Tenecteplase for Acute Ischemic Stroke at 4.5 to 24 Hours: A Meta-Analysis of Randomized Controlled Trials

Abstract

BACKGROUND:

Whether TNK (tenecteplase) benefits patients with acute ischemic stroke treated within 4.5 to 24 hours remains uncertain, and no previous meta-analysis has differentiated between clinical settings where endovascular thrombectomy (EVT) is unavailable or permitted, leading to pooled distinct clinical contexts and obscuring a clear estimation of TNK’s net effect.

METHODS:

We searched for randomized controlled trials comparing intravenous TNK of 0.25 mg/kg with standard care or placebo in adults within 4.5 to 24 hours after acute ischemic stroke onset. The primary outcome was excellent functional outcome (modified Rankin Scale score, 0–1) at 90 days, with additional efficacy and safety end points. A random-effects meta-analysis was performed both overall and within predefined subgroups, stratified by whether EVT was permitted in individual studies (non-EVT versus EVT-permitted).

RESULTS:

Four multicenter randomized controlled trials enrolling 1278 patients were included. TNK significantly increased excellent functional outcome (odds ratio [OR], 1.34 [95% CI, 1.06–1.71]; P=0.02) at 90 days and recanalization (OR, 3.30 [95% CI, 1.59–6.84]; P=0.001) compared with the control group, whereas good functional outcome (modified Rankin Scale score, 0–2), reperfusion, and early neurological improvement did not differ significantly. Subgroup analyses of 596 patients in the non-EVT subgroup showed that TNK significantly improved excellent functional outcome (OR, 1.46 [95% CI, 1.02–2.08]; P=0.04), good functional outcome (OR, 1.50 [95% CI, 1.07–2.09]; P=0.02), recanalization (OR, 6.17 [95% CI, 3.36–11.33]; P<0.00001), and early neurological improvement (OR, 3.21 [95% CI, 1.82–5.66]; P<0.0001). However, in the EVT-permitted subgroup of 682 patients, TNK only improved recanalization (OR, 2.36 [95% CI, 1.34–4.17]; P=0.003). No significant differences were observed between TNK and control in the risks of symptomatic intracerebral hemorrhage or 90-day mortality, either in the overall or subgroup analyses.

CONCLUSIONS:

TNK improves excellent functional outcomes and recanalization in patients with acute ischemic stroke treated within 4.5 to 24 hours, without increasing the risks of symptomatic intracerebral hemorrhage or mortality. Notably, extended-window TNK provides greater additional benefits when EVT is inaccessible, establishing its role as an alternative reperfusion strategy in resource-limited settings.

Ischemic stroke is characterized by high mortality and disability worldwide.¹ In recent years, TNK (tenecteplase) has gradually emerged as a pivotal option for intravenous thrombolysis (IVT) in acute ischemic stroke (AIS).² A recent meta-analysis has confirmed that in patients with AIS receiving IVT within 4.5 hours, TNK significantly increased the proportion of patients achieving excellent functional outcome at 90 days and reduced 3-month disability compared with alteplase.³ However, whether the advantages of TNK can be extended to patients beyond the standard time window (4.5–24 hours) remains a matter of debate, given that studies such as TIMELESS (Thrombolysis in Imaging Eligible, Late Window Patients to Assess the Efficacy and Safety of TNK)⁴ and TRACE-III (TNK Reperfusion Therapy in Acute Ischemic Cerebrovascular Events-III)⁵ trials have not yet arrived at consistent conclusions.

While endovascular therapy (EVT) has become a critical therapeutic modality for large vessel occlusion (LVO) stroke within 24 hours of symptom onset, the global accessibility of EVT stood at only 2.79%, with a mere 0.48% in low- and low-middle-income countries.⁶ Consequently, in resource-limited settings where EVT is unavailable, establishing the independent efficacy of intravenous TNK for extended-window stroke is critical and could potentially significantly broaden the population benefiting from thrombolysis in resource-poor regions. Some prior trials (eg, TRACE-III) enrolled only patients without access to EVT, whereas others such as TIMELESS included a subset who subsequently underwent EVT.^4,7 Given that EVT alone could achieve successful reperfusion in ≈75% of cases,^8,9 any thrombolytic benefit conferred by TNK may be masked by the subsequent efficacy of EVT; this may induce a dilution effect. Notably, no previous meta-analysis has systematically analyzed these 2 distinct study types (non-EVT versus EVT-permitted) as predefined subgroups to isolate TNK-specific effects. Therefore, it is necessary to accurately delineate the authentic efficacy and safety of TNK thrombolysis within 4.5 to 24 hours in these 2 distinct clinical scenarios to provide precise treatment guidance for regions with different medical resource allocations.

