Recombinant human C1-inhibitor prevents non-specific proteolysis by mutant pro-urokinase during optimal fibrinolysis

Abstract

SummaryA single-site mutant of prouPA (M5) spared haemostatic fibrin during thrombolysis in dogs. Zymograms of plasma from these dogs showed an unusual inhibitor complex with C1-inhibitor (C1I). Purified C1I added to human plasma enhanced the fibrinspecificity of M5. In the present study, the effect of recombinant human C1I (recC1I) on high-dose M5 and tPA were compared using fluorescein-labeled standardised clots in a plasma milieu. The shortest time to complete clot lysis (maximum rate) was first determined. This was ∼65% per hour for both activators. By contrast, their top fibrin-specific lysis rate (<20% fibrinogen depletion) was less than half maximum (25–30% per hour). Adding recC1I (250–750 μg/ml) did not affect fibrinolysis, but prevented fibrinogenolysis and plasminogen depletion by M5, raising its fibrin-specific lysis rate to the maximum. With tPA, the recC1I modestly attenuated fibrinogenolysis, raising its fibrin-specific rate to about half the maximum. Consistent with this, the t½ inhibition by C1I was ∼90 min for tPA compared with ∼10 min for tcM5. The t½ of C1I for plasmin was ∼2 min. Zymograms of plasma after clot lysis indicated that recC1I prevented non-specific tcM5 generation from M5, as evidenced by suppression of tcM5:C1I complexes. In conclusion, recC1I raised the fibrin-specificity of M5 in plasma so that a maximum lysis rate could be achieved without fibrinogenolysis. The inhibition by C1I of nonspecific but not fibrin-dependent plasminogen activation could not be duplicated by other serpins. The findings provide a potential means to optimize both the efficacy and safety of thrombolysis.