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

Abstract

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 Lys3003 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 Ϸ600 g/kg M5 versus Ϸ1200 g/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 Ͼ5-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.

A randomized study of prourokinase during primary percutaneous coronary intervention in acute ST‐segment elevation myocardial infarction

“…The sample size of this trial was only 41 which was insufficient to statistically analyse MACE events. As a new thrombolytic drug with fibrin‐selective speciality, prourokinase can be converted into active urokinase and produce thrombolytic effects . PROACT II study has confirmed that intra‐arterial prourokinase can increase recanalization rate without increasing hemorrhagic risk, suggesting prourokinase a safe and effective drug for thrombolysis.…”

“…As a new thrombolytic drug with fibrin-selective speciality, prourokinase can be converted into active urokinase and produce thrombolytic effects. 17,18 PROACT II study has confirmed that intra-arterial prourokinase can increase recanalization rate without increasing hemorrhagic risk, suggesting prourokinase a safe and effective drug for thrombolysis. A Meta-analysis in China has also demonstrated that intravenous administration of prourokinase can improve recanalization rate, decrease MACE events and hemorrhagic complications.…”

C1‐inhibitor prevents non‐specific plasminogen activation by a prourokinase mutant without impeding fibrin‐specific fibrinolysis

“…Recombinant Lys300→His mutant prouPA expressed in Escherichia coli was obtained from Dr. Paolo Sarmientos at Primm (Milan, Italy) and prepared as previously described [8]. Recombinant prouPA expressed in E. coli was obtained from Landing Science and Technology Company, Nanjing, China.…”

“…M5 induced efficient, fibrin‐specific clot lysis in a plasma milieu in vitro and in dogs with venous thromboemboli, in which M5 was associated with no more bleeding than placebo [8]. In a more recent study of arterial thrombi in dogs, M5 and t‐PA induced comparably effective lysis, but blood loss from fresh hemostatic sites was tenfold higher with t‐PA, suggesting that M5 spared hemostatic fibrin.…”

Mutant Prourokinase with Adjunctive C1-Inhibitor Is an Effective and Safer Alternative to tPA in Rat Stroke

“…These led to the development of a single site-directed (Lys300→His) prouPA mutant, M5, which was significantly more stable in plasma at therapeutic doses [18], [19], but otherwise retained the basic mechanism of action of prouPA. Two thrombolytic studies with M5 in dogs with venous or arterial thrombi showed that it was as effective as tPA but caused ten-fold less bleeding from injury sites [20], [21]. In the second of these studies, an unusual plasma inhibitor of two-chain M5 (tcM5) was identified which helped explain the low incidence of bleeding complications.…”