Prourokinase Mutant That Induces Highly Effective Clot Lysis Without Interfering With Hemostasis

• Published in: Circulation research 2002-04, Vol.90 (7), p.757-763
• Main Authors: Liu, Jian-Ning, Liu, Jian-Xia, Liu, Bei-fang, Sun, Ziyong, Zuo, Jian-Ling, Zhang, Pei-xiang, Zhang, Jing, Chen, Yu-hong, Gurewich, Victor
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Biological and medical sciences; Bleeding Time; Blood Coagulation - drug effects; Blood. Blood coagulation. Reticuloendothelial system; Disease Models, Animal; Dogs; Drug Evaluation, Preclinical; Drug Stability; Enzyme Activation - drug effects; Enzyme Activation - genetics; Femoral Vein - drug effects; Fibrin - drug effects; Fibrin - metabolism; Fibrinolysis - drug effects; Hemorrhage - prevention & control; Hemostasis - drug effects; Humans; Macaca mulatta; Male; Medical sciences; Mutagenesis, Site-Directed; Pharmacology. Drug treatments; Plasma - drug effects; Plasma - metabolism; Plasminogen - drug effects; Plasminogen - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Thrombolytic Therapy - methods; Thrombosis - drug therapy; Tissue Plasminogen Activator - pharmacology; Urokinase-Type Plasminogen Activator - genetics; Urokinase-Type Plasminogen Activator - metabolism; Urokinase-Type Plasminogen Activator - pharmacology
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000014825.71092.BD

Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300→His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by ≈600 μg/kg M5 versus ≈1200 μg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.