Publications by authors named "Françoise Dignat-George"

221 Publications

Severe and Irreversible Pancytopenia Associated With SARS-CoV-2 Bone Marrow Infection in a Patient With Waldenstrom Macroglobulinemia.

Clin Lymphoma Myeloma Leuk 2021 Jan 13. Epub 2021 Jan 13.

Hematology and Cellular Therapy Department, Conception University Hospital, Marseille, France; TAGC, INSERM, UMR1090, Aix-Marseille University, Marseille, France; SMARTc Unit, Pharmacokinetics Laboratory, UMR_911 CRO2 Aix-Marseille University, Marseille, France.

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http://dx.doi.org/10.1016/j.clml.2021.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832621PMC
January 2021

Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19.

Blood Adv 2021 02;5(3):628-634

Aix Marseille University, INSERM 1263, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Centre de Recherche en CardioVasculaire et Nutrition (C2VN), Marseille, France.

Coronavirus disease 2019 (COVID-19) has become one of the biggest public health challenges of this century. Severe forms of the disease are associated with a thrombo-inflammatory state that can turn into thrombosis. Because tissue factor (TF) conveyed by extracellular vesicles (EVs) has been implicated in thrombosis, we quantified the EV-TF activity in a cohort of hospitalized patients with COVID-19 (n = 111) and evaluated its link with inflammation, disease severity, and thrombotic events. Patients with severe disease were compared with those who had moderate disease and with patients who had septic shock not related to COVID-19 (n = 218). The EV-TF activity was notably increased in patients with severe COVID-19 compared with that observed in patients with moderate COVID-19 (median, 231 [25th to 75th percentile, 39-761] vs median, 25 [25th to 75th percentile, 12-59] fM; P < .0001); EV-TF was correlated with leukocytes, D-dimer, and inflammation parameters. High EV-TF values were associated with an increased thrombotic risk in multivariable models. Compared with patients who had septic shock, those with COVID-19 were characterized by a distinct coagulopathy profile with significantly higher EV-TF and EV-fibrinolytic activities that were not counterbalanced by an increase in plasminogen activator inhibitor-1 (PAI-1). Thus, this article is the first to describe the dissemination of extreme levels of EV-TF in patients with severe COVID-19, which supports the international recommendations of systematic preventive anticoagulation in hospitalized patients and potential intensification of anticoagulation in patients with severe disease.
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http://dx.doi.org/10.1182/bloodadvances.2020003308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846479PMC
February 2021

Renal SPECT/CT with 99mTc-dimercaptosuccinic acid is a non-invasive predictive marker for the development of interstitial fibrosis in a rat model of renal insufficiency.

Nephrol Dial Transplant 2020 Dec 23. Epub 2020 Dec 23.

Centre Européen de Recherche en Imagerie Médicale (CERIMED), Aix-Marseille Université, France.

Background: CKD increases cardiovascular risk and mortality. Renal fibrosis plays a major role in the progression of CKD but to date, histology remains the gold standard to assess fibrosis. Non-invasive techniques are needed to assess renal parenchymal impairment, and to perform longitudinal evaluation of renal structure. Thus, we evaluated renal isotopic imaging by SPECT/CT with 99mTc-DMSA to monitor renal impairment during renal insufficiency in rats.

Methods: Renal insufficiency was induced by adenine-rich diet (ARD) at 0.25% and 0.5% for 28 days. Renal dysfunction was evaluated by assaying biochemical markers and renal histology. Renal parenchymal impairment was assessed by SPECT/CT isotopic imaging with 99mTc-DMSA on days 0, 7, 14, 21, 28, 35, 49.

Results: Compared to control, ARD rats developed renal dysfunction characterized by increased serum creatinine and BUN, fibrosis and tubulointerstitial damage in kidneys, with dose-dependent effect of the adenine concentration. 99mTc-DMSA SPECT-CT imaging showed a significant decrease in renal uptake over time in 0.25% and 0.5% ARD rats compared to control rats (P = 0.011 and P = 0.0004 respectively). 99mTc-DMSA uptake on D28 was significantly inversely correlated with sirius red staining evaluated on D49 (r = 0.89, P < 0.0001, R2=0.67).

Conclusions: 99mTc-DMSA renal scintigraphy allows a longitudinal follow-up of risk of renal fibrosis in rats. We described that the reduction of renal parenchyma in ARD rats is inversely proportional to newly formed fibrous tissue in the kidney. Our results suggest that 99mTc-DMSA renal scintigraphy may be a useful prognostic non-invasive marker of the development of renal fibrosis in animals and should be tested in humans.
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http://dx.doi.org/10.1093/ndt/gfaa374DOI Listing
December 2020

CD146/sCD146 in the Pathogenesis and Monitoring of Angiogenic and Inflammatory Diseases.

