Efficacy and safety of intracoronary injection of recombinant human prourokinase in patients with slow blood flow/no-reflow during PCI for acute coronary syndrome: a randomised controlled, double-blind, placebo controlled, national multicentre clinical study protocol (PUK-ACS study)

Abstract

Introduction

The coronary slow-flow/no-reflow phenomenon adversely affects clinical outcomes following percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS), demonstrating a substantial association with increased cardiovascular mortality and heart failure incidence. Despite its significant clinical implications, current therapeutic strategies for this condition lack robust guideline recommendations and are not supported by large-scale clinical evidence. Intracoronary administration of recombinant human prourokinase, a novel fibrin-specific thrombolytic, demonstrates potential for improving microvascular dysfunction through targeted fibrinolysis. Nevertheless, current evidence is limited to small-scale clinical studies, lacking large-scale trials to guideline recommendations. This study aims to investigate whether selective intracoronary administration of prourokinase, in addition to conventional intensive antiplatelet therapy, nitrates and calcium channel blockers, can reduce the incidence of cardiovascular events at 12 months post-PCI in patients with ACS experiencing slow flow/no-reflow during the procedure without increasing the incidence of major bleeding events.

Methods and analysis

This is a randomised, double-blind, placebo-controlled study to evaluate the efficacy of low-dose intracoronary administration of prourokinase or placebo via a microcatheter in the infarct-related artery in patients with ACS with slow flow/no-reflow during PCI. The primary endpoint is the incidence of major adverse cardiovascular events (MACE) 12 months after the intervention, defined as the composite of cardiovascular death, recurrent myocardial infarction, hospitalisation for unstable angina, unplanned repeat revascularisation, hospitalisation for heart failure or non-fatal stroke, whichever occurs first. Secondary endpoints include myocardial reperfusion (thrombolysis in myocardial infarction (TIMI) flow grade, TIMI frame count), myocardial infarction severity (ST-segment resolution, infarct area estimated from area under the curve of cardiac troponin T), angina severity (Canadian Cardiovascular Society grading of angina pectoris), cardiac function (left ventricular ejection fraction, New York Heart Association functional classification) and short-term MACE.

Ethics

This study will adhere to the principles outlined in the Declaration of Helsinki and other applicable ethical guidelines. The study protocol has been approved by the Medical Research Ethics Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University (Approval No: SYSKY-2024-735-04).

Dissemination

We will disseminate findings via peer-reviewed journals, international conferences and stakeholder outreach (clinicians, policymakers and patients), supplemented by digital platforms to accelerate knowledge translation and clinical implementation.

Trial registration number

ChiCTR2500103336.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• A strength of our study is that this study is a randomised, double-blind, placebo-controlled, national multicentre trial with a large sample size of 1380 patients.

• The second strength of our study is that the follow-up duration extends to 12 months with systematic assessment of hard clinical endpoints including major adverse cardiovascular events.

• Another strength of our study is that all endpoint events are adjudicated by an independent review committee to reduce detection bias.

• A limitation of our study is that the infarct size is estimated using the area under the curve of cardiac troponin T rather than advanced imaging modalities such as cardiac MRI, which may limit precision.

• Another limitation of our study is that the use of rescue glycoprotein IIb/IIIa inhibitors is permitted but will be adjusted for in multivariable analyses.

Introduction

Acute coronary syndrome (ACS), predominantly resulting from atherosclerotic plaque rupture and consequent thrombus formation, represents a critical cardiovascular emergency.^1–3 Contemporary epidemiological data still reveals an alarming midterm mortality of acute myocardial infarction at a rate of 6.3–18%.^{4 5} While percutaneous coronary intervention (PCI) remains the cornerstone reperfusion therapy for ACS, a substantial proportion of patients (3.65–21.5%) develop the slow-flow/no-reflow post procedure.^6–8 This complication is associated with markedly elevated in-hospital mortality (12.6% vs 3.8% in unaffected patients, p<0.001) and significantly worsened long-term clinical outcomes.^9–11

