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

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 activ...

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Bibliographic Details
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:	eng
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
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Summary:	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.
ISSN:	0009-7330 1524-4571