In this study, we, therefore, meta-analyzed all randomized controlled trials (RCTs) comparing TNK with standard care or placebo in adults within 4.5 to 24 hours after AIS onset to determine the safety and efficacy of ultrawindow TNK therapy. Furthermore, we first performed an EVT-stratified subgroup analysis (non-EVT versus EVT-permitted) to clarify whether the benefit-risk profile differed between the 2 groups. This analysis will not only clarify TNK’s standalone efficacy in resource-limited settings but also optimize combination therapy protocols for EVT-capable centers, ultimately improving stroke outcomes across diverse health care contexts worldwide.

Methods

Data Sharing Statement

Data are available from the corresponding author upon reasonable request.

Overall

This study was conducted in strict adherence to the latest Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement,¹⁰ and its protocol has been registered in the International Prospective Register of Systematic Reviews (registration ID: CRD420251090457). Because this study was a meta-analysis of aggregated data from published trials, neither ethical approval nor informed consent was required.

Eligibility Criteria

This meta-analysis included RCTs that enrolled adult patients with AIS within an extended time window (4.5–24 hours) after symptom onset, who were eligible for IVT based on imaging selection (P: population). The intervention group received TNK at a dose of 0.25 mg/kg (I: intervention), while the comparator group received standard medical treatment or placebo (C: comparator). The main outcomes included at least excellent functional outcomes (modified Rankin Scale [mRS] score, 0–1) at 90 days (O: outcome).

Data Sources and Search Strategy

A systematic literature search was performed in PubMed, Embase, the Cochrane Library, and ClinicalTrials.gov from inception to July 1, 2025, without language restrictions. We also manually searched reference lists of relevant reviews and conference proceedings to identify additional eligible studies. The full search strategy was formulated using the following key terms and combinations: (“ischemic stroke” OR stroke) AND (tenecteplase OR TNK) AND (“beyond 4.5 hours” OR “extended time window” OR “late window” OR “at 4.5 to 24 hours” OR “4.5–24 hours” OR “up to 24 hours” OR “after 4.5 hours”). The full search strategy and the results are detailed in Table S1.

Study Selection, Quality Control, and Bias Assessment

Two reviewers (Z.W. and Jiamin Li) independently screened titles, abstracts, and subsequent full texts against the predefined eligibility criteria. Discrepancies were resolved through consensus reached via discussion, with a third reviewer (Q.M.) consulted when necessary. Two reviewers (Jiameng Li and B.Y.) independently used the Cochrane Collaboration risk-of-bias (RoB 2) tool to evaluate the quality and risk of bias for each included randomized trial across 5 domains: randomization process, deviations from intended interventions, missing outcome data, measurement of the outcome, and selection of the reported results. Each domain was rated as low risk, some concerns, or high risk. Discrepancies were resolved through discussion, and the final judgments were summarized graphically.

Data Extraction

Two reviewers (Z.W. and X. Wang) independently extracted study-level variables into a prepiloted Microsoft Excel form. For each trial, we systematically recorded its formal designation, clinical phase, recruitment country, total number of participating sites, period of enrollment, and the complete set of clinical and neuroimaging eligibility criteria. Within each treatment group (TNK versus control), we extracted aggregated participant data, including sample size (N), age, proportion of male patients, baseline National Institutes of Health Stroke Scale score, baseline infarct core volume, and the percentage of participants who received endovascular thrombectomy (EVT). Furthermore, the efficacy and safety outcomes at all reported follow-up time points were also retrieved.

Outcomes

The efficacy outcomes comprised: (1) excellent functional outcome (mRS score, 0–1 at 90 days), (2) good functional outcome (mRS score, 0–2 at 90 days), (3) reperfusion, (4) recanalization, and (5) early neurological improvement (ENI), with the latter 3 outcomes defined according to the criteria used in the respective source trials. Safety outcomes included: (1) symptomatic intracerebral hemorrhage (sICH), (2) parenchymal hematoma type 1, (3) parenchymal hematoma type 2, (4) poor functional outcome (mRS score, 5–6 at 90 days), and (5) death within 90 days. Detailed definitions of the outcomes across individual studies are provided in Table S2.