Current guidelines offer limited and weak recommendations (Class IIa, Level C) for managing PCI-related slow flow/no-reflow. The 2019 Chinese STEMI Guidelines propose intracoronary tirofiban to reduce no-reflow,¹² while the 2023 ESC ACS Guidelines suggest intravenous glycoprotein (GP) IIb/IIIa inhibitors for thrombotic complications.¹³ However, both lack robust clinical evidence. Additionally, tirofiban, whether administered intracoronary or intravenously, may increase bleeding risk on top of standard dual antiplatelet therapy.¹⁴ Better evidence is urgently needed to optimise no-reflow treatment without raising bleeding complications.

Recombinant human prourokinase is a novel fibrin-specific thrombolytic agent with targeted action and low bleeding risk. It selectively activates fibrin-bound plasminogen, effectively dissolving microvascular thrombi.

In a small-scale randomised controlled trial (RCT), Ge Junbo and his team found that intracoronary administration of rh-prourokinase during primary PCI further improved myocardial reperfusion in patients with ST-segment elevation myocardial infarction (STEMI) compared with the placebo control group, without increasing the risk of major bleeding.¹⁵ However, due to the study’s limited sample size and the lack of further exploration on the impact of different intervention strategies on long-term major adverse cardiovascular events (MACE) as hard endpoints, large-scale RCT evidence is still lacking.

Therefore, the primary objective of this prospective, randomised, double-blind, placebo-controlled multicentre trial is to evaluate the efficacy and safety of intracoronary prourokinase in patients with ACS with slow flow/no-reflow during PCI and its impact on 12-month cardiovascular outcomes. And the secondary objectives of this study are to investigate whether the treatment can improve coronary microcirculation (assessed by corrected thrombolysis in myocardial infarction (TIMI) frame count and ST-segment resolution), enhance myocardial perfusion and reduce MACE at 1-month and 3-month follow-up or improve clinical outcomes, including Canadian Cardiovascular Society (CCS) grading of angina pectoris and New York Heart Association (NYHA) functional classification at 1-month and 12-month follow-up.

Methods

Study design

The PUK-ACS trial is a prospective, randomised, double-blind, placebo-controlled multicentre study enrolling patients with ACS with slow flow/no-reflow during PCI. Participants will be recruited from over 30 centres including Sun Yat-sen Memorial Hospital and collaborating centres.

The detailed inclusion and exclusion criteria are shown in box 1.

Box 1. Eligible criteria.

Inclusion criteria

1. Age ≥18 years and ≤75 years, regardless of gender.

2. Patients diagnosed with ACS* undergoing PCI.

3. Occurrence of slow-flow/no-reflow phenomenon during PCI: After reopening the occluded coronary artery, TIMI flow in the culprit vessel remains below grade 3 (TIMI flow 0–1 indicates no-reflow, while TIMI flow 2 indicates slow-flow).

4. Haemodynamically stable (SBP >90 mm Hg without inotropes).

5. Sign the informed consent form.

Exclusion criteria

1. Patients who have undergone prior intravenous thrombolysis.

2. History of intracranial haemorrhage.

3. Ischaemic stroke within the past 3 months.

4. Suspected aortic dissection.

5. Active bleeding or bleeding risk factors (excluding normal physiological menstruation).

6. Major head or facial trauma within the past 3 months.

7. Severe or poorly controlled hypertension (systolic blood pressure >180 mm Hg or diastolic blood pressure >110 mm Hg).

8. Major trauma, major surgery or non-compressible vascular bleeding within the past 3 weeks.

9. Pre-existing severe hepatic or renal dysfunction (ALT >3 × upper limit of normal or eGFR <30 mL/min/1.73m²).

10. Pregnant or lactating women, or women with potential for pregnancy.

11. Known allergy or contraindication to the study drug or contrast agent.

12. Patients with malignancy.

13. Concurrent participation in another clinical trial.

A total of 1380 patients will be randomised at 1:1 to receive intracoronary administration of either prourokinase (20 mg) or placebo based on the standard therapy. Follow-up will be conducted at 1, 3, 6, 9 and 12 months post-PCI to assess symptoms, ECG, biomarkers and cardiovascular events. The trial flow chart is shown in figure 1. The detailed study design schedule is provided in online supplemental table 2.

