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

Summary.  Background:  Prourokinase (prouPA) is unstable in plasma at therapeutic concentrations. A mutant form, M5, made more stable by reducing its intrinsic activity was therefore developed. Activation to two‐chain M5 (tcM5) induced a higher catalytic activity than two‐chain urokinase plasminogen activator (tcuPA), implicating an active site functional difference. Consistent with this, an unusual tcM5 complex with plasma C1‐inhibitor was recently described in dog and human plasma. The effect of C1‐inhibitor on fibrinolysis and fibrinogenolysis by M5 is the subject of this study. Methods and results:  Zymograms of tcM5 and tcuPA incubated in plasma revealed prominent tcM5–C1‐inhibitor complexes, which formed within 5 min. The inhibition rate by purified human C1‐inhibitor (250 μg mL −1 ) was about 7‐fold faster for tcM5 than it was for tcuPA (10 μg mL −1 ). The effect of the inhibitor on the stability of M5 and prouPA was determined by incubating them in plasma at high concentrations (10–20 μg mL −1 ) ± C1‐inhibitor supplementation. Above 10 μg mL −1 , depletion of all plasma plasminogen occurred, indicating plasmin generation and tcM5/tcuPA formation. With supplemental C1‐inhibitor, M5 stability was restored but not prouPA stability. Clot lysis by M5 ± supplemental C1‐inhibitor showed no attenuation of the rate of fibrinolysis, whereas fibrinogenolysis was prevented by C1‐inhibitor. Moreover, because of higher dose‐tolerance, the rate of fibrin‐specific lysis reached that achievable by non‐specific fibrinolysis without inhibitor. Conclusions:  Plasma C1‐inhibitor stabilized M5 in its proenzyme configuration in plasma by inhibiting tcM5 and thereby non‐specific plasminogen activation. At the same time, fibrin‐specific plasminogen activation remained unimpaired. This unusual dissociation of effects has significant implications for improving the safety and efficacy of fibrinolysis.