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Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish
BACKGROUND: Blood clotting in humans is initiated by the binding of tissue factor to activated coagulation factor VII (FVIIa) in the plasma. Previous studies have reported that hepsin and factor VII (FVII)‐activating protease are responsible for generating FVIIa. OBJECTIVES: We aimed to identify oth...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590325/ https://www.ncbi.nlm.nih.gov/pubmed/33134781 http://dx.doi.org/10.1002/rth2.12428 |
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author | Khandekar, Gauri Iyer, Neha Jagadeeswaran, Pudur |
author_facet | Khandekar, Gauri Iyer, Neha Jagadeeswaran, Pudur |
author_sort | Khandekar, Gauri |
collection | PubMed |
description | BACKGROUND: Blood clotting in humans is initiated by the binding of tissue factor to activated coagulation factor VII (FVIIa) in the plasma. Previous studies have reported that hepsin and factor VII (FVII)‐activating protease are responsible for generating FVIIa. OBJECTIVES: We aimed to identify other proteases that may activate FVII using zebrafish as a model. METHODS: We screened 179 genes encoding serine protease domains using the piggyback knockdown method to identify genes involved in the activation of zebrafish Fvii. A prolonged kinetic prothrombin time (kPT) assay was used to detect gene knockdown effects. RESULTS: In the primary screen, 21 genes showed prolonged kPT. In the secondary screen, 14 of 21 genes showed positive results. In the tertiary screen, all 14 genes showed prolonged kPT. These 14 genes were knocked down again to estimate relative levels of zebrafish Fviia. Six genes, including known genes, such as f10 and novel prostasin and hepatocyte growth factor B (hgfb), showed lower Fviia levels. Fvii levels were affected only by the knockdown of f7 and not by the knockdown of the other five genes. CONCLUSIONS: Prostasin and hgfb are involved in generating Fviia. We hypothesize that prostasin exerts serine protease activity directly or indirectly to activate Fvii. As Hgfb has a mutated serine protease domain, it may not cleave Fvii but may bind to Fvii to induce autoactivation. The approach developed here may be extended to design other large‐scale knockdown screens. |
format | Online Article Text |
id | pubmed-7590325 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-75903252020-10-30 Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish Khandekar, Gauri Iyer, Neha Jagadeeswaran, Pudur Res Pract Thromb Haemost Original Articles ‐ Hemostasis BACKGROUND: Blood clotting in humans is initiated by the binding of tissue factor to activated coagulation factor VII (FVIIa) in the plasma. Previous studies have reported that hepsin and factor VII (FVII)‐activating protease are responsible for generating FVIIa. OBJECTIVES: We aimed to identify other proteases that may activate FVII using zebrafish as a model. METHODS: We screened 179 genes encoding serine protease domains using the piggyback knockdown method to identify genes involved in the activation of zebrafish Fvii. A prolonged kinetic prothrombin time (kPT) assay was used to detect gene knockdown effects. RESULTS: In the primary screen, 21 genes showed prolonged kPT. In the secondary screen, 14 of 21 genes showed positive results. In the tertiary screen, all 14 genes showed prolonged kPT. These 14 genes were knocked down again to estimate relative levels of zebrafish Fviia. Six genes, including known genes, such as f10 and novel prostasin and hepatocyte growth factor B (hgfb), showed lower Fviia levels. Fvii levels were affected only by the knockdown of f7 and not by the knockdown of the other five genes. CONCLUSIONS: Prostasin and hgfb are involved in generating Fviia. We hypothesize that prostasin exerts serine protease activity directly or indirectly to activate Fvii. As Hgfb has a mutated serine protease domain, it may not cleave Fvii but may bind to Fvii to induce autoactivation. The approach developed here may be extended to design other large‐scale knockdown screens. John Wiley and Sons Inc. 2020-09-22 /pmc/articles/PMC7590325/ /pubmed/33134781 http://dx.doi.org/10.1002/rth2.12428 Text en © 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH) This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Original Articles ‐ Hemostasis Khandekar, Gauri Iyer, Neha Jagadeeswaran, Pudur Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish |
title | Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish |
title_full | Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish |
title_fullStr | Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish |
title_full_unstemmed | Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish |
title_short | Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish |
title_sort | prostasin and hepatocyte growth factor b in factor viia generation: serine protease knockdowns in zebrafish |
topic | Original Articles ‐ Hemostasis |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590325/ https://www.ncbi.nlm.nih.gov/pubmed/33134781 http://dx.doi.org/10.1002/rth2.12428 |
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