A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance

Sylvie Cointe (1, 2, 3) , Karim Harti Souab (4) , Tarik Bouriche (5) , Loris Vallier (3) , Amandine Bonifay (3) , Coralie Judicone (5) , Stephane Robert (3) , Romain Armand (4) , Philippe Poncelet (5) , Jacques Albanèse (4) , Francoise Dignat-George (3, 2) , Romaric Lacroix (3, 2)

1 Laboratoire d'hématologie biologique [Hôpital de la Timone - Hôpital Nord - APHM]
2 LA CONCEPTION - Hôpital de la Conception [CHU - APHM]
3 C2VN - Centre recherche en CardioVasculaire et Nutrition = Center for CardioVascular and Nutrition research
4 TIMONE - Hôpital de la Timone [CHU - APHM]
5 BIOCYTEX

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 (epsilon-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) x 10(-3)/min, p = 0.02; 1.4 [1-1.6] vs 5.2 [2.2-16] A(405) x 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.

Mots clés: microvesicles; septic shock

Mots clés: Extracellular vesicles; fibrinolysis; immunomagnetic separation

Domaines: Sciences du Vivant [q-bio]

Sylvie Cointe, Karim Harti Souab, Tarik Bouriche, Loris Vallier, Amandine Bonifay, et al.. A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance. Journal of Extracellular Vesicles, 2018, 7 (1), pp.1494482. ⟨10.1080/20013078.2018.1494482⟩. ⟨hal-02060265⟩