Statistical Analysis

Dichotomous outcomes were presented as odds ratios (ORs) with 95% CIs. Considering the clinical and methodological heterogeneity among the included studies, effect sizes were pooled using the random-effects model. The heterogeneity between studies was evaluated using the Cochran Q test, with P<0.1 indicating statistical significance. In addition, the extent of heterogeneity was quantified using the I² statistic, with values exceeding 75% being classified as indicative of high heterogeneity.¹¹ Subgroup analyses were performed based on whether EVT treatment was allowed in individual studies (non-EVT versus EVT-permitted), the neuroimaging modality used (diffusion-weighted imaging [DWI]-fluid-attenuated inversion recovery [FLAIR] mismatch versus magnetic resonance imaging [MRI]/computed tomography [CT] perfusion), and study geographic region (China versus the United States and Canada). Subgroup effects were compared using the χ²-based interaction tests, with P<0.05 considered statistically significant. Two reviewers (Z.W. and Jiamin Li) independently assessed the certainty of evidence obtained from the findings of our meta-analysis using the Grading of Recommendations, Assessment, Development and Evaluation approach via GRADEpro GDT. Due to the inclusion of <10 trials, funnel plots and the Egger regression test were not conducted. Statistical analyses were conducted using Review Manager 5.4.1. All statistical tests were 2-sided, with P<0.05 considered statistically significant.

Results

Study Selection and Characteristics

The systematic review initially identified 188 records from 4 databases: PubMed (n=67), Embase (n=62), Cochrane Library (n=45), and ClinicalTrials.gov (n=14). After removing 63 duplicate records, 125 studies underwent screening. Among these, 117 records were excluded due to nonrelevance (n=45), nonclinical trials (n=53), non-RCT design (n=11), or treatment initiation beyond the 4.5-hour window (n=8). Eight full-text articles were assessed for eligibility, with 4 excluded due to incomplete studies (n=3) or wrong population (n=1). Ultimately, 4 RCTs (ROSE-TNK [MRI-Guided Thrombolysis for Stroke Beyond Time Window by TNK],¹² TIMELESS,⁴ TRACE-III,⁵ and CHABLIS-T II [Chinese Acute Tissue-Based Imaging Selection for Lysis in Stroke-TNK II]⁷) fulfilled the inclusion criteria and comprised a total of 1278 patients (TNK, n=643; control, n=635; Figure 1). The included trials all evaluated the efficacy and safety of TNK versus standard medical treatment or placebo in patients with AIS presenting within 4.5 to 24 hours from symptom onset or last known well time.

Figure 1.

Flowchart of the systematic review. RCT indicates randomized controlled trial.

All studies were multicenter, prospective, parallel-group trials conducted in China (ROSE-TNK, TRACE-III, and CHABLIS-T II) or in the United States and Canada (TIMELESS), with patient enrollment spanning 2019 to 2023. Among the 4 included studies, the intervention groups all received TNK at a dose of 0.25 mg/kg. For the comparator groups, standard medical treatment was administered in 3 trials (ROSE-TNK, TRACE-III, and CHABLIS-T II), while a placebo was given in the TIMELESS trial. The included studies enrolled adult patients (aged ≥18 years, with an upper age limit of 80 years in ROSE-TNK and CHABLIS-T II) who presented with AIS within 4.5 to 24 hours of onset. Eligible patients had National Institutes of Health Stroke Scale scores ranging from ≥5 (TIMELESS) to 6 to 25 (ROSE-TNK, TRACE-III) and prestroke mRS scores of 0 to 1 (ROSE-TNK and TRACE-III) or 0 to 2 (TIMELESS and CHABLIS-T II). The neuroimaging inclusion criteria differed across the studies. ROSE-TNK required a DWI-FLAIR mismatch but did not mandate confirmation of LVO. TIMELESS, TRACE-III, and CHABLIS-T II incorporated screening for LVO and utilized CT or MRI perfusion to confirm the presence of salvageable tissue. Patients receiving TNK were predominantly male (46.5%–77.5%), with median ages of 62.7 to 72 years. Baseline National Institutes of Health Stroke Scale scores were mild-to-moderate (median, 7.5–12), and infarct core volumes varied considerably (0.32–16.4 mL). Control groups exhibited a similar sex distribution (46.5%–70.8% male), age (62.8–73 years), and National Institutes of Health Stroke Scale scores (median, 7–12) to the TNK groups. Infarct core volumes were comparable to those in the treatment arms (0.40–14.9 mL), with ROSE-TNK again enrolling patients with the smallest lesions. Notably, included trials exhibited significant variability in EVT utilization. TIMELESS showed the highest EVT rates (TNK: 77.2%; control: 77.4%), and CHABLIS-T II had moderate EVT utilization (TNK: 53.2%; control: 56.6%). In contrast, ROSE-TNK and TRACE-III explicitly excluded EVT. All trials reported standardized outcome measures including the 90-day mRS distribution and safety end points such as sICH. The key characteristics of the included studies are summarized in Table 1 and Table S3, while an overview of outcome measures across all studies is presented in Table S4.

