Tenecteplase and the Future of Thrombolytics: A Practical Update for the Evolving Stroke Era

Tissue plasminogen activator (tPA), a naturally occurring serine protease produced by endothelial cells, promotes fibrinolysis by converting plasminogen to plasmin at sites of fibrin-rich thrombi.¹ Alteplase (Activase; Genentech, South San Francisco, CA), a recombinant form of tPA, is engineered to mimic the endogenous enzyme. Alteplase exhibits fibrin specificity by preferentially activating plasminogen that is bound to fibrin within thrombi, rather than circulating plasminogen.¹ At the site of the thrombus, it cleaves fibrin crosslinks, generating fibrin degradation products and facilitating thrombus dissolution.¹

The efficacy of alteplase is significantly influenced by the age and composition of the thrombus. Fresh thrombi are more susceptible to fibrinolysis, owing to greater exposure of fibrin-binding sites, less established fibrin crosslinking, reduced fibrotic remodeling, and higher concentrations of endogenous plasminogen.² As thrombi age, these factors shift, rendering the clot more resistant to enzymatic degradation.² These time-sensitive changes help explain why earlier administration of alteplase is associated with better clinical outcomes and higher recanalization rates.²

Pharmacologic and Logistical Limitations of Alteplase
Alteplase has proven efficacy, but is limited by its short half-life of <6 minutes.¹ Pharmacokinetic studies show that alteplase serum levels rise quickly after bolus administration but decline sharply due to rapid plasma clearance (550–690 mL/min), necessitating an immediate continuous infusion to maintain a steady serum concentration.³ Even delays of >5 minutes between bolus and infusion result in a precipitous drop in drug concentration.⁴ Even under optimal conditions, the steady-state concentration may be low due to the drug’s rapid clearance. This creates logistical challenges in busy emergency departments and for individuals undergoing a “drip-and-ship” transfer to thrombectomy-capable centers.

The issues with low alteplase serum concentration also affect penetrance into the thrombus. Because its diffusion into thrombi is limited by rapid serum clearance and low effective serum concentrations, alteplase primarily interfaces with the thrombus surface.⁵ This limits the drug’s ability to interface with the deep fibrin matrix, reducing the likelihood of breaking up the thrombus. These limitations led to interest in exploring alternative thrombolytic agents for acute reperfusion therapy.

Optimizing Thrombolysis Through Molecular Design
Tenecteplase (TNKase; Genentech, South San Francisco, CA) (TNK) offers multiple pharmacokinetic and pharmacodynamic advantages for the treatment of individuals with ischemic stroke. TNK was engineered with 3 key point mutations to resist inactivation by plasminogen activator inhibitor-1 (PAI-1), limit systemic degradation, and enhance enzyme stability.^5,6 These modifications result in a significantly longer half-life, enabling one-time bolus administration and reducing infusion-related logistical complexity.⁵ TNK achieves higher effective serum concentrations than alteplase, allowing deeper penetration into the clot and stronger fibrin binding, especially in large vessel occlusions (LVOs).⁷ Its superior fibrin specificity (~15-fold higher) and markedly reduced susceptibility to PAI-1 (by ~80-fold) may further reduce systemic activation of plasminogen and the risk of hemorrhagic complications.⁶ The Figure illustrates the mechanistic differences between alteplase and TNK.

Figure. Mechanisms of action of alteplase and tenecteplase in ischemic stroke. Alteplase binds to fibrin within the thrombus and catalyzes the conversion of plasminogen to plasmin, initiating fibrinolysis. It has relatively low fibrin specificity and is susceptible to inhibition by plasminogen activator inhibitor-1 (PAI-1). Alteplase exhibits primarily superficial infiltration into the clot and is associated with greater systemic fibrinolytic activity (A). Tenecteplase demonstrates ~15-fold higher specificity for fibrin-bound plasminogen and an 80-fold reduction in binding to PAI-1, leading to greater resistance to inactivation. This allows for more localized, efficient thrombolysis with deeper penetration into the clot matrix (B).
Created in BioRender. Bugbee, E. (2025) https://BioRender.com/ffzdxag

Due to robust evidence from randomized controlled trials (RCTs), TNK has been adopted into many international guidelines as a noninferior and safe substitute for alteplase, including the Canadian Stroke Best Practice Recommendations, the European Stroke Organisation recommendations, and the National Clinical Guideline for Stroke for the UK and Ireland.^{6, 8-10} A recent meta-analysis of RCTs studying TNK vs alteplase demonstrated a small improved chance of excellent functional outcomes with TNK when thrombolysis is administered within the 4.5-hour window, with comparable rates of adverse events.¹¹ The greater fibrin specificity of TNK provides a rationale for potentially lower hemorrhagic risk, but clinical data have not shown a significant difference in the risk of symptomatic intracerebral hemorrhage compared with alteplase.¹²