Figure 1. Study flow chart and schedule of study enrolment and interventions. ACS, acute coronary syndrome; PCI, percutaneous coronary intervention.

Randomisation

Eligible patients will be randomised via a computer-generated sequence, with allocation concealed from investigators, data collectors, and statisticians to ensure blinding.

Emergency unblinding procedure

If a subject exhibits TIMI grade 0 flow with severe haemodynamic instability or experiences a serious adverse event requiring emergency treatment, the investigator may unblind the treatment assignment to facilitate clinical management. The unblinding must be documented in detail, including time, reason and actions taken. The unblinding must be reported immediately to the independent review committee (IRC) and the sponsor. The subject will then be withdrawn from the trial and reported as a dropout. The monitor must be informed, and the event summarised with justification, date, and signature in the final report.

Interventions

Prourokinase or placebo was manually injected via a microcatheter proximal to the target lesion.

Conventional therapy:

1. In the absence of contraindications, all enrolled patients should receive the following medications routinely according to current Chinese and international guidelines for the diagnosis and treatment of acute coronary syndromes:

   1. Aspirin.

   2. Clopidogrel or ticagrelor.

   3. Intraprocedural heparin (administered at the guideline-recommended dose).

   4. Vasodilators: such as nitroglycerin/nicorandil/adenosine/verapamil, etc.

2. Other pharmacological treatments:

In the absence of contraindications, all enrolled patients should receive routine treatment with beta-blockers, angiotensin receptor-neprilysin inhibitor/angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker and statins, etc, according to current domestic and international guidelines for the diagnosis and treatment of acute coronary syndromes.

The use of open-labelled urokinase or prourokinase for intracoronary administration is prohibited during the procedure.

Rescue therapy: If TIMI flow grade does not recover after the administration of the study drug and is accompanied by haemodynamic instability, a GP IIb/IIIa receptor antagonist (such as tirofiban) may be administered intravenously or intracoronarily. The use of rescue GP IIb/IIIa inhibitors will be recorded prospectively.

Outcomes

Primary endpoint

12-month MACE rate, including cardiovascular death (including death due to stroke), all coronary events (recurrent myocardial infarction, hospitalisation for unstable angina or unplanned repeat revascularisation), as well as hospitalisation for heart failure and non-fatal stroke, with the first occurrence considered as the endpoint.

Secondary endpoints

1. TIMI flow grade after PCI.

2. Corrected TIMI frame count (CTFC)^{16 17} after PCI, defined according to the method described by Gibson et al. Briefly, images are acquired at 30 frames per second and the frame count is recorded from the moment contrast agent enters the coronary artery and fully occupies the proximal segment until it reaches predefined distal landmarks. The first frame is defined as when contrast completely fills the proximal coronary artery, and the last frame is when contrast reaches the distal landmark of that artery.

3. ST-segment resolution: Measured at 1 day and 7 (±2) days (before discharge) after PCI. For STEMI, it is determined using the lead with the greatest ST-segment elevation.

4. Measurement of cardiac troponin T (cTnT) and Creatine Kinase-MB (CK-MB) at 0 hours, 24 hours, 48 hours and 7 (±2) days (before discharge) after PCI.

5. Echocardiographic assessment of left ventricular ejection fraction (LVEF) and serum N-terminal pro-B-type Natriuretic Peptide levels at 7 (±2) days and 12 months (±15 days) after PCI.

6. Estimation of myocardial infarct size using the area under the curve (AUC) of cTnT measured at 0 hours, 24 hours, 48 hours and 7 days after PCI.

