Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1

Current antifibrinolytic agents reduce blood loss by inhibiting plasmin active sites (e.g., aprotinin) or by preventing plasminogen/tissue plasminogen activator (tPA) binding to fibrin clots (e.g., ε-aminocaproic acid and tranexamic acid); however, they have adverse side effects. Here, we expressed 60-residue ((NH2)NAE…IEK(COOH)) Kunitz domain1 (KD1) mutants of human tissue factor pathway inhibitor type-2 that inhibit plasmin as well as plasminogen activation. A single (KD1-L17R-K(COOH)) and a double mutant (KD1-Y11T/L17R- K(COOH)) were expressed in Escherichia coli as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-K(COOH) was also expressed in Pichia pastoris. KD1-Y11T/L17R-K(COOH) inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with K(d) of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-K(COOH) and KD1-Y11T/L17R-K(COOH) did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-K(COOH) was better than that of KD1-L17R-K(COOH) and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-K(COOH) was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-K(COOH) did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-K(COOH) is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using Pichia.