Table 1.

Characteristics of the Included Studies

Quality Control of Included Studies

The methodological quality of the included studies was rigorously evaluated using RoB 2.0 across 5 critical domains. Two studies (ROSE-TNK and TRACE-III) demonstrated low risk of bias across all domains. The TIMELESS trial showed some concerns in the selection of reported results (D5), while CHABLIS-T II exhibited some concerns in deviations from intended interventions (D2), with both maintaining low risk in all other domains. The overall risk-of-bias assessments indicated some concerns for TIMELESS and CHABLIS-T II, while the remaining 2 studies were rated as low risk. None of the included studies was judged to have a high risk of bias. These assessments support the overall robustness of the findings though the identified concerns in specific domains should be considered (Figure 2).

Figure 2.

Risk-of-bias assessment for the included studies. CHABLIS-T II indicates Chinese Acute Tissue-Based Imaging Selection for Lysis in Stroke-Tenecteplase II; ROSE-TNK, MRI-Guided Thrombolysis for Stroke Beyond Time Window by TNK; TIMELESS, Thrombolysis in Imaging Eligible, Late Window Patients to Assess the Efficacy and Safety of Tenecteplase; and TRACE-III, Tenecteplase Reperfusion Therapy in Acute Ischemic Cerebrovascular Events-III.

Efficacy Outcomes

Overall

A meta-analysis was conducted incorporating the 4 included studies, with efficacy and safety outcomes synthesized and summarized in Table 2, and the pooled 90-day mRS distribution across the 4 included studies is presented in Figure 3. Specifically, in all 4 included studies, the proportion of patients achieving an excellent functional outcome (mRS score, 0–1) at 90 days was 35.1% (225/641) in the TNK group versus 28.9% (183/634) in the control group. Pooled analysis revealed that TNK significantly increased the odds of achieving an excellent functional outcome compared with the control group (OR, 1.34 [95% CI, 1.06–1.71]; P=0.02), with no statistical heterogeneity observed across studies (Cochran Q test: P=0.80; I²=0%). For good functional outcome (mRS score, 0–2) at 90 days, the TNK group showed a higher proportion of 47.0% (301/641) compared with 42.7% (271/634) in control group, with an OR of 1.16 (95% CI, 0.84–1.60; P=0.38; Cochran Q test: P=0.14; I²=45%), which was not statistically significant (Table 2; Figure 4). Reperfusion outcomes were reported exclusively in TIMELESS and TRACE-III. The pooled analysis indicated an OR of 1.33 for TNK versus control (95% CI, 0.69–2.54; P=0.39; Cochran Q test: P=0.05; I²=74%), indicating no statistically significant difference (Table 2; Figure S1). Recanalization was evaluated in TIMELESS, TRACE-III, and CHABLIS-T II. TNK significantly improved the odds of successful vessel recanalization compared with the control group (OR, 3.30 [95% CI, 1.59–6.84]; P=0.001), with statistical heterogeneity observed across studies (Cochran Q test: P=0.007; I²=80%). The end point of ENI was systematically evaluated in ROSE-TNK, TRACE-III, and CHABLIS-T II trials. The proportion of patients achieving ENI was numerically greater in the TNK group than in the control group, but the difference did not reach statistical significance (OR, 2.12 [95% CI, 0.81–5.55]; P=0.13; Cochran Q test: P=0.01; I²=77%; Table 2; Figure 5).

Table 2.

Overview of Analyses for the Efficacy and Safety Outcomes

Figure 3.

Distribution of scores on the modified Rankin Scale at 90 days. TNK indicates tenecteplase.

Figure 4.

