A novel fibrin-targeted urokinase fusion protein enhances thrombolytic efficacy with reduced bleeding risk

Introduction Acute thrombotic events such as ischemic stroke, ST-elevation myocardial infarction (STEMI), and pulmonary embolism are leading causes of morbidity and mortality worldwide. Current thrombolytic treatment strategies primarily rely on recombinant tissue-type plasminogen activator (rtPA) variants such as alteplase and tenecteplase, although their clinical utility is restricted by a narrow therapeutic window, incomplete clot dissolution, and bleeding risk. To overcome these limitations, we engineered a novel fibrin-targeted fibrinolytic construct by fusing urokinase-type plasminogen activator (uPA) to a fibrin-specific single-domain antibody (sdAb), enabling targeted thrombolysis. Method Anti-fibrin sdAbs were generated using phage display technology. Phage libraries were constructed from mRNA isolated from B cells of llamas previously immunised with fibrin degradation products. A fibrin-specific sdAb demonstrating no cross-reactivity with fibrinogen was selected and genetically fused to uPA. The resulting fusion protein, termed anti-fibrin uPA (aFn-uPA), was validated through the following in vitro the tests: clot lysis time, plasmin generation, static and under flow thrombolysis monitoring. Furthermore, its thrombolytic efficacy was compared with rtPA in a murine stroke model at the Stroke-Alliance Laboratory. Results In vitro, aFn-uPA demonstrated greater thrombolytic potential than both rtPA and uPA. At equimolar concentrations, clot lysis times for aFn-uPA were 21-44% shorter than those for rtPA ([ Fig. 1 ]). Whereas uPA alone showed no clot lysis nor plasmin generation, aFn-uPA generated a dose-dependent plasmin curve, with plasmin peak and endogenous plasmin potential values comparable to those of rtPA. In vitro thrombolysis confirmed superior efficacy of aFn-uPA in dissolving pre-formed thrombi compared to rtPA under both static and flow conditions. In vivo, aFn-uPA was as effective as rtPA in restoring cerebral blood flow in a specialized murine model of ischemic stroke designed to assess fibrinolytic activity. Fig. 1 aFn-uPA induces faster clot lysis than rtPA and uPA. Clot lysis assays were performed using normal pooled plasma with recombinant tissue-type plasminogen activator (rtPA, solid line), urokinase-type plasminogen activator (uPA, dotted line), and fibrin-targeted urokinase-type plasminogen activator (aFn-uPA, dashed line) at concentrations of 2 nM (teal) and 4 nM (pink). Absorbance at 405 nm was recorded over 100 minutes to monitor clot dissolution kinetics. Conclusion We developed a fibrin-targeted uPA fusion protein that exhibited enhanced fibrinolytic capacity across multiple assays. In clot lysis assays, aFn-uPA showed significantly greater fibrinolytic potency than rtPA and uPA, while plasmin generation was comparable to rtPA. Thrombolysis assays further confirmed its superior ability to dissolve pre-formed clots. In a murine model of ischemic stroke, aFn-uPA achieved effective thrombolysis with a low incidence of bleeding complications, highlighting its potential as a safe and efficacious thrombolytic agent. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany