BRGM Nancy Corinne Merly

Towards a less stringent groundwater body objective? Feedback from a French heavily industrial and urban study area

Authors: C. Merly (1) (BRGM, 151 Bd Stalingrad, Orléans) , M. Grémont (2) , P. Vigouroux (2) , M Bouzit (2) , B. Clozel (1) , L. Cadilhac (3) , C Schlosser (3) (1) BRGM, Direction Régionale Auvergne Rhône Alpes, 151 Bd Stalingrad, 69626 Villeurbanne, France (2) BRGM, D3E, NRE, 1039 Rue de Pinville, 34000 Montpellier (3) Agence de l'Eau Rhône Méditerranée Corse, 2 Allée de Lodz, 69007 Lyon Groundwater Directive 2006/118/EC (Daughter to Water Framework Directive 2000/60/EC) establishes specific measures in order to prevent and control groundwater pollution. It specifically requires to assess plumes originating from punctual sources such as those due to industrial activities, in order to check if they do not spread and degrade the chemical state of the groundwater bodies. In 2012-2013, BRGM (French Geological Survey) supported by the AERMC (Water Agency on the Mediterranean Rhone River basin) carried out a study to assess the impact of industrial activities on the quality of groundwater bodies in the Rhône river basin. It enables to identify 10 groundwater bodies which were at risk of not reaching the groundwater environmental quality target set by the French application of the Groundwater Framework Directive. Some of these groundwater bodies impacted by persistent and widely spread contaminants such as chlorinated solvents are very unlikely to recover groundwater quality standard by 2027. In this context, AERMC and the BRGM initiated an innovative project in order to define a methodological framework to justify for an exemption of the current groundwater quality objectives and the possibility to propose a less stringent groundwater objective.

The methodology was developed for a French groundwater body severely impacted by anthropogenic contaminants (chlorinated solvents and pesticides) where urban and industrial pressures are significant. The study included four phases:

-1) Detailed description of the quantitative and qualitative hydrogeology of the study area; This step enabled to a) make the inventory of substances concerned by GWFD and for which an exemption case can be developed, b) describe the understanding of the hydrogeological functioning of the zone (flow direction, interface groundwater and surface waters), c) identify the substances for which a significant impact on groundwater quality was observed, d) identify and describe sources zones and finally e) give recommendation on groundwater monitoring. -2) Inventory of remedial actions (including costs) and definition of scenario; This steps aimed at a) making an inventory of current remedial actions, b) suggesting other potential remedial actions for development of scenario and c) development of scenario aiming at analyzing the situation from various perspectives and on which assessment methods were applied.

-3) Application of assessment tools for the justifications for exemption; Notably, two main methods were applied here: a) Assessment of actors payment capacity and b) cost-benefits analyse (including a full inventory of benefits originating from the current and a good status groundwater body). -4) Definition of a less stringent groundwater quality objectives; this steps is planned in near-future.

This project gives great methodological feedback on technical, regulatory and governance aspects. Regarding technical aspects, among others, data availability and study boundaries were discussed. As for regulation, the stand points differences between contaminated land management and French declination of the groundwater directive requirements were tackled. At last but not least, the governance of the project involved many types of stakeholders with whom a common vision was attempted to be developed throughout the running of the project.


Marc Romana
email: marc.romana@inserm.fr

Philippe Connes

Nigel S Key

Microparticles in sickle cell disease

Keywords: sickle cell disease, microparticles, physiopathology

come

Introduction

Sickle cell disease (SCD) is a group of genetic disorders that have in common the production of the abnormal hemoglobin S (HbS) instead of hemoglobin A. HbS occurs as a result of a single base mutation in exon 1 of the -globin gene that causes the substitution of valine for glutamic acid at the sixth position of the -globin molecule. Sickle cell anemia (SCA) refers to the disorder produced by homozygous HbS inheritance, whereas co-inheritance of HbS and hemoglobin C or -thalassemia are at the origin of the two other sickle cell syndromes most commonly encountered, i.e. hemoglobin SC (HbSC) disease and hemoglobin S/-thalassemia (HbS/-thal) [START_REF] Rees | Sickle-cell disease[END_REF].

