| Summary: | 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 (<sub>NH2</sub>NAE…IEK<sub>COOH</sub>) 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<sub>COOH</sub>) and a double mutant (KD1-Y11T/L17R- K<sub>COOH</sub>) were expressed in <i>Escherichia coli</i> as His-tagged constructs, each with enterokinase cleavage sites. KD1-Y11T/L17R-K<sub>COOH</sub> was also expressed in <i>Pichia pastoris</i>. KD1-Y11T/L17R-K<sub>COOH</sub> inhibited plasmin comparably to aprotinin and bound to the kringle domains of plasminogen/plasmin and tPA with <i>K<sub>d</sub></i> of ~50 nM and ~35 nM, respectively. Importantly, compared to aprotinin, KD1-L17R-K<sub>COOH</sub> and KD1-Y11T/L17R-K<sub>COOH</sub> did not inhibit kallikrein. Moreover, the antifibrinolytic potential of KD1-Y11T/L17R-K<sub>COOH</sub> was better than that of KD1-L17R-K<sub>COOH</sub> and similar to that of aprotinin in plasma clot-lysis assays. In thromboelastography experiments, KD1-Y11T/L17R-K<sub>COOH</sub> was shown to inhibit fibrinolysis in a dose dependent manner and was comparable to aprotinin at a higher concentration. Further, KD1-Y11T/L17R-K<sub>COOH</sub> did not induce cytotoxicity in primary human endothelial cells or fibroblasts. We conclude that KD1-Y11T/L17R-K<sub>COOH</sub> is comparable to aprotinin, the most potent known inhibitor of plasmin and can be produced in large amounts using <i>Pichia</i>.
|
|---|