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

Authors

Kanagasabai Vadivel, University of California, Los Angeles
Anne K. Zaiss, University of California, Los Angeles
Yogesh Kumar, University of California, Los Angeles
Frank M. Fabian, University of Nebraska - Lincoln
Ayman E.A. Ismail, University of Nebraska - Lincoln
Mark A. Arbing, University of California, Los Angeles
Wallace G. Buchholz, University of Nebraska - Lincoln
William H. Velander, University of Nebraska - LincolnFollow
S Paul Bajaj, University of California, Los Angeles

Date of this Version

11-17-2020

Document Type

Article

Citation

J. Clin. Med. 2020, 9, 3684; doi:10.3390/jcm9113684

Comments

CC-BY

Abstract

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-KCOOH 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.