A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance. Issue 1 (1st December 2018)

• Record Type: Journal Article
• Authors: 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
• Is Part Of: Journal of extracellular vesicles. Volume 7:Issue 1(2018)
• Publication Date: 2018-12-01
• Subjects: Extracellular vesicles -- microvesicles -- fibrinolysis -- immunomagnetic separation -- septic shock; Cells -- Mechanical properties -- Periodicals; Transport Vesicles; Cells -- Mechanical properties; Periodicals; Fulltext; Internet Resources; Periodicals; Electronic journals; Periodicals; 571.63
• DOI: 10.1080/20013078.2018.1494482
• Languages: English
• ISSNs: 2001-3078

Abstract

ABSTRACT: 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.