Towards Effective and Safe Thrombolysis and Thromboprophylaxis: Preclinical Testing of a Novel Antibody-Targeted Recombinant Plasminogen Activator Directed Against Activated Platelets

• Published in: Circulation research Vol. 114; no. 7; pp. 1083 - 1093
• Main Authors: Wang, Xiaowei, Palasubramaniam, Jathushan, Gkanatsas, Yannik, Hohmann, Jan David, Westein, Erik, Kanojia, Ruchi, Alt, Karen, Huang, Dexing, Jia, Fu, Ahrens, Ingo, Medcalf, Robert L, Peter, Karlheinz, Hagemeyer, Christoph E
• Format: Journal Article
• Language: English
• Published: United States American Heart Association, Inc 28.03.2014
• Subjects: Animals; Blood Platelets - drug effects; Blood Platelets - immunology; Carotid Arteries - diagnostic imaging; CHO Cells; Cricetinae; Cricetulus; Drug Evaluation, Preclinical; Fibrinolytic Agents - adverse effects; Fibrinolytic Agents - therapeutic use; Integrin alpha2 - immunology; Mice; Mice, Inbred C57BL; Plasminogen - metabolism; Platelet Activation; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - therapeutic use; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; Single-Chain Antibodies - therapeutic use; Thromboembolism - drug therapy; Thromboembolism - prevention & control; Thrombolytic Therapy; Ultrasonography; Urokinase-Type Plasminogen Activator - genetics; Urokinase-Type Plasminogen Activator - therapeutic use; urokinase-type plasminogen activator; thrombolytic therapy; blood platelets; thrombosis
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/CIRCRESAHA.114.302514

RATIONALE:Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated. OBJECTIVE:We developed a novel targeted fibrinolytic drug that is directed against activated platelets. METHODS AND RESULTS:We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet–integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5–scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5–scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5–scuPA as compared with the same dose of a mutated, nontargeting scFv–scuPA or vehicle. The ability of scFvSCE5–scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen. CONCLUSIONS:Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.