Biomedicines 2020 Dec 10;8(12). Epub 2020 Dec 10.

Hematology Department, Center for CardioVascular and Nutrition Research C2VN, Faculty of Pharmacy, Timone Campus, Aix-Marseille University, Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de Recherche Pour L'agriculture, L'alimentation et L'environnement (INRAE), 13005 Marseille, France.

CD146 is a cell adhesion molecule expressed on endothelial cells, as well as on other cells such as mesenchymal stem cells and Th17 lymphocytes. This protein also exists in a soluble form, whereby it can be detected in biological fluids, including the serum or the cerebrospinal fluid (CSF). Some studies have highlighted the significance of CD146 and its soluble form in angiogenesis and inflammation, having been shown to contribute to the pathogenesis of many inflammatory autoimmune diseases, such as systemic sclerosis, mellitus diabetes, rheumatoid arthritis, inflammatory bowel diseases, and multiple sclerosis. In this review, we will focus on how CD146 and sCD146 contribute to the pathogenesis of the aforementioned autoimmune diseases and discuss the relevance of considering it as a biomarker in these pathologies.
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http://dx.doi.org/10.3390/biomedicines8120592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764286PMC
December 2020

Multifaceted role of extracellular vesicles in atherosclerosis.

Atherosclerosis 2021 02 11;319:121-131. Epub 2020 Nov 11.

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

Extracellular vesicles (EVs) are small vesicles released by the majority of cells in response to cell activation or death stimuli. They are grouped as small EVs or exosomes, large EVs such as microvesicles (MVs) and apoptotic bodies, resulting from distinct mechanisms of generation. EVs are released into the extracellular space, in most human biological fluids and tissues, including atherosclerotic plaques. They transport complex cargo of bioactive molecules, including proteins, lipids and genetic material and are therefore involved in pathophysiological pathways of cell-cell communication. Indeed, EVs are involved in several processes such as inflammation, coagulation, vascular dysfunction, angiogenesis and senescence, contributing to the initiation and progression of atherothrombotic diseases. Consequently, they behave as a determinant of atherosclerotic plaque vulnerability leading to major cardiovascular disorders. Over the last decade, the field of EVs research has grown, highlighting their involvement in atherosclerosis. However, limitations in both detection methodologies and standardisation have hindered implementation of EVs in the clinical settings. This review summarizes the effect of EVs in atherosclerosis development, progression and severity, with specific attention devoted to their ambivalent roles in senescence and hemostasis. This review will also highlight the role of MVs as multifaceted messengers, able to promote or to attenuate atherosclerosis progression. Finally, we will discuss the main technical challenges and prerequisites of standardization for driving EVs to the clinics and delineate their relevance as emergent biomarkers and innovative therapeutic approaches in atherosclerosis.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.11.006DOI Listing
February 2021

Technical and biological review of authorized medical devices for platelets-rich plasma preparation in the field of regenerative medicine.

Platelets 2021 Feb 6;32(2):200-208. Epub 2020 Nov 6.

Cell Therapy Laboratory, Hôpital De La Conception, AP-HM, Marseille, France.

Platelet-rich plasma (PRP) has seen increased interest and utilization over the past decade, particularly in the field of musculoskeletal disease. This growth has been accompanied by the development of medical devices to realize PRP preparation which includes blood collection, centrifugation, and PRP isolation. The final PRP composition is directly influenced by this preparation step and absence of biological quality control led to a lack of comparability between PRP products that could explain the large variability in the clinical benefit of PRP reported in literature. To circumvent this issue, the scientific community developed different PRP classifications but none of them have been adopted. The goal of this review is to furnish both technical and biological characteristics from PRP commercial systems. On review of 1379 studies, 105 studies were selected according to inclusion criteria for technical analysis and led to the identification of 50 commercial systems that have been classified in three technical categories based on the blood harvesting technique (tubes, syringes or bags). Twelve studies were selected and sufficiently describe biological characteristics from only 14 commercial systems from the 50 identified in the technical analysis. Inclusion of duplicates characterization from a same PRP system lead to the final analysis of 36 PRP preparations that met the inclusion criteria of the biological analysis. All these PRP preparations have been classified among the seven existing classifications. Comparison from all biological parameters and classifications revealed a large heterogeneity among the available current PRP commercial systems. Index of biological sensitivity of classifications to distinguish PRP preparations were also variable. Although these findings should help clinicians in selecting a system that meets their specific needs, this also raises the question to standardize the parameters to biologically define PRP preparation among users and to systematically performed PRP qualification when used.
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http://dx.doi.org/10.1080/09537104.2020.1832653DOI Listing
February 2021

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

Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods.

