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...
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MDPI AG
2020-11-01
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| Series: | Journal of Clinical Medicine |
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| Online Access: | https://www.mdpi.com/2077-0383/9/11/3684 |
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| author | Kanagasabai Vadivel Anne K. Zaiss Yogesh Kumar Frank M. Fabian Ayman E. A. Ismail Mark A. Arbing Wallace G. Buchholz William H. Velander S. Paul Bajaj |
| author_facet | Kanagasabai Vadivel Anne K. Zaiss Yogesh Kumar Frank M. Fabian Ayman E. A. Ismail Mark A. Arbing Wallace G. Buchholz William H. Velander S. Paul Bajaj |
| author_sort | Kanagasabai Vadivel |
| collection | DOAJ |
| description | 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>. |
| first_indexed | 2024-03-10T14:47:51Z |
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| spelling | doaj.art-8225ff1b319d465197425cdb0dec2bd32023-11-20T21:14:17ZengMDPI AGJournal of Clinical Medicine2077-03832020-11-01911368410.3390/jcm9113684Enhanced Antifibrinolytic Efficacy of a Plasmin-Specific Kunitz-Inhibitor (60-Residue Y11T/L17R with C-Terminal IEK) of Human Tissue Factor Pathway Inhibitor Type-2 Domain1Kanagasabai Vadivel0Anne K. Zaiss1Yogesh Kumar2Frank M. Fabian3Ayman E. A. Ismail4Mark A. Arbing5Wallace G. Buchholz6William H. Velander7S. Paul Bajaj8Department of Orthopedic Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USADepartment of Orthopedic Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USADepartment of Orthopedic Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USADepartment of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, NE 68588, USADepartment of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, NE 68588, USAProtein Expression Technology Center, UCLA-DOE Institute, University of California, Los Angeles, CA 90095, USADepartment of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, NE 68588, USADepartment of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, NE 68588, USADepartment of Orthopedic Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USACurrent 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>.https://www.mdpi.com/2077-0383/9/11/3684aprotininε-aminocaproic acidtranexamic acidantifibrinolyticsthromboelastography |
| spellingShingle | Kanagasabai Vadivel Anne K. Zaiss Yogesh Kumar Frank M. Fabian Ayman E. A. Ismail Mark A. Arbing Wallace G. Buchholz William H. Velander S. Paul Bajaj 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 Journal of Clinical Medicine aprotinin ε-aminocaproic acid tranexamic acid antifibrinolytics thromboelastography |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | aprotinin ε-aminocaproic acid tranexamic acid antifibrinolytics thromboelastography |
| url | https://www.mdpi.com/2077-0383/9/11/3684 |
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