title: Prourokinase mutant that induces highly effective clot lysis without interfering with hemostasis.
abstract: 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 approximately 600 microg/kg M5 versus approximately 1200 microg/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.
authors.affiliation: Vascular Research Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215-5400, USA. jnliu@prodigy.net
authors.forename: Jian-Ning
authors.initials: JN
authors.lastname: Liu
authors.forename: Jian-Xia
authors.initials: JX
authors.lastname: Liu
authors.forename: Bei-fang
authors.initials: BF
authors.lastname: Liu Bf
authors.forename: Ziyong
authors.initials: Z
authors.lastname: Sun
authors.forename: Jian-Ling
authors.initials: JL
authors.lastname: Zuo
authors.forename: Pei-xiang
authors.initials: PX
authors.lastname: Zhang Px
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Yu-hong
authors.initials: YH
authors.lastname: Chen Yh
authors.forename: Victor
authors.initials: V
authors.lastname: Gurewich
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001760:Bleeding Time; D001777:Blood Coagulation / Q000187:drug effects*; D004195:Disease Models, Animal; D004285:Dogs; D004353:Drug Evaluation, Preclinical; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005268:Femoral Vein / Q000187:drug effects; D005337:Fibrin / Q000187:drug effects / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects*; D006470:Hemorrhage / Q000517:prevention & control; D006487:Hemostasis / Q000187:drug effects*; D006801:Humans; D008253:Macaca mulatta; D008297:Male; D016297:Mutagenesis, Site-Directed; D010949:Plasma / Q000187:drug effects / Q000378:metabolism; D010958:Plasminogen / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015912:Thrombolytic Therapy / Q000379:methods*; D013927:Thrombosis / Q000188:drug therapy*; D010959:Tissue Plasminogen Activator / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D011994:Recombinant Proteins; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase