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(Circulation Research. 2002;90:757.)
© 2002 American Heart Association, Inc.

Molecular Medicine

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

Jian-Ning Liu, Jian-Xia Liu, Bei-fang Liu, Ziyong Sun, Jian-Ling Zuo, Pei-xiang Zhang, Jing Zhang, Yu-hong Chen, Victor Gurewich

From the Vascular Research Laboratory (J.-N.L., Z.S., V.G.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass; the Institute of Molecular Medicine (J.-N.L., J.-X.L., B.L., Z.S., P.Z., J.Z., Y.C.), Nanjing University, Nanjing, China; and Suzhou Medical College (J.-X.L., J.-L.Z.), Suzhou, China.

Correspondence to Dr Jian-Ning Liu, Vascular Research Laboratory/BUR/554G, BI-Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215-5400. E-mail jnliu{at}prodigy.net

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 Lys300His 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.

Key Words: fibrinolysis • bleeding • animals • plasminogen • activators