Fibrin Selective Alteplase with Improved Thrombolysis and Inhibition Resistance Engineered by Rational Design

AIMSThrombolytic enzymes represent an accessible and streamlined treatment for acute ischemic stroke, yet current FDA-approved agents, alteplase and tenecteplase, are limited by incomplete recanalization, hemorrhagic risk, and neurotoxicity. We aimed to develop a multidisciplinary workflow for rational design and multi-level testing of thrombolytic proteins to generate a next-generation thrombolytic with improved potency and safety. METHODS AND RESULTSOur pipeline integrated in silico design, biochemical characterization, in vitro thrombolysis, and in vivo validation. Computational screening included rational mutagenesis to reduce side effects, reconstructed ancestral sequences, and database-mined homologs. Selected candidates were biochemically assessed for enzymatic activity, fibrin stimulation, selectivity and affinity, inhibition resistance, and clot penetrability. The most promising variants underwent in vitro clot thrombolysis and in vivo efficacy and safety studies in rats. Brnoteplase emerged as the lead candidate, exhibiting 80-fold enhanced fibrin selectivity, 4-fold higher inhibition resistance, and superior clot penetrability compared to alteplase. In vitro, Brnoteplase demonstrated deeper intrathrombus penetration and a progressive dose-response profile, unlike alteplase, which declined at higher concentrations. In vivo, Brnoteplase achieved higher thrombolysis rates and recanalization frequency across all doses, with extended biological half-life enabling bolus administration. Safety assessments revealed the lowest incidence of severe hemorrhagic transformation and minimal hemispheric asymmetry. CONCLUSIONSWe established a robust pipeline for efficient generation and selection of novel thrombolytics and identified Brnoteplase as a promising candidate combining improved fibrin selectivity, inhibition resistance, biological half-life, and clot penetration. These features translate into higher thrombolytic efficacy and a promising safety profile in vivo, warranting further preclinical evaluation to confirm its potential as a next-generation thrombolytic for acute ischemic stroke.