Publications by authors named "Philippe Poncelet"

10 Publications

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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

Tissue factor pathway inhibitor primes monocytes for antiphospholipid antibody-induced thrombosis.

Blood 2019 10 21;134(14):1119-1131. Epub 2019 Aug 21.

Center for Thrombosis and Hemostasis and.

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 β1 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.
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http://dx.doi.org/10.1182/blood.2019001530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776793PMC
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

Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode.

Int J Cardiol 2017 Sep;243:216-222

IHU Liryc, Electrophysiology and Heart Modeling Institute, fondation Bordeaux Université, 33600, Pessac, France. Electronic address:

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, αIIbβ3, 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.
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http://dx.doi.org/10.1016/j.ijcard.2017.03.068DOI Listing
September 2017

Tips and tricks for flow cytometry-based analysis and counting of microparticles.

Transfus Apher Sci 2015 Oct 27;53(2):110-26. Epub 2015 Oct 27.

Hematology Laboratory, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), CHU UCL Namur, Université catholique de Louvain, Yvoir, Belgium.

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.
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http://dx.doi.org/10.1016/j.transci.2015.10.008DOI Listing
October 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

Phase I studies of AVE9633, an anti-CD33 antibody-maytansinoid conjugate, in adult patients with relapsed/refractory acute myeloid leukemia.

Invest New Drugs 2012 Jun 26;30(3):1121-31. Epub 2011 Apr 26.

Hematology and Oncology Department, Saint-Antoine Hospital, AP-HP, Paris, France.

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.
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http://dx.doi.org/10.1007/s10637-011-9670-0DOI Listing
June 2012

Overcoming limitations of microparticle measurement by flow cytometry.

Semin Thromb Hemost 2010 Nov 3;36(8):807-18. Epub 2010 Nov 3.

UMR-S608 INSERM, F-Marseille, and Faculté de Pharmacie, Université de la Méditerranée, F-Marseille, France.

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.
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http://dx.doi.org/10.1055/s-0030-1267034DOI Listing
November 2010
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