Cells 2020 09 24;9(10). Epub 2020 Sep 24.

Cell Therapy Department, Hôpital de la Conception, AP-HM, INSERM CIC BT 1409, 13005 Marseille, France.

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.
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http://dx.doi.org/10.3390/cells9102158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598595PMC
September 2020

Circulating Endothelial Cells as a Marker of Endothelial Injury in Severe COVID -19.

J Infect Dis 2020 Nov;222(11):1789-1793

Aix-Marseille University, INSERM 1263, INRAE, C2VN, Marseille, France.

Beside the commonly described pulmonary expression of the coronavirus disease 2019 (COVID-19), major vascular events have been reported. The objective of this study was to investigate whether increased levels of circulating endothelial cells (CECs) might be associated with severe forms of COVID-19. Ninety-nine patients with COVID-19 were enrolled in this retrospective study. Patients in the intensive care units (ICU) had significantly higher CEC counts than non-ICU patients and the extent of endothelial injury was correlated with putative markers of disease severity and inflammatory cytokines. Together, these data provide in vivo evidence that endothelial injury is a key feature of COVID-19.
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http://dx.doi.org/10.1093/infdis/jiaa528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454721PMC
November 2020

Mathematical modeling of peripheral blood neutrophil kinetics to predict CLAD after lung transplantation.

Transpl Immunol 2020 10 23;62:101321. Epub 2020 Jul 23.

Aix-Marseille Univ, APHM, INSERM UMR1068, CNRS UMR7258, SMARTc-CRCM, Marseille, France.

Background: The presence of neutrophils in the lung was identified as a factor associated with CLAD but requires invasive samples. The aim of this study was to assess the kinetics of peripheral blood neutrophils after lung transplantation as early predictor of CLAD.

Methods: We retrospectively included all recipients transplanted in our center between 2009 and 2014. Kinetics of blood neutrophils were evaluated to predict early CLAD by mathematical modeling using unadjusted and adjusted analyses.

Results: 103 patients were included, 80 in the stable group and 23 in the CLAD group. Bacterial infections at 1 year were associated with CLAD occurrence. Neutrophils demonstrated a high increase postoperatively and then a progressive decrease until normal range. Recipients with CLAD had higher neutrophil counts (mixed effect coefficient beta over 3 years = +1.36 G/L, 95% Confidence Interval [0.99-1.92], p < .001). A coefficient of celerity (S for speed) was calculated to model the kinetics of return to the norm before CLAD occurrence. After adjustment, lower values of S (slower decrease of neutrophils) were associated with CLAD (Odds Ratio = 0.26, 95% Confidence Interval [0.08-0.66], p = .01).

Conclusion: A slower return to the normal range of blood neutrophils was early associated with CLAD occurrence.
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http://dx.doi.org/10.1016/j.trim.2020.101321DOI Listing
October 2020

Sera From Patients With Minimal Change Disease Increase Endothelial Permeability to Sodium.

Kidney Int Rep 2020 Jul 20;5(7):1071-1075. Epub 2020 Apr 20.

Aix-Marseille University, Centre de recherche en CardioVasculaire et Nutrition, Institut national de la santé et de la recherche médicale 1263, Institut national de recherche pour l'agriculture, l'alimentation et l'environnement 1260, Marseille, France.

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http://dx.doi.org/10.1016/j.ekir.2020.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335967PMC
July 2020

Platelet-rich plasma preparations in sports rehabilitation: Where we started and where we should go.

Ann Phys Rehabil Med 2020 Oct 18:101414. Epub 2020 Oct 18.

Cell therapy laboratory, Inserm CIC BT 1409, hôpital de la Conception, AP-HM, Marseille, France; Inserm, Inra, C2VN, Aix Marseille université, Marseille, France.

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http://dx.doi.org/10.1016/j.rehab.2020.06.003DOI Listing
October 2020

Uremic Toxic Blood-Brain Barrier Disruption Mediated by AhR Activation Leads to Cognitive Impairment during Experimental Renal Dysfunction.

J Am Soc Nephrol 2020 07 11;31(7):1509-1521. Epub 2020 Jun 11.

Centre Européen de recherche en Imagerie Médicale, Aix Marseille Université, Centre National de la Recherche Scientifique, Marseille, France

Background: Uremic toxicity may play a role in the elevated risk of developing cognitive impairment found among patients with CKD. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AhR), which is widely expressed in the central nervous system and which we previously identified as the receptor of indoxyl sulfate in endothelial cells.

Methods: To characterize involvement of uremic toxins in cerebral and neurobehavioral abnormalities in three rat models of CKD, we induced CKD in rats by an adenine-rich diet or by 5/6 nephrectomy; we also used AhR knockout mice overloaded with indoxyl sulfate in drinking water. We assessed neurologic deficits by neurobehavioral tests and blood-brain barrier disruption by SPECT/CT imaging after injection of Tc-DTPA, an imaging marker of blood-brain barrier permeability.

