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 (_NH2NAE … 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.