TNK treatment is associated with various workflow advantages. Real-world evidence shows that door-to-needle times are shorter with TNK treatment compared with alteplase due to the ease of bolus-only administration.¹² This ease of use also facilitates thrombolytic delivery in mobile stroke units—specialized ambulances equipped with onboard CT scanners—enabling faster, prehospital treatment initiation.⁶

Optimal TNK Dose
Various clinical trials are assessing TNK dose optimization. Robust data support a TNK dose of 0.25 mg/kg. Phase 3 trial results (the second Alteplase-Tenecteplase Trial Evaluation for Stroke Thrombolysis [ATTEST-2, NCT02814409], Tenecteplase Reperfusion Therapy in Acute Ischemic Cerebrovascular Events-II [TRACE-2, NCT04797013], and Tenecteplase vs Alteplase for Patients With Acute Ischemic Stroke [ORIGINAL, NCT04915729]) demonstrated noninferiority of TNK vs alteplase treatment for 90-day outcomes. Further support for this dosing came from the Canadian Alteplase Compared to Tenecteplase in Patients With Acute Ischemic Stroke (AcT, NCT038889249) trial, a multicenter RCT that confirmed the noninferiority of TNK at 0.25 mg/kg compared with alteplase in a broad population of people who experienced a stroke.¹³ Early trials, including the Study of Tenecteplase in Acute Ischemic Stroke (TNK-S2B, NCT00252239), found that 0.4 mg/kg TNK increased symptomatic intracerebral hemorrhage (sICH) risk compared with standard-dose alteplase, and subsequent trials (The Norwegian Tenecteplase Stroke Trial 2 [NOR-TEST 2, NCT03854500], Determining the Optimal Dose of Tenecteplase Before Endovascular Therapy for Ischaemic Stroke [EXTEND-IA TNK Part 2, NCT03340493]) reinforced signals of harm at this dose.¹⁴

At the other end of the spectrum, the lowest dose of TNK studied, 0.1 mg/kg, demonstrated the most favorable safety profile. In the TNK-2SB study, the 0.1 mg/kg dose showed acceptable outcomes, and TNK-Tissue-Type Plasminogen Activator Evaluation for Minor Ischemic Stroke With Proven Occlusion (TEMPO-1, NCT01654445), a trial evaluating TNK in minor strokes (National Institutes of Health Stroke Scale score <5, last seen well <12 hours), reported 0% sICH in the 0.1 mg/kg arm, compared with 4% in the 0.25 mg/kg arm.^14,15 However, results of the Tenecteplase vs Alteplase for Acute Ischaemic Stroke (TAAIS, ACTRN12608000466347) trial demonstrated superior radiographic reperfusion in the 0.25 mg/kg arm compared with the 0.1 mg/kg arm.¹⁶ To balance safety and efficacy, the ACT-GLOBAL THROMBOLYSIS (ACT-WHEN-001) Domain Within the ACT-GLOBAL Adaptive Platform Trial (ACT-WHEN, NCT06320431), a substudy of A Multi-Factorial, Multi-Arm, Multi-Stage, Randomised, Global Adaptive Platform Trial for Stroke (ACT-GLOBAL, NCT06352632), is testing an intermediate TNK dose of 0.18 mg/kg (ie, two-thirds of 0.25 mg/kg). Results of the Enhanced Control of Hypertension and Thrombolysis Stroke Study (ENCHANTED, NCT01422616) showed that treatment with 0.6 mg/kg alteplase reduced sICH vs treatment with 0.9 mg/kg, but did not meet noninferiority for functional outcomes. Analysis of subgroup data demonstrated better safety in individuals on antiplatelets. This two-thirds TNK strategy aims to pair the safety of 0.1 mg/kg with the efficacy seen at 0.25 mg/kg to optimize outcomes across a broader population of individuals with stroke.¹⁷

Expanding the Thrombolysis Window: TNK at the Edge
Nearly a decade ago, individuals with stroke presenting beyond the 4.5-hour window were deemed ineligible for thrombolysis under time-based guidelines, excluding a majority of people with stroke from potential treatment. This paradigm began to shift with results of the Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke (WAKE-UP, NCT01525290) and Extending the Time for Thrombolysis in Emergency Neurological Deficits (EXTEND, NCT01492725) trials, which demonstrated that imaging-based selection could safely expand treatment eligibility.¹⁵ WAKE-UP used MRI diffusion–fluid-attenuated inversion recovery mismatch to identify individuals with stroke likely within the therapeutic window despite unknown onset. The EXTEND trial applied CT or MRI perfusion–diffusion mismatch to extend alteplase administration in people with stroke up to 9 hours postonset, showing improved outcomes in individuals with salvageable tissue.¹⁸

These landmark studies laid the foundation for a transition from rigid, time-based eligibility to a more tissue-based approach. Given its favorable pharmacokinetics, TNK has emerged as an attractive treatment candidate for extended-window thrombolysis. Two trials evaluated TNK in extended windows: Tenecteplase in Wake-up Ischaemic Stroke Trial (TWIST, NCT03181360), which treated wake-up stroke within 4.5 hours of awakening using noncontrast CT without perfusion selection, and Tenecteplase in Stroke Patients Between 4.5 and 24 Hours (TIMELESS, NCT03785678), which treated patients 4.5 to 24 hours after last known well using CT or MRI perfusion selection.

