalization rate after NAC vs. saline infusion, especially in the case of concomitant treatment with an αIIbβ3 inhibitor, which suggested a synergistic action between these two treatments (13). The NAC for thrombolysis in acute stroke (NAC-S) trial (ClinicalTrials.gov: NCT04920448) is an ongoing phase 2 randomized study that assesses the safety of NAC combined with IV tPA in an AIS setting.

Three ex vivo studies found that recombinant DNAse 1 accelerated tPA-induced thrombolysis, whereas tPA alone was ineffective (7–9). These results indicate that co-administration of DNAse 1 and tPA could be of interest in the setting of AIS with large vessel occlusion. These results must be confirmed in future clinical studies. Recently, advances have been made regarding the role of DNase 1 as an adjuvant to tPA for thrombolysis of AIS thrombi. The ability of DNase 1 to improve tPA-mediated thrombolysis is linked to a potentiation of fibrinolysis, as indicated by increased release of plasmin-generated D-dimers in the presence of DNase 1 (44). As a result of these findings, a first clinical trial to assess the safety and efficacy of DNase 1 in patients with AIS who received IV tPA and are eligible for EVT (NETs-TARGET study, ClinicalTrials.gov: NCT04785066) will be launched in the coming months. Of note, the enzyme responsible for the NET formation, peptidylarginine deiminase type IV, was recently found released together with NETs into the extracellular milieu, thus leading to citrullination and inactivation of ADAMTS13 and impairing vWF multimer cleavage (72). This observation suggests that NETosis may result in increased resistance to not only tPA but also ADAMTS13. Therefore, peptidylarginine deiminase type IV inhibitors, several already developed (73), may be of clinical interest to improve the efficacy of thrombolytic cocktails combining tPA, ADAMTS13, and DNase 1.