7. Change in CCS angina grade at 1 and 12 months after PCI.

8. Change in LVEF measured by echocardiography after PCI.

9. Change in NYHA functional classification at 1 and 12 months after PCI.

10. MACE at 1 and 3 months after PCI.

Safety endpoints

1. Primary safety endpoint: Perioperative bleeding (TIMI bleeding classification)^18–20 event rate.

   1. Major bleeding: Any symptomatic intracranial haemorrhage, or clinically overt signs of haemorrhage (including imaging) associated with a drop in haemoglobin of ≥50 g/L (or when the haemoglobin concentration is not available, an absolute drop in haematocrit of ≥15%).

   2. Minor bleeding: Any clinically overt sign of haemorrhage (including imaging) that is associated with a fall in haemoglobin concentration of 30 to <50 g/dL (or, when haemoglobin concentration is not available, a fall in haematocrit of 9 to <15%).

   3. Minimal bleeding: Clinical signs of bleeding (including imaging evidence) with a decrease in haemoglobin concentration <30 g/dL or a decrease in haematocrit <9%.

2. Secondary safety endpoints: Serum creatinine, alanine aminotransferase, aspartate aminotransferase, total serum bilirubin, white blood cell count, neutrophil count, platelet count and haemoglobin will be collected from subjects at hospital enrolment and at each follow-up time point. Vital signs and physical examinations will be performed to assess the safety of the drug use.

Independent review committee

The assessment of the primary endpoints in this trial will be based on the evaluations provided by the IRC. The IRC is an independent body comprised of experts tasked with adjudicating endpoint events in accordance with prespecified protocol criteria. This ensures consistent and accurate endpoint assessment through the uniform application of standardised guidelines, thereby reducing bias that may arise from variability in event evaluation across different centres.

Members of the IRC will be selected based on their expertise in relevant clinical trial areas. All members must remain independent of both the investigators and the sponsor, may not participate directly in the conduct of the trial and must be free of any conflicts of interest that could affect their impartiality or independent judgement.

In accordance with standardised case report form (CRF) completion guidelines, all CRFs must be completed by designated and appropriately trained research personnel to ensure data completeness and accuracy. To further enhance data integrity, an independent data manager performs verification checks. Should any discrepancies or questions related to CRF entries be identified, the data manager generates a data resolution query, which is forwarded to the investigator. The investigator is required to verify the data against source documents and provide a timely response. Based on the investigator’s feedback, the data manager subsequently updates and confirms the data. No formal interim analysis is planned for this study.

Sample size calculation

Sample size was determined by the primary endpoint of 12-month MACE rate, hypothesising superior efficacy for recombinant prourokinase versus control. With one-sided α=0.025, 80% power, and 1:1 randomisation. According to previous literature,^21–24 primary endpoint of 12-month MACE rate was determined as 13% for the control group and 8.8% for the prourokinase group according to previous studies. Power Analysis and Sample Size V.15.0 software analysis determined that 587 participants per group were required, which was adjusted to 690 per group to account for an anticipated 15% attrition rate.

Statistical analysis

Continuous variables will be assessed for normality and presented accordingly: normally distributed variables as mean±SD, and non-normally distributed variables as median and IQR. Categorical variables will be summarised as frequencies and percentages. Between-group comparisons will be conducted using the χ² test (or Fisher’s exact test, if applicable) for categorical variables. For continuous variables, the Student’s t-test will be applied for normally distributed data, while the Mann-Whitney U test will be used for non-normally distributed data. The primary analysis for the primary endpoint will be conducted using the intention-to-treat population, which includes all randomised patients regardless of whether they received the assigned treatment. A per-protocol analysis will be performed as a sensitivity analysis. Time-to-event end points will be analysed using the Kaplan-Meier method. The comparison between groups will be performed using log-rank test. The HRs and their corresponding 95% CIs will be estimated using a Cox proportional hazards model. To account for the potential confounding effect of rescue GP IIb/IIIa inhibitor use, a multivariable Cox regression analysis including rescue GP IIb/IIIa use will be performed as a covariate to adjust for its potential impact on the primary endpoint. Because no provision for correction for multiplicity will be planned when tests for the secondary or other outcomes are conducted, results will be reported as point estimates and 95% CIs. The widths of the CIs have not been adjusted for multiplicity and should not be used to infer definitive treatment effects for secondary outcomes. All statistical analyses will be performed using SPSS V.25.0 software. A one-sided p value <0.025 will be considered statistically significant for the superiority analysis of the primary endpoint. For all other analyses (including secondary endpoints and safety analyses), two-sided p values <0.05 will be considered statistically significant.