Forest plots of excellent functional outcome and good functional outcome. Forest plots of excellent functional outcome (A) and good functional outcome (B). CHABLIS-T II indicates Chinese Acute Tissue-Based Imaging Selection for Lysis in Stroke-Tenecteplase II; EVT, endovascular treatment; ROSE-TNK, MRI-Guided Thrombolysis for Stroke Beyond Time Window by TNK; TIMELESS, Thrombolysis in Imaging Eligible, Late Window Patients to Assess the Efficacy and Safety of Tenecteplase; TNK, tenecteplase; and TRACE-III, Tenecteplase Reperfusion Therapy in Acute Ischemic Cerebrovascular Events-III.

Figure 5.

Forest plots of recanalization and early neurological improvement (ENI). Forest plots of recanalization (A) and ENI (B). CHABLIS-T II indicates Chinese Acute Tissue-Based Imaging Selection for Lysis in Stroke-Tenecteplase II; EVT, endovascular treatment; ROSE-TNK, MRI-Guided Thrombolysis for Stroke Beyond Time Window by TNK; TIMELESS, Thrombolysis in Imaging Eligible, Late Window Patients to Assess the Efficacy and Safety of Tenecteplase; TNK, tenecteplase; and TRACE-III, Tenecteplase Reperfusion Therapy in Acute Ischemic Cerebrovascular Events-III.

Non-EVT Versus EVT-Permitted Subgroups

In ROSE-TNK and TRACE-III, EVT was protocol-prohibited, resulting in an exclusively non-EVT population for subgroup analysis. Subgroup analyses within this non-EVT cohort indicated that TNK was associated with favorable outcomes across the 4 end points. For excellent functional outcome, TNK increased the odds by 46% (OR, 1.46 [95% CI, 1.02–2.08]; P=0.04; Cochran Q test: P=0.50; I²=0%). The 50% higher odds of good functional outcome were observed in the TNK group (OR, 1.50 [95% CI, 1.07–2.09]; P=0.02; Cochran Q test: P=0.66; I²=0%; Table 2; Figure 4). TNK group conferred a marked increase in the likelihood of recanalization (OR, 6.17 [95% CI, 3.36–11.33]; P<0.00001). A significant benefit was also observed for ENI in the TNK group (OR, 3.21 [95% CI, 1.82–5.66]; P<0.0001) with no heterogeneity (Cochran Q test: P=0.55; I²=0%; Table 2; Figure 5).

In the EVT-permitted subgroup analysis encompassing TIMELESS and CHABLIS-T II, TNK was associated with a statistically significant improvement in recanalization (OR, 2.36 [95% CI, 1.34–4.17]; P=0.003; Cochran Q test: P=0.15; I²=53%; Table 2; Figure 5). Conversely, no significant treatment effects of TNK were detected for excellent functional outcome (OR, 1.25 [95% CI, 0.91–1.73]; P=0.17; Cochran Q test: P=0.70; I²=0%), good functional outcome (OR, 0.95 [95% CI, 0.61–1.50]; P=0.84; Cochran Q test: P=0.15; I²=51%), or ENI (OR, 0.92 [95% CI, 0.49–1.73]; P=0.80; Table 2; Figures 4 and 5).

DWI-FLAIR Mismatch Versus MRI/CT Perfusion Subgroups

Neuroimaging-based subgroup analyses showed that patients selected by DWI-FLAIR mismatch achieved a significant gain in ENI with TNK (OR, 4.68 [95% CI, 1.19–18.34]; P=0.03). In contrast, those screened by MRI/CT perfusion imaging demonstrated a clear improvement in excellent functional outcome (OR, 1.36 [95% CI, 1.06–1.75]; P=0.01), with no significant heterogeneity observed (Cochran Q test: P=0.68; I²=0%; Table S5).

China Versus the US and Canada Subgroups

Among trials conducted in China (ROSE-TNK, TRACE-III, and CHABLIS-T II), TNK significantly improved excellent functional outcome (OR, 1.36 [95% CI, 1.01–1.83]; P=0.04), reperfusion (OR, 1.88 [95% CI, 1.13–3.14]; P=0.02), and recanalization (OR, 4.61 [95% CI, 2.53–8.40]; P<0.00001). In the TIMELESS trial conducted in the United States and Canada, TNK was associated with higher odds of recanalization (OR, 1.86 [95% CI, 1.19–2.89]; P=0.006; Table S6).