The results of both studies confirmed the safety of TNK treatment in their respective populations, but failed to demonstrate significant functional improvement compared with controls.¹⁵ In contrast, the Tenecteplase Reperfusion Therapy in Acute Ischemic Cerebrovascular Events-III (TRACE III, NCT05141305) trial enrolled individuals with perfusion mismatch within 4.5 to 24 hours of stroke onset who did not undergo thrombectomy, and reported improved functional outcomes in individuals treated with TNK.¹⁵ Complementing these results, a meta-analysis of RCTs assessing intravenous thrombolysis beyond 4.5 hours in individuals not undergoing thrombectomy found that TNK treatment was associated with higher odds of achieving excellent functional outcomes compared with alteplase.¹⁸

TNK Use in People on Anticoagulation
About 1 in 5 thrombolysis-eligible individuals with acute ischemic stroke (AIS) are taking a direct oral anticoagulant (DOAC).¹³ Although guidelines list recent DOAC use as a relative contraindication, emerging data challenge this notion. A multicenter international registry found lower sICH rates among DOAC-treated individuals who received thrombolysis vs controls without DOAC.¹⁹ A 2023 meta-analysis likewise reported lower sICH rates and better outcomes after thrombolysis despite recent DOAC ingestion and measurable drug levels.²⁰ Ongoing studies, including ACT-WHEN and Safety and Efficacy of Intravenous Thrombolysis in Patients With Ischemic Stroke and Direct Oral Anticoagulants Intake (DO-IT, NCT06571149), should clarify the safety profile of thrombolysis in DOAC-treated individuals and may inform future guideline updates.^17,21

A major barrier to safe thrombolysis in this population remains the lack of reliable bedside assessment of anticoagulant effects. Conventional international normalized ratio testing is inadequate for assessment of DOAC effects. Point-of-care alternatives, such as DOAC dipsticks, anti-Xa assays, or thrombin time testing, show promise, but are limited by availability.²² Mass spectrometry remains the most accurate testing method to assess anticoagulant effects, but is impractical due to time constraints.²² As diagnostics improve, stroke protocols must adapt to integrate point-of-care testing and individualized risk assessment.

Meanwhile, the field of anticoagulation is evolving, with factor XIa inhibitors such as asundexian (Bayer AG,  Leverkusen, Germany) and milvexian (Bristol Myers Squibb, New York, NY; Janssen Pharmaceutica NV, Beerse, Belgium) currently being evaluated in late-phase trials.²³ These agents exhibit robust efficacy in preventing venous and arterial thrombosis, coupled with a favorable bleeding profile, having a minimal impact on hemostasis even with dual-antiplatelet therapy.²³ The adoption of factor XIa inhibitors for the treatment of atrial fibrillation and stroke prevention raises key questions about the compatibility of these agents with thrombolysis.

What’s Next on the Horizon for Reperfusion Therapy?
Combining thrombolysis with neuroprotection is an emerging strategy to reduce reperfusion injury and improve outcomes in individuals with AIS. A leading treatment candidate in this population is nerinetide (NoNO Inc., Toronto, Ontario, Canada), a neuroprotective peptide that disrupts postsynaptic density protein 95 (PSD-95) signaling.²⁴ In preclinical models, nerinetide reduced infarct size, particularly when administered within 3 hours of ischemia onset and in the setting of reperfusion.²⁵ However, 3 clinical trials (Safety and Efficacy of Nerinetide [NA-1] in Subjects Undergoing Endovascular Thrombectomy for Stroke [ESCAPE-NA1, NCT02930018], Efficacy and Safety of Nerinetide in Participants With Acute Ischemic Stroke Undergoing Endovascular Thrombectomy Excluding Thrombolysis [ESCAPE-NEXT, NCT04462536], and Field Randomization of Nerinetide [NA-1] Therapy in Early Responders [FRONTIER, NCT02315443]) did not meet their primary end points. These trials diverged from animal models by extending the treatment window to 12 hours and including individuals with permanent vessel occlusion. In the ESCAPE-NA1 trial, more than half of the participants received alteplase before nerinetide; subsequent studies revealed that plasmin generated by alteplase cleaves nerinetide, eliminating its therapeutic effect.²⁴ A post hoc meta-analysis of individuals treated within 3 hours of symptom onset showed that nerinetide was associated with significant improvements in functional outcomes, stroke progression, and infarct volume, supporting its potential benefit in early reperfusion settings.²⁴ To test this hypothesis prospectively, the NoNO-42 Trial in Acute Ischemic Stroke Patients Selected for Thrombolysis With or Without Endovascular Thrombectomy (ACT-42, NCT06403267), a domain of the ongoing ACT-GLOBAL adaptive platform trial, is currently evaluating nerinetide in individuals presenting within 3 hours of ischemic stroke onset.¹⁷

