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A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance
Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052415/ https://www.ncbi.nlm.nih.gov/pubmed/30034644 http://dx.doi.org/10.1080/20013078.2018.1494482 |
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author | Cointe, Sylvie Harti Souab, Karim Bouriche, Tarik Vallier, Loris Bonifay, Amandine Judicone, Coralie Robert, Stéphane Armand, Romain Poncelet, Philippe Albanese, Jacques Dignat-George, Françoise Lacroix, Romaric |
author_facet | Cointe, Sylvie Harti Souab, Karim Bouriche, Tarik Vallier, Loris Bonifay, Amandine Judicone, Coralie Robert, Stéphane Armand, Romain Poncelet, Philippe Albanese, Jacques Dignat-George, Françoise Lacroix, Romaric |
author_sort | Cointe, Sylvie |
collection | PubMed |
description | Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (ε-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8–3.0] vs 3.1 [1.7–18] A(405) × 10(−3)/min, p = 0.02; 1.4 [1–1.6] vs 5.2 [2.2–16] A(405) × 10(−3)/min, p = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance. |
format | Online Article Text |
id | pubmed-6052415 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-60524152018-07-20 A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance Cointe, Sylvie Harti Souab, Karim Bouriche, Tarik Vallier, Loris Bonifay, Amandine Judicone, Coralie Robert, Stéphane Armand, Romain Poncelet, Philippe Albanese, Jacques Dignat-George, Françoise Lacroix, Romaric J Extracell Vesicles Research Article Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (ε-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8–3.0] vs 3.1 [1.7–18] A(405) × 10(−3)/min, p = 0.02; 1.4 [1–1.6] vs 5.2 [2.2–16] A(405) × 10(−3)/min, p = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance. Taylor & Francis 2018-07-16 /pmc/articles/PMC6052415/ /pubmed/30034644 http://dx.doi.org/10.1080/20013078.2018.1494482 Text en © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Cointe, Sylvie Harti Souab, Karim Bouriche, Tarik Vallier, Loris Bonifay, Amandine Judicone, Coralie Robert, Stéphane Armand, Romain Poncelet, Philippe Albanese, Jacques Dignat-George, Françoise Lacroix, Romaric A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance |
title | A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance |
title_full | A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance |
title_fullStr | A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance |
title_full_unstemmed | A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance |
title_short | A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance |
title_sort | new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052415/ https://www.ncbi.nlm.nih.gov/pubmed/30034644 http://dx.doi.org/10.1080/20013078.2018.1494482 |
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