Safety Outcomes

In the assessment of safety outcomes, no statistically significant differences were observed between the TNK group and the control group (Table 2). This was supported by the ORs for sICH (OR, 1.68 [95% CI, 0.80–3.52]; P=0.17; Cochran Q test: P=0.48; I²=0%; Figure 6), parenchymal hematoma type 1 (OR, 3.60 [95% CI, 0.87–14.95]; P=0.08; Cochran Q test: P=0.83; I²=0%; Figure S2), parenchymal hematoma type 2 (OR, 2.03 [95% CI, 0.98–4.22]; P=0.06; Cochran Q test: P=0.75; I²=0%; Figure S3), poor functional outcome (mRS score, 5–6; OR, 1.00 [95% CI, 0.73–1.36]; P=0.99; Cochran Q test: P=0.33; I²=13%; Figure S4), and death within 90 days (OR, 1.04 [95% CI, 0.76–1.43]; P=0.81; Cochran Q test: P=0.64; I²=0%; Figure S5), none of which reached statistical significance. Furthermore, subgroup analyses of both non-EVT and EVT-permitted populations showed no significant differences between TNK and control groups in sICH or death within 90 days (Table 2; Figure 6; Figures S2 through S5). Similarly, subgroup analyses based on neuroimaging criteria (DWI-FLAIR mismatch versus MRI/CT perfusion) and study region (China versus the United States and Canada) also revealed no significant differences in safety outcomes (Tables S5 and S6).

Figure 6.

Forest plot of symptomatic intracerebral hemorrhage (sICH). CHABLIS-T II indicates Chinese Acute Tissue-Based Imaging Selection for Lysis in Stroke-Tenecteplase II; EVT, endovascular treatment; ROSE-TNK, MRI-Guided Thrombolysis for Stroke Beyond Time Window by TNK; TIMELESS, Thrombolysis in Imaging Eligible, Late Window Patients to Assess the Efficacy and Safety of Tenecteplase; TNK, tenecteplase; and TRACE-III, Tenecteplase Reperfusion Therapy in Acute Ischemic Cerebrovascular Events-III.

Grading of Evidence

The overall certainty of evidence for each outcome, graded according to the Grading of Recommendations, Assessment, Development and Evaluation approach, is presented in Table S7.

Discussion

Main Findings

In our study, we found that TNK significantly improved excellent functional outcome at 90 days and recanalization versus control, without increasing the risks of sICH or mortality. Notably, in the non-EVT subgroup, TNK improved excellent functional outcome, good functional outcome, recanalization, and ENI, whereas the benefit was confined to recanalization in the EVT-permitted subgroup. These findings not only support TNK use as an alternative reperfusion therapy for extended-window stroke in resource-limited settings without EVT access but also underscore the need for future studies to optimize TNK-EVT combination strategies in eligible patients.

Comparison With Previous Studies

Intravenous TNK has been extensively studied in the context of AIS. A recent meta-analysis of 11 RCTs revealed that TNK, when administered within 4.5 hours of symptom onset, was superior to alteplase in achieving an mRS score of 0 to 1 at 3 months.³ The release of pivotal trials such as TIMELESS and TRACE-III has prompted a few systematic reviews to appraise the efficacy and safety of TNK within the extended therapeutic time window.^13–16 However, these systematic reviews were constrained by methodological limitations, leading to a clinical gap in the available evidence. Specifically, Palaiodimou et al¹⁵ did not include TRACE-III, thus failing to capture the largest cohort of patients in whom EVT was not planned. Ifzaal et al,¹⁴ despite centering their research on the 4.5 to 24-hour time window for stroke treatment, incorporated studies that focused on wake-up strokes, as well as those restricted to the 4.5 to 6-hour time window. This inclusion strategy has markedly increased clinical heterogeneity and obscured the 4.5 to 24-hour late-window treatment effect of TNK.¹⁴ Crucially, no prior meta-analysis conducted EVT-stratified analyses but rather pooled EVT and non-EVT patients, potentially masking differential TNK effects across treatment pathways. To address these gaps, we conducted a meta-analysis of 4 RCTs (ROSE-TNK, TIMELESS, TRACE-III, and CHABLIS-T II) that compared TNK with standard care or placebo in adults within 4.5 to 24 hours after AIS onset, aiming to determine the safety and efficacy of ultrawindow TNK therapy. In addition, we first performed an EVT-stratified subgroup analysis (non-EVT versus EVT-permitted) to clarify whether the benefit-risk profile differed between the 2 groups, generating scenario-specific evidence for late-window AIS therapy.

