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1.
Blood ; 134(14): 1119-1131, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31434703

RESUMO

Antiphospholipid antibodies (aPLs) with complex lipid and/or protein reactivities cause complement-dependent thrombosis and pregnancy complications. Although cross-reactivities with coagulation regulatory proteins contribute to the risk for developing thrombosis in patients with antiphospholipid syndrome, the majority of pathogenic aPLs retain reactivity with membrane lipid components and rapidly induce reactive oxygen species-dependent proinflammatory signaling and tissue factor (TF) procoagulant activation. Here, we show that lipid-reactive aPLs activate a common species-conserved TF signaling pathway. aPLs dissociate an inhibited TF coagulation initiation complex on the cell surface of monocytes, thereby liberating factor Xa for thrombin generation and protease activated receptor 1/2 heterodimer signaling. In addition to proteolytic signaling, aPLs promote complement- and protein disulfide isomerase-dependent TF-integrin ß1 trafficking that translocates aPLs and NADPH oxidase to the endosome. Cell surface TF pathway inhibitor (TFPI) synthesized by monocytes is required for TF inhibition, and disabling TFPI prevents aPL signaling, indicating a paradoxical prothrombotic role for TFPI. Myeloid cell-specific TFPI inactivation has no effect on models of arterial or venous thrombus development, but remarkably prevents experimental aPL-induced thrombosis in mice. Thus, the physiological control of TF primes monocytes for rapid aPL pathogenic signaling and thrombosis amplification in an unexpected crosstalk between complement activation and coagulation signaling.


Assuntos
Anticorpos Antifosfolipídeos/imunologia , Monócitos/imunologia , Tromboplastina/imunologia , Trombose/imunologia , Animais , Coagulação Sanguínea , Células Cultivadas , Feminino , Humanos , Lipoproteínas/imunologia , Masculino , Camundongos Endogâmicos C57BL , Monócitos/patologia , Transdução de Sinais , Trombose/sangue , Trombose/patologia
2.
Thromb Res ; 182: 64-74, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31450010

RESUMO

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.

3.
J Extracell Vesicles ; 7(1): 1494482, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30034644

RESUMO

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.

4.
Int J Cardiol ; 243: 216-222, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28747025

RESUMO

BACKGROUND: Thrombotic risk constitutes a major complication of atrial fibrillation (AF). Platelets and microparticles (MPs) are important for hemostasis and thrombosis, however their participation during AF is not well known. The aim of this study was to characterize platelet function and MPs procoagulant and fibrinolytic activity in AF patients and to determine the effects of an acute-AF episode. METHODS: Blood was collected from paroxysmal (21) and persistent (16) AF patients referred for AF catheter ablation. Ten patients in sinus rhythm for 10days were induced in AF allowing comparisons of left atrium samples before and after induction. Platelet aggregation with ADP, TRAP, collagen, and ristocetin was studied. Platelet surface expression of PAR-1, αIIbß3, GPIb and P-selectin were evaluated by flow cytometry, and MPs-associated procoagulant and fibrinolytic activity levels were determined by functional assays. RESULTS: A specific reduction in platelet aggregation to TRAP, activating the thrombin receptor PAR-1, was found in all AF patients. No differences in platelet receptor expression were found. Yet, after acute-induced AF, the platelet response was improved. Furthermore, a significant decrease of left atrium tissue factor-dependent procoagulant activity of MPs was observed. CONCLUSION: Acute episodes of AF results in a decrease in MPs-associated tissue factor activity, possibly corresponding to consumption, which in turn favors coagulation and the local production of thrombin. A decreased platelet basal aggregation to TRAP may result from PAR1 desensitization, whereas the improved response after an induced episode of AF suggests activation of coagulation and PAR1 re-sensitization.