The ACT-WHEN substudy of the ongoing ACT-GLOBAL trial is poised to answer several important questions in thrombolysis. Current data suggest minimal clinical differences between 0.1 mg/kg and 0.25 mg/kg dosing, and large trials indicate that a two-thirds dose may be as safe as, and possibly more effective than, the standard dose in individuals on antiplatelet therapy. ACT-WHEN will also clarify the best approach for individuals taking DOACs by comparing 3 strategies: no thrombolysis, two-thirds-dose TNK, and standard-dose TNK.¹⁷

The ACT-WHEN sub-arm will also test whether to give thrombolysis before thrombectomy for individuals with LVO.¹⁷ Bridging thrombolysis with TNK may increase early recanalization, but risks distal embolization beyond thrombectomy reach, and any benefit may be time-dependent, with some meta-analyses favoring earlier presenters (<120 minutes). In Endovascular Thrombectomy With Versus Without Intravenous rhTNK‐tPA in Stroke (BRIDGE-TNK, NCT04733742), an open-label trial in China, people with LVO within 4.5 hours randomized to TNK plus thrombectomy had better 90-day outcomes than those receiving thrombectomy alone.²⁶ ACT-WHEN will add dose-stratified evidence to guide bridging decisions.¹⁷

Intra-arterial thrombolysis is a catheter-directed, low-dose thrombolytic given during thrombectomy to clear residual or distal clots. Results of early studies were inconsistent, but recent trials (Intra‐Arterial Recombinant Human TNK Tissue‐Type Plasminogen Activator Thrombolysis for Acute Large Vascular Occlusion After Successful Mechanical Thrombectomy Recanalization [ANGEL-TNK, NCT05624190], Intra‐Arterial Tenecteplase After Successful Endovascular Recanalisation in Patients With Acute Posterior Circulation Arterial Occlusion [ATTENTION-IA, NCT05684172], Intraarterial Alteplase versus Placebo After Mechanical Thrombectomy [CHOICE, NCT03876119], Optimal Dosage of Adjunctive Intra‐Arterial Tenecteplase Following Successful Endovascular Thrombectomy in Patients With Large Vessel Occlusion Acute Ischemic Stroke [DATE, ChiCTR2400080624], Intra‐Arterial Alteplase for Acute Ischaemic Stroke After Mechanical Thrombectomy [PEARL, NCT05856851], Adjunctive Intra‐Arterial Tenecteplase Following Near‐Complete to Complete Reperfusion for Large Vessel Occlusion Stroke [POST-TNK, ChiCTR2200064809], and Adjunctive Intra‐Arterial Urokinase After Near‐Complete to Complete Reperfusion for Acute Ischemic Stroke [POST-UK, ChiCTR2200065617]) showed promising results. A pooled meta-analysis of these trials demonstrated that intra-arterial thrombolysis after thrombectomy increased the odds of excellent 90-day outcomes without significant increases in sICH.²⁷ The REACT sub-arm of the ongoing ACT-GLOBAL adaptive platform trial is being performed to evaluate intra-arterial TNK and alteplase across all post–endovascular thrombectomy reperfusion grades. 

Heterogeneous thrombus composition drives variable lysis. Strategies targeting distinct elements of the thrombotic process (eg, DNase for neutrophil extracellular traps, P2Y12 and thrombin inhibitors, and von Willebrand factor–directed approaches) have shown mixed results, and other strategies are being investigated in ongoing trials.²⁸ Stroke care is entering a new, rapidly advancing era, with an encouraging outlook for thrombolytic therapy.

Conclusion
The evolution from alteplase to TNK treatment represents a key advance in thrombolytic therapy for individuals with AIS. TNK offers substantial pharmacologic and logistical advantages. Across RCTs and real-world cohorts, outcomes are generally at least comparable to those with alteplase, with signals of better functional recovery in some analyses at the 0.25 mg/kg dose and a similar safety profile. Nonetheless, several uncertainties remain. Optimal dosing, selection criteria for imaging-guided late windows, and safe use alongside contemporary antithrombotics are still being defined, with trials such as ACT-WHEN expected to clarify these points. Parallel lines of investigation are testing combination strategies with neuroprotective agents. Taken together, current evidence supports TNK as a credible first-line reperfusion option while these questions are being resolved.