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


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 microg mL(-1)) was about 7-fold faster for tcM5 than it was for tcuPA (10 microg 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 microg mL(-1)) +/- C1-inhibitor supplementation. Above 10 microg 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.
Pannell
R
R
Vascular Research Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Kung
W
W
Gurewich
V
V
D003174:Complement C1 Inactivator Proteins / Q000494:pharmacology*; D050718:Complement C1 Inhibitor Protein; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism*; D005342:Fibrinolysis; D006801:Humans; D010958:Plasminogen / Q000187:drug effects* / Q000378:metabolism; D015843:Serpins / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; C469952:SERPING1 protein, human; D015843:Serpins; D005337:Fibrin; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator