Publications by authors named "Coralie Judicone"

8 Publications

  • Page 1 of 1

Granulocyte microvesicles with a high plasmin generation capacity promote clot lysis and improve outcome in septic shock.

Blood 2022 Jan 13. Epub 2022 Jan 13.

AP-HM, France.

Microvesicles (MVs) have previously been shown to exert profibrinolytic capacity, which is increased in patients with septic shock (SS) with a favorable outcome. We therefore hypothesized that the plasmin generation capacity (PGC) could confer to MVs a protective effect supported by their capacity to lyse a thrombus, and we investigated the mechanisms involved. Using a MV-PGC kinetic assay, ELISA and flow cytometry, we found that granulocyte MVs (Gran-MVs) from SS patients display a heterogeneous PGC profile driven by the uPA (urokinase)/uPAR system. In vitro, these MVs lyse a thrombus according to their MV-PGC levels in a uPA/uPAR-dependent manner, as shown in a fluorescent clot lysis test and a lysis front retraction assay. Fibrinolytic activators conveyed by MVs contribute to approximately 30% of the plasma plasminogenolytic capacity of SS patients. In a murine model of SS, the injection of high PGC Gran-MVs significantly improved mouse survival and reduced the number of thrombi in vital organs. This was associated with a modification of the mouse coagulation and fibrinolysis properties toward a more fibrinolytic profile. Interestingly, mouse survival was not improved when soluble uPA was injected. Finally, using a multiplex array on plasma from SS patients, we found that neutrophil elastase correlates with the effect of high-PGC-capacity plasma and modulates the Gran-MV plasmin generation capacity by cleaving uPA-PAI-1 complexes. In conclusion, we show that high PGC level displayed by Gran-MVs reduce thrombus formation and improve survival conferring to Gran-MVs a protective role in a murine model of sepsis.
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http://dx.doi.org/10.1182/blood.2021013328DOI Listing
January 2022

A new hybrid immunocapture bioassay with improved reproducibility to measure tissue factor-dependent procoagulant activity of microvesicles from body fluids.

Thromb Res 2020 12 21;196:414-424. Epub 2020 Sep 21.

BioCytex, Research and Technology Department, Marseille, France. Electronic address:

Background: The procoagulant activity of tissue factor-bearing microvesicles (MV-TF) has been associated with the risk of developing venous thrombosis in cancer patients. However, MV-TF assays are limited either by i) a lack of specificity, ii) a low sensitivity, or iii) a lack of repeatability when high-speed centrifugation (HS-C) is used to isolate MV. Therefore, our objective was to develop a new hybrid "capture-bioassay" with improved reproducibility combining MV immunocapture from biofluids and measurement of their TF activity.

Materials And Methods: Factor Xa generation and flow cytometry assays were used to evaluate IMS beads performance, and to select the most effective capture antibodies. The analytical performance between IMS-based and HS-C-based assays was evaluated with various models of plasma samples (from LPS-activated blood, spiked with tumoral MV, or with saliva MV) and different biofluids (buffer, plasma, saliva, and pleural fluid).

Results: Combining both CD29 and CD59 antibodies on IMS beads was as efficient as HS-C to isolate plasmatic PS+ MV. The IMS-based strategy gave significantly higher levels of MV-TF activity than HS-C in tumor MV spiked buffer, and both pleural fluids and saliva samples. Surprisingly, lower TF values were measured in plasma due to TFPI (TF pathway inhibitor) non-specifically adsorbed onto beads. This was overcome by adding a TFPI-blocking antibody. After optimization, the new IMS-based assay significantly improved reproducibility of MV-TF bioassay versus the HS-C-based assay without losing specificity and sensitivity. In addition, this approach could identify the cellular origin of MV-TF in various biological fluids.

Conclusion: Compared to HS-C, the IMS-based measurement of MV-TF activity in body fluids improves reproducibility and makes the assay compatible with clinical practice. It can facilitate future automation.
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http://dx.doi.org/10.1016/j.thromres.2020.09.020DOI Listing
December 2020

Increasing the sensitivity of the human microvesicle tissue factor activity assay.

Thromb Res 2019 Oct 19;182:64-74. Epub 2019 Jul 19.

Aix Marseille Univ, INSERM, INRA, C2VN, Marseille, France; Department of Hematology and Vascular Biology, CHU La Conception, APHM, Marseille, France.

Introduction: The TF-FVIIa complex is the primary activator of coagulation. Elevated levels of microvesicle (MV) bearing tissue factor (TF)-dependent procoagulant activity are detectable in patients with an increased risk of thrombosis. Several methods have been described to measure MV TF activity but they are hampered by limited sensitivity and specificity. The aim of this work was to increase the sensitivity of the MV TF activity assay (called Chapel Hill assay).

Material And Methods: Improvements of the MV TF activity assay included i/ speed and time of centrifugation, ii/ use of a more potent inhibitory anti-TF antibody iii/ use of FVII and a fluorogenic substrate to increase specificity.