Patient and public involvement

There was no patient or public involvement in the design and conduct of this study.

Trial stage

• June 2025–August 2026: Patient recruitment.

• September 2026–September 2027: Completion of follow-up.

• October 2027–June 2028: Data analysis. The first patient was enrolled on 18 September 2025.

Discussion

The PUK-ACS trial investigates whether intracoronary administration of recombinant prourokinase can improve myocardial perfusion and cardiovascular outcomes in patients with ACS experiencing slow-flow/no-reflow phenomenon during PCI. The study comprehensively evaluates multiple clinical endpoints including TIMI flow recovery, ST-segment resolution, myocardial infarction size estimated by cTnT AUC, LVEF by echocardiography, patients’ clinical angina symptoms, cardiac functional status, as well as the occurrence of both short-term and long-term MACE.

PCI is currently the first-line reperfusion therapy recommended by clinical guidelines for patients with ACS. However, after successful opening of the culprit vessel, approximately 3.65–21.5% of patients with ACS still develop slow-flow or no-reflow phenomenon.^6–8 This pathophysiological complication markedly elevates the risk of malignant arrhythmia, worsens myocardial ischaemia, extends the infarct zone, increases heart failure incidence and substantially compromises both short-term survival and long-term prognosis.^9–11 A large-scale clinical study involving 291 380 patients with acute myocardial infarction demonstrated that patients with coronary slow-flow/no-reflow had markedly higher in-hospital mortality compared with controls (12.6% vs 3.8%, adjusted OR 2.20, p<0.001).¹¹ Therefore, active prevention, early identification and proper management of slow-flow/no-reflow phenomenon following PCI in patients with ACS are of critical importance.

Current guidelines provide limited treatment recommendations for PCI-related slow-flow/no-reflow phenomenon, with low recommendation classes and evidence levels. The 2023 European Society of Cardiology guidelines recommend intravenous GP IIb/IIIa receptor antagonists when no-reflow or thrombotic complications occur during PCI (Class IIa, Level C).¹³ However, this recommendation is supported by limited clinical evidence, resulting in its low recommendation classification (Level C) in current guidelines. Moreover, both intracoronary and intravenous administration of tirofiban may increase bleeding risks when combined with conventional dual antiplatelet therapy.¹⁴ Therefore, there is an urgent need for additional clinical evidence to identify better treatment approaches for coronary slow-flow/no-reflow during PCI without increasing bleeding risks.

Recombinant human prourokinase is a specific plasminogen activator produced through genetic engineering technology, currently mainly used for thrombolytic therapy in patients with STEMI.²⁵ As a fibrin-specific thrombolytic agent, it demonstrates superior clinical performance characterised by targeted thrombus selectivity, excellent recanalisation efficacy, significantly reduced risks of systemic haemorrhage or vital organ bleeding complications, and a favourable safety profile—collectively establishing it as a next-generation thrombolytic with optimal efficacy-safety balance. One of the primary mechanisms underlying coronary slow-flow/no-reflow in patients with ACS is microcirculatory embolism caused by thrombus fragments from acute thrombosis circulating to the distal coronary arteries. After entering the bloodstream, prourokinase binds to plasminogen adsorbed on thrombus fibrin, leading to partial dissolution of fibrin clots.⁹ The exposed E-fragments after fibrinolysis trigger a cascade amplification effect of prourokinase, rapidly activating the plasminogen bound to these fragments and increasing its activity by 500-fold, thereby achieving thrombolytic effects that relieve coronary microcirculatory obstruction and improve myocardial perfusion. In a small RCT conducted by Ge Junbo, intracoronary administration of rh-prourokinase during emergency PCI was found to further improve myocardial reperfusion in patients with STEMI compared with placebo control, without increasing major bleeding risks.¹⁵ However, this study had a small sample size and did not further explore the impact of different intervention strategies on long-term hard cardiovascular endpoints (MACE). Therefore, there remains a lack of evidence from large-scale RCTs.

