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Thromb Res ; 182: 64-74, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31450010


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.

J Extracell Vesicles ; 7(1): 1494482, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30034644


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] A405 × 10-3/min, p = 0.02; 1.4 [1-1.6] vs 5.2 [2.2-16] A405 × 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.

Semin Thromb Hemost ; 43(2): 129-134, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27923263


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.

Micropartículas Derivadas de Células/metabolismo , Fibrinólise/fisiologia , Hemostasia/fisiologia , Humanos
Oncotarget ; 7(3): 3357-66, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26689993


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.

Biomarcadores Tumorais/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Molécula de Adesão da Célula Epitelial/metabolismo , Neoplasias/fisiopatologia , Derrame Pleural Maligno/diagnóstico , Micropartículas Derivadas de Células/patologia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Derrame Pleural Maligno/metabolismo , Estudos Prospectivos
Br J Haematol ; 171(4): 615-24, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26205481


The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 ß-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 ß-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 ß-thalassaemia major patients.

Plaquetas/fisiologia , Transfusão de Sangue , Micropartículas Derivadas de Células/fisiologia , Trombofilia/sangue , Talassemia beta/sangue , Adolescente , Adulto , Autoanticorpos/sangue , Autoanticorpos/imunologia , Plaquetas/ultraestrutura , Micropartículas Derivadas de Células/classificação , Terapia Combinada , Diabetes Mellitus/etiologia , Membrana Eritrocítica/ultraestrutura , Feminino , Hemoglobina Fetal/imunologia , Citometria de Fluxo , Humanos , Hipogonadismo/etiologia , Ferro/sangue , Sobrecarga de Ferro/sangue , Sobrecarga de Ferro/etiologia , Masculino , Lipídeos de Membrana/sangue , Pessoa de Meia-Idade , Estresse Oxidativo , Fosfatidilserinas/sangue , Risco , Esplenectomia , Trombofilia/etiologia , Reação Transfusional , Adulto Jovem , Talassemia beta/complicações , Talassemia beta/cirurgia , Talassemia beta/terapia
Arterioscler Thromb Vasc Biol ; 32(4): 1054-8, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22328775


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.

Micropartículas Derivadas de Células/patologia , Doença das Coronárias/patologia , Citometria de Fluxo , Biomarcadores/sangue , Calibragem , Micropartículas Derivadas de Células/química , Doença das Coronárias/sangue , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Tamanho da Partícula , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Sensibilidade e Especificidade