Results: In CKD rats, we found cognitive impairment in the novel object recognition test, the object location task, and social memory tests and an increase of blood-brain barrier permeability associated with renal dysfunction. We found a significant correlation between Tc-DTPA content in brain and both the discrimination index in the novel object recognition test and indoxyl sulfate concentrations in serum. When we added indoxyl sulfate to the drinking water of rats fed an adenine-rich diet, we found an increase in indoxyl sulfate concentrations in serum associated with a stronger impairment in cognition and a higher permeability of the blood-brain barrier. In addition, non-CKD AhR knockout mice were protected against indoxyl sulfate-induced blood-brain barrier disruption and cognitive impairment.

Conclusions: AhR activation by indoxyl sulfate, a uremic toxin, leads to blood-brain barrier disruption associated with cognitive impairment in animal models of CKD.
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http://dx.doi.org/10.1681/ASN.2019070728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350986PMC
July 2020

Commentary about mesenchymal stem cells and scarred vocal folds.

Stem Cell Res Ther 2020 05 7;11(1):173. Epub 2020 May 7.

Therapy Department, Aix Marseille Univ, APHM, INSERM, CBT-1409, La Conception University Hospital, Cell, Marseille, France.

A commentary to "Hertegård, S., Nagubothu, S.R., Malmström, E. et al. Treatment of vocal fold scarring with autologous bone marrow-derived human mesenchymal stromal cells - first phase I/II human clinical study. Stem Cell Res Ther 11, 128 (2020)" concerning the surgical intervention including a scar resection, the use of the Voice Handicap Index, the surgical and regulatory points of view regarding the inclusion of patients with laryngeal carcinomas history, and the side effects of bone marrow harvesting.
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http://dx.doi.org/10.1186/s13287-020-01693-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206705PMC
May 2020

Perirenal Adipose Tissue Displays an Age-Dependent Inflammatory Signature Associated With Early Graft Dysfunction of Marginal Kidney Transplants.

Front Immunol 2020 17;11:445. Epub 2020 Mar 17.

C2VN, INSERM 1263, Aix-Marseille Univ, INRAE, Marseille, France.

Better understanding of the contribution of donor aging and comorbidity factors of expanded criteria donors (ECD) to the clinical outcome of a transplant is a challenge in kidney transplantation. We investigated whether the features of donor-derived stromal vascular fraction of perirenal adipose tissue (PRAT-SVF) could be indicative of the deleterious impact of the ECD microenvironment on a renal transplant. A comparative analysis of cellular components, transcriptomic and vasculogenic profiles was performed in PRAT-SVF obtained from 22 optimal donors and 31 ECD deceased donors. We then investigated whether these parameters could be associated with donor aging and early allograft dysfunction. When compared with the PRAT-SVF of non-ECD donors, ECD PRAT-SVF displayed a lower proportion of stromal cells, a higher proportion of inflammatory NK cells. The global RNA sequencing approach indicated a differential molecular signature in the PRAT-SVF of ECD donors characterized by the over-expression of CXCL1 and IL1-β inflammatory transcripts. The vasculogenic activity of PRAT-SVF was highly variable but was not significantly affected in marginal donors. Periorgan recruitment of monocytes/macrophages and NK cells in PRAT-SVF was associated with donor aging. The presence of NK cell infiltrates was associated with lower PRAT-SVF angiogenic activity and with early allograft dysfunction evaluated on day 7 and at 1 month post-transplant. Our results indicate that human NK cell subsets are differentially recruited in the periorgan environment of aging kidney transplants. We provide novel evidence that PRAT-SVF represents a non-invasive and timely source of donor material with potential value to assess inflammatory features that impact organ quality and function.
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http://dx.doi.org/10.3389/fimmu.2020.00445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089962PMC
March 2021

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

J Extracell Vesicles 2020 3;9(1):1713526. Epub 2020 Feb 3.

Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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http://dx.doi.org/10.1080/20013078.2020.1713526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034442PMC
February 2020

Feasibility of First Injection of Autologous Adipose Tissue-Derived Stromal Vascular Fraction in Human Scarred Vocal Folds: A Nonrandomized Controlled Trial.

JAMA Otolaryngol Head Neck Surg 2020 04;146(4):355-363

Department of Oto-Rhino-Laryngology and Head and Neck Surgery, Assistance Publique-Hôpitaux de Marseille, La Conception University Hospital, Marseille, France.