In a key prior study, Huang et al (ERUPTION trial)¹⁵ demonstrated that intracoronary prourokinase (20 mg) during primary PCI significantly improved final CTFC (27.1±14.2 vs 34.6±18.3 frames, p<0.001), complete ST-segment resolution at 2 hours (65.3% vs 43.7%, p=0.008) and peak CK-MB levels (307.8 vs 442.5 U/L, p=0.006) compared with placebo, without increasing major bleeding (1.8% vs 0%, p=0.343).

However, that study had several limitations: (1) follow-up was only 30 days with a surrogate primary endpoint (CTFC) rather than hard clinical outcomes; (2) the sample size (n=345) was insufficient for clinical event detection; (3) the population was restricted to patients with STEMI; and (4) the single-blind design may have introduced bias.

The PUK-ACS trial addresses these gaps by extending follow-up to 12 months with MACE as the primary endpoint, enrolling 1380 patients, covering the full ACS spectrum (STEMI, non-STEMI and unstable angina) and employing a double-blind design with independent endpoint adjudication.

The PUK-ACS trial evaluates whether intracoronary prourokinase improves myocardial perfusion and long-term outcomes in patients with ACS with slow flow/no-reflow during PCI. Despite PCI being the gold standard for ACS, no-reflow remains a major challenge, increasing mortality and heart failure risk. Current tirofiban recommendations lack strong evidence and carry bleeding risks. Intracoronary administration of prourokinase may offer a safer, more effective alternative.

Comparison with prior RCT

First, prior trial primarily recruited patients with STEMI,¹⁵ while this study expands the study population to patients with ACS and the sample size was increased to enhance statistical power. Second, previous studies were limited to 30-day follow-up periods and used CTFC as the primary endpoint, without evaluating the impact of intervention on long-term prognosis. In contrast, our study features a total 12-month follow-up period and rigorously evaluates the intervention’s impact on MACE as hard clinical endpoints. Therefore, the PUK-ACS study aims to comprehensively evaluate the safety and efficacy of this intervention.

Limitations

While the current study provides valuable clinical insights, certain methodological constraints should be acknowledged. First, the assessment of infarct size through ECG parameters and conventional biomarkers may lack the precision offered by advanced imaging modalities like cardiac MRI with late gadolinium enhancement.²⁶ Second, the predetermined sample size, while adequate for primary endpoint analysis, may be underpowered to adequately explore potential differential treatment benefits across various clinical subgroups, such as patients with different culprit vessel locations (eg, left anterior descending artery vs right coronary artery vs left circumflex artery) or distinct ACS subtypes (STEMI vs non-STEMI vs unstable angina).

Ethics and dissemination

Ethics

Approved by the Medical Research Ethics Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University (SYSKY-2024-735-04). As of the time of protocol submission, a total of 29 participating centres have secured local ethical approvals. For centres that are still undergoing administrative review or will be activated later according to the project’s enrolment timeline, local ethical approval will be obtained prior to the initiation of patient recruitment at each site. A complete list of participating centres, their ethics approval status and all institutional review boards that have granted approval so far is provided in online supplemental table 1.

Dissemination

Results will be published in journals, presented at conferences, and shared via social media to maximise impact.