Results: The specificity of the MV TF activity assay was demonstrated by the absence of activity on MV derived from a knock-out-TF cell line using an anti-human TF monoclonal antibody called SBTF-1, which shows a higher TF inhibitory effect than the anti-human TF monoclonal antibody called HTF-1. Experiments using blood from healthy individuals, stimulated or not by LPS, or plasma spiked with 3 different levels of MV, demonstrated that the new assay was more sensitive and this allowed detection of MV TF activity in platelet free plasma (PFP) samples from healthy individuals. However, the assay was limited by an inter-assay variability, mainly due to the centrifugation step.

Conclusions: We have improved the sensitivity of the MV TF activity assay without losing specificity. This new assay could be used to evaluate levels of TF-positive MV as a potential biomarker of thrombotic risk in patients.
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http://dx.doi.org/10.1016/j.thromres.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825876PMC
October 2019

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

J Extracell Vesicles 2018 16;7(1):1494482. Epub 2018 Jul 16.

Aix-Marseille Université, C2VN, UMR-1263, INSERM, INRA 1260, UFR de Pharmacie, Marseille, France.

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] 3.1 [1.7-18]  × 10/min,  = 0.02; 1.4 [1-1.6] 5.2 [2.2-16]  × 10/min,  = 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.
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http://dx.doi.org/10.1080/20013078.2018.1494482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052415PMC
July 2018

Microparticles and Fibrinolysis.

Semin Thromb Hemost 2017 Mar 6;43(2):129-134. Epub 2016 Dec 6.

Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Microparticles (MPs) are submicronic vesicles which are formed by budding of the cellular membrane of virtually any cell type in response to cell activation or apoptosis. Both circulating MPs and MPs generated within tissues harbor molecules with a large repertoire of biological activities and transfer material to target cells. Depending on their cellular origin, the stimuli triggering their formation, or their localization, they may participate in the maintenance of organ or vascular homeostasis as well as inducing dysfunction. MPs have mostly been described as having procoagulant properties. However, the fact that some MP subsets are able to efficiently generate plasmin suggests that the role of MPs in hemostasis is more complex than initially thought. In this review, we summarize key findings showing that MPs provide a heterogeneous catalytic surface for plasmin generation, according to their cellular origin. We further address the specific features of the MP-dependent fibrinolytic system. Potential consequences of this MP-associated fibrinolytic activity in pathology are illustrated in cancer.
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http://dx.doi.org/10.1055/s-0036-1592301DOI Listing
March 2017

Detection of EpCAM-positive microparticles in pleural fluid: A new approach to mini-invasively identify patients with malignant pleural effusions.

Oncotarget 2016 Jan;7(3):3357-66

VRCM, UMR-S1076, Aix-Marseille Université, INSERM, Faculté de Pharmacie, Marseille, France.

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions.
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http://dx.doi.org/10.18632/oncotarget.6581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823111PMC
January 2016

Platelet and not erythrocyte microparticles are procoagulant in transfused thalassaemia major patients.

Br J Haematol 2015 Nov 24;171(4):615-24. Epub 2015 Jul 24.

Centre de Référence Maladies Rares Thalassémies, Marseille-Lyon, Service d'Hémato-Oncologie Pédiatrique, Hôpital de la Timone, APHM, Marseille, France.

The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 β-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte- and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P < 0·001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P = 0·001) and platelet microparticle level (P < 0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when β-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused β-thalassaemia major patients.
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http://dx.doi.org/10.1111/bjh.13609DOI Listing
November 2015

High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles--brief report.

Arterioscler Thromb Vasc Biol 2012 Apr 9;32(4):1054-8. Epub 2012 Feb 9.

UMR Institut National de la Santé et de la Recherche Médicale/Aix-Marseille Université, Faculté de Pharmacie, France.

Objective: Cellular microparticles (MP) are promising biomarkers in many pathological situations. Although flow cytometry (FCM) is widely used for their measurement, it has raised controversies because the smallest MP size falls below the detection limit of standard FCM (sd-FCM). Following recent technological improvements leading to high sensitivity FCM (hs-FCM), our objectives were (1) to evaluate the potential of hs-FCM for extended MP detection, (2) to set up a standardized protocol for MP enumeration, and (3) to compare MP counts obtained with both sensitivity levels.

Methods And Results: Compared with sd-FCM, hs-FCM displayed improved forward scatter resolution and lower background noise, allowing us to discriminate previously undetectable small MP in plasma samples. Using fluorescent beads with appropriate sizes (0.1/0.3/0.5/0.9 μm) and relative amounts, a new standardized hs-FCM MP protocol was set up and provided reproducible MP counts. Applied to coronary patient samples, it resulted into 8- to 20-fold increases in MP counts as compared with sd-FCM. Interestingly, the ratio between small and large MP varied according to clinical status but also depending on MP subset, suggesting access to new biological information.

Conclusions: Recent improvements in FCM provide access to previously undetectable MP and represent a new opportunity to enhance their impact as biomarkers in clinical practice.
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http://dx.doi.org/10.1161/ATVBAHA.111.244616DOI Listing
April 2012
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