Importance: Patients with scarred vocal folds, whether congenitally or after phonosurgery, often exhibit dysphonia that negatively affects daily life and is difficult to treat. The autologous adipose tissue-derived stromal vascular fraction (ADSVF) is a readily accessible source of cells with angiogenic, anti-inflammatory, immunomodulatory, and regenerative properties.

Objective: To evaluate the feasibility and tolerability of local injections of autologous ADSVF in patients with scarred vocal folds.

Design, Setting, And Participants: CELLCORDES (Innovative Treatment for Scarred Vocal Cords by Local Injection of Autologous Stromal Vascular Fraction) is a prospective, open-label, single-arm, single-center, nonrandomized controlled trial with a 12-month follow-up and patient enrollment from April 1, 2016, to June 30, 2017. Eight patients with severe dysphonia attributable to vocal fold scarring associated with a congenital malformation or resulting from microsurgical sequelae (voice handicap index score >60 of 120) completed the study. Data analysis was performed from September 1, 2018, to January 1, 2019.

Interventions: Injection of ADSVF into 1 or 2 vocal folds.

Main Outcomes And Measures: The primary outcomes were feasibility and the number and severity of adverse events associated with ADSVF-based therapy. The secondary outcomes were changes in vocal assessment, videolaryngostroboscopy, self-evaluation of dysphonia, and quality of life at 1, 6, and 12 months after cell therapy.

Results: Seven women and 1 man (mean [SD] age, 44.6 [10.4] years) were enrolled in this study. Adverse events associated with liposuction and ADSVF injection occurred; most of them resolved spontaneously. One patient received minor treatment to drain local bruising, and another experienced a minor contour defect at the liposuction site. At 12 months, the voice handicap index score was improved in all patients, with a mean (SD) improvement from baseline of 40.1 (21.5) points. Seven patients (88%) were considered to be responders, defined as improvement by 18 points or more in the voice handicap index score (the minimum clinically important difference).

Conclusions And Relevance: The findings suggest that autologous ADSVF injection in scarred vocal folds is feasible and tolerable. The findings require confirmation in a randomized clinical trial with a larger population.

Trial Registration: ClinicalTrials.gov Identifier: NCT02622464.
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http://dx.doi.org/10.1001/jamaoto.2019.4328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163407PMC
April 2020

Therapeutic targeting of soluble CD146/MCAM with the M2J-1 monoclonal antibody prevents metastasis development and procoagulant activity in CD146-positive invasive tumors.

Int J Cancer 2020 09 22;147(6):1666-1679. Epub 2020 Feb 22.

INSERM, INRAE, C2VN, UFR Pharmacie, Aix-Marseille University, Marseille, France.

Initially discovered in human melanoma, CD146/MCAM is expressed on many tumors and is correlated with cancer progression and metastasis. However, targeting CD146 remains challenging since it is also expressed on other cell types, as vessel cells, where it displays important physiological functions. We previously demonstrated that CD146 is shed as a soluble form (sCD146) that vectorizes the effects of membrane CD146 on tumor angiogenesis, growth and survival. We thus generated a novel monoclonal antibody, the M2J-1 mAb, which specifically targets sCD146, but not membrane CD146, and counteracts these effects. In our study, we analyzed the effects of sCD146 on the dissemination and the associated procoagulant phenotype in two highly invasive human CD146-positive cancer cell lines (ovarian and melanoma). Results show that sCD146 induced epithelial to mesenchymal transition, favored the generation of cancer stem cells and increased the membrane expression of tissue factor. Treatment of cancer cells with sCD146 in two experimental models (subcutaneous xenografting and intracardiac injection of cancer cells in nude mice) led to increased tumor dissemination and procoagulant activity. The M2J-1 mAb drastically reduced metastasis but also procoagulant activity, in particular by decreasing the number of circulating tumor microparticles, and blocked the relevant signaling pathways as demonstrated by RNA expression profiling experiments. Thus, our findings demonstrate that sCD146 mediates important pro-metastatic and procoagulant effects in two CD146-positive tumors. Targeting sCD146 with the newly generated M2J-1 mAb could constitute an innovative strategy for preventing dissemination and thromboembolism in many CD146-positive tumors.
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http://dx.doi.org/10.1002/ijc.32909DOI Listing
September 2020

Ticagrelor attenuates the increase of extracellular vesicle concentrations in plasma after acute myocardial infarction compared to clopidogrel.

J Thromb Haemost 2020 03 9;18(3):609-623. Epub 2020 Jan 9.

First Chair and Department of Cardiology, Medical University of Warsaw, Poland.

Background: Platelet P2Y12 antagonist ticagrelor reduces mortality after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets, leukocytes, and endothelial cells release proinflammatory and prothrombotic extracellular vesicles (EVs), we hypothesized that the release of EVs is more efficiently inhibited by ticagrelor compared to clopidogrel.