Potential Implications for Clinical Practice

Overall: Efficacy and Safety of TNK in Extended IVT Time Windows for AIS

Currently, the time window for IVT in AIS remains primarily restricted to 4.5 or 6 hours after symptom onset, with clinical practice adopting a cautious stance toward IVT in patients beyond this window. Although studies such as TRACE-III have provided preliminary evidence supporting the extension of the time window for TNK to 24 hours,⁵ international guidelines have not yet established clear recommendations for TNK use in patients beyond the standard window,^17,18 resulting in missed treatment opportunities for some potentially eligible individuals. Our systematic analysis of existing RCTs revealed that, following rigorous imaging screening, TNK significantly increased the odds of achieving an excellent functional outcome (mRS score, 0–1) at 90 days in patients with AIS treated within 4.5 to 24 hours, consistent with prior studies.^14,15 Notably, our meta-analysis is the first to confirm that TNK thrombolysis within the 4.5 to 24-hour window significantly enhanced vascular recanalization rates, irrespective of subsequent EVT. The advantages of TNK, including its high fibrin specificity, robust resistance to plasminogen activator inhibitor-1, and prolonged half-life, may underpin its sustained thrombolytic efficacy in the delayed time window.² While our study did not replicate the positive results reported by Aladawi et al¹⁶ regarding good functional outcome (mRS score, 0–2) at 90 days and ENI, this discrepancy may be attributed to the inclusion of neutral results from CHABLIS-T II and the increased heterogeneity inherent in the expanded cohort. Nevertheless, the overall evidence supports the value of TNK in selected patients beyond the standard time window. Moreover, TNK administered within the 4.5 to 24-hour window demonstrated favorable safety profiles, with no increased risk of sICH or 90-day mortality. Therefore, based on available evidence, we conclude that appropriately screened patients with AIS with imaging-confirmed perfusion mismatch presenting within the 4.5 to 24-hour time window may derive benefit from IVT with TNK (0.25 mg/kg). Future high-quality studies are warranted to further refine optimal patient selection criteria, thereby expanding the therapeutic benefits of TNK IVT in patients beyond the standard time window.

Non-EVT Settings: Expanding Reperfusion Therapy in Resource-Limited Regions

EVT performed within 24 hours of symptom onset confers substantial prognostic benefit in patients with AIS with LVO.¹⁹ However, timely initiation of EVT remains a global challenge, affecting even some developed countries, while resource limitations further exacerbate access to EVT in low- and middle-income countries.⁶ Consequently, IVT remains the sole treatment option for the majority of patients with AIS. Our study revealed that, within the non-EVT subgroup (comprising the ROSE-TNK and TRACE-III trials), TNK administered within the 4.5 to 24-hour window demonstrated robust benefits across multiple end points, including excellent functional outcome, good functional outcome, successful recanalization, and ENI. These findings underscore the standalone efficacy of TNK in settings where EVT is unavailable. A recent meta-analysis of 8 RCTs comparing IVT beyond 4.5 hours versus standard care in AIS revealed that TNK-treated patients achieved even superior excellent functional outcomes (OR, 1.47 [95% CI, 1.06–2.04]) compared with those receiving alteplase (OR, 1.38 [95% CI, 1.08–1.78]).²⁰ TRACE-III was a phase III clinical trial involving 58 centers across China. While its overall design was comparable to that of the TIMELESS trial, TRACE-III specifically excluded patients scheduled for EVT. This study demonstrated that in patients with AIS with anterior circulation LVO who presented within 4.5 to 24 hours of symptom onset and were unavailable for EVT, intravenous TNK significantly improved the proportion of patients achieving an mRS score of 0 to 1 at 90 days.⁵ As the first study worldwide to extend the time window for IVT from the traditional 4.5 hours to 24 hours, TRACE-III represents a landmark achievement and has prompted updates to Chinese guidelines on reperfusion therapy for AIS.^21,22

Our subgroup analysis of the non-EVT population further validated these findings, supporting the use of TNK as an alternative reperfusion therapy for patients with stroke in the extended time window within resource-limited settings without access to EVT. These findings are particularly relevant for low- and middle-income countries, where EVT accessibility remains severely limited. In such regions, TNK could serve as a standalone reperfusion strategy, significantly expanding the population eligible for thrombolysis and reducing disability burden. Furthermore, both ROSE-TNK and TRACE-III were conducted in China. Future international multicenter clinical trials are warranted to verify the generalizability of these findings in a more diverse global population.