Assuntos
Fibrilação Atrial/sangue , Fibrilação Atrial/terapia , Plaquetas/fisiologia , Micropartículas Derivadas de Células/metabolismo , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Fibrilação Atrial/diagnóstico , Plaquetas/efeitos dos fármacos , Ablação por Cateter/métodos , Micropartículas Derivadas de Células/efeitos dos fármacos , Estudos de Coortes , Feminino , Humanos , Cetoprofeno/farmacologia , Cetoprofeno/uso terapêutico , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos
5.
Transfus Apher Sci ; 53(2): 110-26, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26603057

RESUMO

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Citometria de Fluxo/métodos , Animais , Humanos
6.
Arterioscler Thromb Vasc Biol ; 32(4): 1054-8, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22328775

RESUMO

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.


Assuntos
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
7.
Invest New Drugs ; 30(3): 1121-31, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21519855

RESUMO

The efficacy of anti-CD33 immunoconjugates had been previously demonstrated for gemtuzumab-ozogamicin. AVE9633 is an anti-CD33-maytansine conjugate created by ImmunoGen Inc. Phase I trials of AVE9633 were performed in patients with AML to evaluate tolerability, pharmacokinetics and pharmacodynamics. Three phase I studies of AVE9633 were performed in 54 patients with refractory/relapsed AML, evaluating drug infusion on day 1 of a 21-day cycle (Day 1 study), day 1 and 8 (Day 1/8 study) and day 1, 4 and 7 (Day 1/4/7 study) of a 28-day cycle. Toxicity was mainly allergic reaction during infusion (3 grade 3 bronchospasms). DLT was reached for the D1-D7 schedule at 150 mg/sqm (1 keratitis, 1 liver toxicity), and the MTD was set at 130 mg/sqm for this schedule. In the two other phases I, the DLT was not reached. In the Day 1/8 study, CD33 on peripheral blasts was saturated and down-modulated for doses of 75 mg/m(2) × 2 or higher, which was correlated with WBC kinetics and plasma levels of AVE9633. Decrease of DM4/CD33 ratio on the blasts surface between day 1 and 8 was the rational for evaluating day 1/4/7 schedule. This induced relatively constant DM4/CD33 levels over the first 8 days, however no activity was noted. One CRp, one PR and biological activity in five other patients were observed in this study. The Day 1 and Day 1/4/7 studies were early discontinued because of drug inactivity at doses significantly higher than CD33 -saturating doses. No myelossuppression was observed at any trial of AVE9633. The pharmacokinetics/pharmacodynamics data obtained in these studies will provide very useful information for the design of the next generation of immunoconjugates.


Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Antineoplásicos/administração & dosagem , Imunoconjugados/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Maitansina/análogos & derivados , Maitansina/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Espasmo Brônquico/induzido quimicamente , Hipersensibilidade a Drogas/etiologia , Feminino , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Infusões Intravenosas , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/metabolismo , Masculino , Maitansina/efeitos adversos , Maitansina/farmacocinética , Pessoa de Meia-Idade , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico
8.
Semin Thromb Hemost ; 36(8): 807-18, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21049381

RESUMO

Circulating microparticles are submicron vesicles released from cell membranes in response to activation or apoptosis. Acknowledgment of their role both as markers and pathogenic effectors in thrombosis, inflammation, and the spread of cancer has increased the interest of their measurement in clinical practice. However, assessment of their clinical use is impeded by technological issues. Among the different methodologies available, flow cytometry is the most commonly used technique. This review addresses flow cytometry limitations in microparticle measurement that may be subdivided into three domains: sizing, probing, and counting. This article also covers the various standardization strategies and technological improvements that have been proposed to overcome these limitations. New tools using size-calibrated beads and recent progress in instrumentation have opened new avenues to improve detection of microparticle populations of smaller sizes. Significant optimization in microparticle detection is also expected from the use of new fluorescent dyes and the establishment of practical recommendations. Finally, absolute counting of microparticles will also benefit from adapted bead-based strategies or, alternatively, from the generalized availability of volumetric systems. Overall, recent technological improvements maintain flow cytometry as a highly competitive analytical method to measure microparticles. Challenging these evolutions in pathological situations is a mandatory step to validate their real impact in clinical practice.


Assuntos
Micropartículas Derivadas de Células , Citometria de Fluxo/métodos , Animais , Plaquetas , Citometria de Fluxo/instrumentação , Humanos , Tamanho da Partícula
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