Objectives: We compared EV concentrations and EV procoagulant activity in plasma of patients after AMI treated with ticagrelor or clopidogrel.

Methods: After percutaneous coronary intervention, 60 patients with first AMI were randomized to ticagrelor or clopidogrel. Flow cytometry was used to determine concentrations of EVs from activated platelets (CD61 , CD62p ), fibrinogen , phosphatidylserine (PS ), leukocytes (CD45 ), endothelial cells (CD31 , 146 ), and erythrocytes (CD235a ) in plasma at randomization, after 72 hours and 6 months of treatment. A fibrin generation test was used to determine EV procoagulant activity.

Results: Concentrations of platelet, fibrinogen , PS , leukocyte, and erythrocyte EVs increased 6 months after AMI compared to the acute phase of AMI (P ≤ .03). Concentrations of platelet EVs were lower on ticagrelor compared to clopidogrel after 6 months (P = .03). Concentrations of fibrinogen , PS , and leukocyte EVs were lower on ticagrelor compared to clopidogrel both after 72 hours and 6 months (P ≤ .03). Concentrations of endothelial EVs and EV procoagulant activity did not differ between patient groups and over time (P ≥ .17).

Conclusions: Ticagrelor attenuates the increase of EV concentrations in plasma after acute myocardial infarction compared to clopidogrel. The ongoing release of EVs despite antiplatelet therapy might explain recurrent thrombotic events after AMI and worse clinical outcomes on clopidogrel compared to ticagrelor.
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http://dx.doi.org/10.1111/jth.14689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065161PMC
March 2020

Adipose-Derived Stem Cells from Systemic Sclerosis Patients Maintain Pro-Angiogenic and Antifibrotic Paracrine Effects In Vitro.

J Clin Med 2019 Nov 14;8(11). Epub 2019 Nov 14.

Aix Marseille University, INSERM, INRA, C2VN, 13005 Marseille, France.

Innovative therapies based on autologous adipose-derived stem/stromal cells (ASC) are currently being evaluated for treatment of systemic sclerosis (SSc). Although paracrine angiogenic and antifibrotic effects are considered the predominant mechanisms of ASC therapeutic potential, the impact of SSc on ASC paracrine functions remains controversial. In this study, phenotype, senescence, differentiation potential, and molecular profile were determined in ASC from SSc patients (SSc-ASC) ( = 7) and healthy donors (HD-ASC) ( = 7). ASC were co-cultured in indirect models with dermal fibroblasts (DF) from SSc patients or endothelial cells to assess their pro-angiogenic and antifibrotic paracrine effects. The angiogenic activity of endothelial cells was measured in vitro using tube formation and spheroid assays. DF collagen and alpha smooth muscle actin (αSMA) content were quantified after five days of co-culture with ASC. Differentiation capacity, senescence, and mRNA profiles did not differ significantly between SSc-ASC and HD-ASC. SSc-ASC retained the ability to stimulate angiogenesis through paracrine mechanisms; however, functional assays revealed reduced potential compared to HD-ASC. DF fibrosis markers were significantly decreased after co-culture with SSc-ASC. Together, these results indicate that SSc effects do not significantly compromise the angiogenic and the antifibrotic paracrine properties of ASC, thereby supporting further development of ASC-based autologous therapies for SSc treatment.
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http://dx.doi.org/10.3390/jcm8111979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912239PMC
November 2019

The Interaction of Platelets with Colorectal Cancer Cells Inhibits Tumor Growth but Promotes Metastasis.

Cancer Res 2020 01 14;80(2):291-303. Epub 2019 Nov 14.

Aix Marseille Univ, INSERM, INRA, Marseille, France.

Platelets promote metastasis, however, their role in tumor growth remains controversial. Here, we investigated the effect of platelet interactions with colorectal tumor cells. Platelets extravasated into the tumor microenvironment and interacted with tumor cells in a cadherin-6-dependent manner. The interaction induced platelet spreading, release of their granule content, and the generation of three types of microparticles (iMP) that expressed platelet markers, tumor markers, or both. The presence of iMPs was confirmed in colorectal cancer tissue specimens. Platelets significantly reduced tumor growth and increased intratumoral macrophages. This was mediated by iMP recruitment of macrophages via the chemoattractants RANTES, MIF, CCL2, and CXCL12 and activation of their tumor cell killing capacity through IFNγ and IL4, which led to cell-cycle arrest of tumor cells in a p21-dependent manner. In contrast, in the bloodstream, iMPs activated endothelial cells and platelets and induced epithelial-to-mesenchymal transition of tumor cells, promoting metastasis. Altogether, these results indicate that depending on the environment, local or bloodstream, the consequences of the interactions between platelets and a tumor may promote or prevent cancer progression. SIGNIFICANCE: Tumor cell interaction with platelets produces chimeric extracellular vesicles that suppress primary tumor growth by activating tumor-eliminating macrophages, while promoting metastasis through EMT and endothelial activation.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1181DOI Listing
January 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

Microvesicles and Cancer Associated Thrombosis.