EVT-Permitted Settings: Role of TNK in Extended Windows Amid EVT Access

In our study, the EVT-permitted subgroup (TIMELESS and CHABLIS-T II) showed that TNK’s benefits were primarily confined to vascular recanalization, with no significant improvement in functional outcomes. This might be attributed to the high proportion of patients with AIS who received EVT (77.3% in TIMELESS and 54.9% in CHABLIS-T II), potentially overshadowing the incremental benefits of TNK administered within the 4.5 to 24-hour window.^4,7 In addition, the short time intervals between thrombolysis and EVT in these studies might have diluted the effect of TNK. It is important to highlight that the TIMELESS trial demonstrated that among patients with LVO who have immediate access to EVT, the administration of TNK beyond the standard time window does not yield incremental benefits.⁴ Similarly, the CHABLIS-T II study confirmed that TNK thrombolysis administered beyond the standard time window failed to improve patients’ functional prognosis but did enhance reperfusion rates.⁷ However, TNK’s role in EVT-capable centers should not be dismissed. A clinical trial involving 39 stroke centers in China confirmed that, for patients with AIS with LVO presenting within 4.5 hours of onset, intravenous TNK combined with EVT significantly improved functional outcomes compared with thrombectomy alone, with a favorable safety profile.²³ Current guidelines recommend TNK as a bridging therapy within the standard time window of AIS due to its practical advantages, such as single-bolus administration and higher fibrin specificity.^17,24 Ongoing phase III clinical trials such as ETERNAL-LVO (Extending the Time Window for Tenecteplase by Effective Reperfusion in Patients With Large Vessel Occlusion; NCT04454788) and POST-ETERNAL (Extending the Time Window for Tenecteplase by Recanalization of Basilar Artery Occlusion in Posterior Circulation Stroke; NCT05105633) will provide additional evidence-based data regarding the efficacy and safety of TNK-based bridging therapy in extended time windows. Notably, future studies should explore optimal protocols for combining TNK with EVT in the extended window, including patient selection criteria and timing strategies, to maximize synergistic effects.

Limitations

This meta-analysis has several limitations. First, only 4 RCTs were included, resulting in a relatively small sample size that may reduce the robustness of conclusions, particularly in subgroup analyses. Notably, within the non-EVT subgroup, patients from the TRACE-III trial accounted for as much as 86.6% of the total, which may lead to the pooled treatment effects being dominated by this single study. Second, both the TIMELESS and CHABLIS-T II trials enrolled a subset of patients with stroke who underwent EVT. However, as neither trial reported outcomes stratified by EVT treatment status, our subgroup analysis was limited to a comparison between trials that entirely excluded EVT and those that permitted it (ie, non-EVT versus EVT-permitted subgroups). Third, heterogeneity was observed across the trials with regard to neuroimaging selection criteria, EVT utilization rates, and outcome definitions, all of which could contribute to between-study variability. All analyses used random-effects models to address these variations. Fourth, the included trials were predominantly from China (3 of 4 studies), and whether these findings can be extended to other populations warrants additional investigation. Finally, potential publication bias could not be formally assessed due to the limited number of studies.

Conclusions

TNK improves excellent functional outcomes and recanalization in patients with AIS treated within 4.5 to 24 hours, without increasing the risks of sICH or mortality. Notably, extended-window TNK provides greater additional benefits when EVT is inaccessible. These findings support TNK use as an alternative reperfusion therapy for extended-window stroke in resource-limited settings without EVT access while highlighting the need for future studies to optimize TNK-EVT combination strategies in eligible patients.

ARTICLE INFORMATION

Author Contributions

Dr Ma contributed to the study conception and design, and was responsible for critical review of the manuscript and its final editing. Z. Wang contributed to the study design and drafted the article. Z. Wang, Jiamin Li, and X. Wang participated in methodological operations. B. Yuan and Jiameng Li participated in data analysis and interpretation. All authors have read and approved the final version of the article.

Sources of Funding

This study was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (grants 2024ZD0527600 and 2024ZD0527603) and the National Key Research and Development Program of China (grant 2023YFC2506504).

Disclosures

None.

Supplemental Material

Tables S1–S7

Figures S1–S5

Supplementary Material

str-57-050-s001.pdf ^{(1.8MB, pdf)}

str-57-050-s002.pdf ^{(68.9KB, pdf)}