Semin Thromb Hemost 2019 Sep 20;45(6):593-603. Epub 2019 Aug 20.

Aix-Marseille University, INSERM, INRA, C2VN UMR_S1263, UFR de Pharmacie, Marseille, France.

Microvesicles (MVs) are small membrane enclosed structures released into the extracellular space by virtually all cell types. Their composition varies according to the cell origin and the stimulus which caused their formation. They harbor functional molecules and participate in intercellular communication. Endothelium, inflammatory cells, and cancer cells produce procoagulant MVs which contribute to cancer-associated thrombosis (CAT) in animal models. The tissue factor (TF) conveyed by these MVs was shown to play a key role in different animal models of experimental CAT. Alternatively, other molecular mechanisms involving polyphosphates or phosphatidylethanolamine could also be involved. In clinical practice, an association between an increase in the number of TF-positive or the procoagulant activity of these MVs and the occurrence of CAT has indeed been demonstrated in pancreatic-biliary cancers, suggesting that they could behave as a biomarker predictive for CAT. However, to date, this association was not confirmed in other types of cancer. Potential causes explaining this limited associated between MVs and CAT are (1) the diversity of mechanisms associating MVs and different types of cancer; (2) a more complex role of MVs in hemostasis integrating their anticoagulant and fibrinolytic activity; and (3) the lack of sensitivity, reproducibility, and standardization of current methodologies permitting measurement of MVs. Each of these hypotheses constitutes an interesting exploration path for a future reassessment of the clinical interest of the MVs in CAT.
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http://dx.doi.org/10.1055/s-0039-1693476DOI Listing
September 2019

Platelets, Thrombo-Inflammation, and Cancer: Collaborating With the Enemy.

Front Immunol 2019 31;10:1805. Epub 2019 Jul 31.

Aix Marseille Univ, INSERM 1263, INRA 1260, Center for CardioVascular and Nutrition Research (C2VN), Marseille, France.

Platelets are small anucleate cells present in the blood stream, their typical role in primary hemostasis has been well-described. However, new evidence suggests that they have critically important roles in cancer progression and inflammation. Cancer cells can activate platelets, thus using them as physical shields from blood shear forces and natural killer (NK) cells. The activated platelets may also regulate hematopoietic and immune cell migration toward the tumor site; therefore, contributing to the cancer-associated inflammation. The activation of platelets by cancer cells may also contribute to metastasis and cancer progression by stimulating deep venous thrombosis and neutrophil extracellular trap formations (NETs) that "hide" cancer cells. We strived to review the current literature to dissect the role of platelets in cancer-associated thrombosis and tumor microenvironment inflammation.
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http://dx.doi.org/10.3389/fimmu.2019.01805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684752PMC
September 2020

Involvement of Platelets in Cancers.

Semin Thromb Hemost 2019 Sep 5;45(6):569-575. Epub 2019 Aug 5.

Aix Marseille Univ, INSERM, INRA, C2VN, Marseille, France.

Cancer-related venous thromboembolism (VTE) is frequent and constitutes the second leading cause of death in patients with cancer. High platelet count is one of independent predictive factors of cancer-associated VTE. Besides the implication of platelets in cancer-associated VTE, recent clinical and experimental evidences support that platelets play several roles in the progression of malignancies and inversely, cancer can also influence platelet count and activity. The objective of this report is to review the current literature regarding the role of platelets in cancer through experimental results and population-based studies. Platelets are implicated in cancer progression and metastasis through proangiogenic factors (growth factors and signaling pathways), antiangiogenic factors (angiostatin, endostatin, thrombospondin-1), and matrix metalloproteinases. In addition, platelets are involved in cancer-associated thrombosis and thus tumor cell-induced platelet activation, through anionic phospholipids on their surface, released soluble factors, such as P-selectin, CD40 ligand, platelet factor 4, thrombospondin-1 or beta-thromboglobulin, tumor cell procoagulant proteins (tissue factor, urokinase-type plasminogen activator, plasminogen activator inhibitor type 1), and microparticles. Due to these different mechanisms, platelets may represent a potential therapeutic target. The main current treatments against platelets are: (1) acetylsalicylic acid (aspirin) and nonsteroidal anti-inflammatory drugs, nonselective cyclo-oxygenase (COX)-1 and COX-2 inhibitors, which are associated with decreased cancer incidence and better overall survival and (2) irreversible inhibitor of P2Y12 subtype which decreases cancer incidence. Platelets are key players in tumor growth, metastasis, and cancer-associated thrombosis. This multifaceted role identifies them as a relevant therapeutic target for prevention of cancer occurrence and treatment of cancer.
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http://dx.doi.org/10.1055/s-0039-1693475DOI Listing
September 2019

Extracellular vesicles from T cells overexpress miR-146b-5p in HIV-1 infection and repress endothelial activation.

Sci Rep 2019 07 16;9(1):10299. Epub 2019 Jul 16.

Aix Marseille Univ, INSERM, C2VN, Marseille, France.

Human immunodeficiency virus type 1 (HIV-1) infection promotes a generalized activation of host responses that involves not only CD4 T cells, but also cells of the microenvironment, which are not directly infected, such as endothelial cells. The mechanisms triggering HIV-1-associated vascular alterations remain poorly understood. Extracellular vesicles (EVs), implicated in cell-to-cell communication, have been recently described as carriers of microRNAs (miRNAs). Here, we show that miR-146b-5p is upregulated in both CD4 T cells, CD4 T cell-derived EVs and circulating EVs obtained from antiretroviral therapy-naive HIV-1-infected patients. We further demonstrate that EVs from T cell line overexpressing miR-146b-5p mimics (miR-146b-EVs): 1) protect their miRNA cargo from RNase degradation, 2) transfer miR-146b-5p mimics into endothelial cells and 3) reduce endothelial inflammatory responses in vitro and in vivo in the lungs of mice through the downregulation of nuclear factor-κB-responsive molecules. These data advance our understanding on chronic inflammatory responses affecting endothelial homeostasis, in infectious and non-infectious diseases and pave the way for potential new anti-inflammatory strategies.
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http://dx.doi.org/10.1038/s41598-019-44743-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635508PMC
July 2019

FCGR3A and FCGR2A Genotypes Differentially Impact Allograft Rejection and Patients' Survival After Lung Transplant.

Front Immunol 2019 12;10:1208. Epub 2019 Jun 12.

Établissement Français du Sang PACA-Corse 13005, Marseille, France.

Fc gamma receptors (FcγRs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of and can impact the expression level, IgG affinity and function of the CD32 and CD16 FcγRs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether and polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of ([131R/H], rs1801274) and ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, < 0.0001, 95% CI 2.37-9.61), but it could not be associated with the level of CD16- mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, = 0.024, 95% CI 1.08-3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of LTRs and may be of use to stratify patients at higher risk of experiencing graft rejection. Furthermore, these data suggest that in the LTx setting, specific mechanisms of humoral alloreactivity, which cannot be solely explained by the complement and CD16-mediated pathogenic effects of DSA, may be involved in the development of acute and chronic lung allograft rejection.
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http://dx.doi.org/10.3389/fimmu.2019.01208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582937PMC
October 2020

Toward standardization of assays measuring extracellular vesicle-associated tissue factor activity.

J Thromb Haemost 2019 08 23;17(8):1261-1264. Epub 2019 Jun 23.

Clinical Division of Haematology and Haemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.

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http://dx.doi.org/10.1111/jth.14481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851965PMC
August 2019

Thrombosis Risk Associated with Head and Neck Cancer: A Review.

Int J Mol Sci 2019 Jun 11;20(11). Epub 2019 Jun 11.

Aix Marseille Univ, INSERM 1263, INRA, Center for CardioVascular and Nutrition Research (C2VN), 27 Boulevard Jean Moulin, 13385 Marseille, France.

Venous thromboembolism (VTE) is a common complication for cancer patients. VTE-associated risk varies according to the type of tumor disease. Head and neck cancer is a common cancer worldwide, and most tumors are squamous cell carcinomas due to tobacco and alcohol abuse. The risk of VTE associated with head and neck (H&N) cancer is considered empirically low, but despite the high incidence of H&N cancer, few data are available on this cancer; thus, it is difficult to state the risk of VTE. Our review aims to clarify this situation and tries to assess the real VTE risk associated with H&N cancer. We report that most clinical studies have concluded that there is a very low thrombosis risk associated with H&N cancer. Even with the biases that often exist, this clinical review seems to confirm that the risk of VTE was empirically hypothesized. Furthermore, we highlight that H&N cancer has all the biological features of a cancer associated with a high thrombosis risk, including a strong expression of procoagulant proteins, modified thrombosis/fibrinolysis mechanisms, and secretions of procoagulant microparticles and procoagulant cytokines. Thus, this is a paradoxical situation, and some undiscovered mechanisms that could explain this clinical biological ambivalence might exist.
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http://dx.doi.org/10.3390/ijms20112838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600456PMC
June 2019