In deoxygenated conditions and after a delay period, HbS molecules polymerize inducing the sickling of affected red blood cells (RBC) and leading to decreased deformability and increased fragility. Sickle RBC, which exhibit abnormal adhesive properties to endothelial cells [START_REF] Parise | Erythrocyte adhesion in sickle cell disease[END_REF], do not easily traverse the microcirculation, causing frequent vaso-occlusive episodes [START_REF] Frenette | Sickle cell vasoocclusion: heterotypic, multicellular aggregations driven by leukocyte adhesion[END_REF]. Vaso-occlusion is not only due to sickling of RBC, but also to endothelial-leukocyte-RBC interactions, and is enhanced by inflammatory processes and endothelial-leukocyte-RBC interactions [START_REF] Zhang | Neutrophils, platelets, and inflammatory pathways at the nexus of sickle cell disease pathophysiology[END_REF][START_REF] Kaul | Hypoxia/reoxygenation causes inflammatory response in sickle transgenic mice but not in normal mice[END_REF]. Vascular obstruction and ischemia are followed by a restoration of blood flow, which further provokes reperfusion injury. These cycles of hypoxia-reperfusion cause oxidant and inflammatory stress, which increase the expression of several endothelial cell adhesion molecules [START_REF] Kaul | Hypoxia/reoxygenation causes inflammatory response in sickle transgenic mice but not in normal mice[END_REF]. Furthermore, sickle RBC exhibit a reduced lifespan associated with intravascular hemolysis, leading to the release of free Hb and heme into the blood. These bioactive molecules are responsible for the decrease in nitric oxide (NO) bioavailability, which may be involved in several sickle complications such as priapism, leg ulcer, pulmonary hypertension and stroke [START_REF] Kato | Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance, priapism, leg ulceration, pulmonary hypertension, and death on patients with sickle cell disease[END_REF][START_REF] Kato | Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes[END_REF]. Currently, hydroxyurea (HU) is the only drug known to reduce the occurrence of some complications such as painful vaso-occlusive crisis (VOC), acute chest syndrome (ACS) and the need for transfusion [START_REF] Charache | Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia[END_REF][START_REF] Steinberg | Effect of hydroxyurea on mortality and morbidity in adult sickle cell anemia: risks and benefits up to 9 years of treatment[END_REF]. Originally prescribed to SCA patients because of its ability to induce expression of fetal hemoglobin (which is known to alleviate HbS polymerization), it has subsequently been shown to possess pleotropic effect that in aggregate contribute to its therapeutic efficacy [START_REF] Kato | New insights into sickle cell disease: mechanisms and investigational therapies[END_REF].

Several of the pathophysiological pathways known to be associated with SCD, such as oxidative and inflammation stress, may activate circulating blood cells and lead to the release of sub-micron vesicles, named microparticles (MP) [START_REF] Hebbel | Microparticles in sickle cell anaemia: promise and pitfalls[END_REF]. During the last two decades, a large number of studies in the SCD field have been performed to better describe the quantitative and qualitative profile of MPs in SCD patients and their involvement in SCD pathophysiological processes. In this review, we will focus on the characteristics and functional properties of plasma MPs and present evidence that MPs could be both biomarkers and bio-effectors in SCD.

Microparticles: definition and genesis

MPs fall under the umbrella term of extracellular vesicles (EV), which is used to designate all types of vesicles released from cells, the best characterized being exosomes, apoptotic vesicles (also called apoptotic bodies), and MPs [START_REF] György | Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles[END_REF]. These various types of EV differ in regards to several characteristics such as size, density, morphology, composition (protein, lipid and nucleic acid) as well as sub-cellular origin [START_REF] György | Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles[END_REF]. It should be noted that the overlap of some of these parameters between EV sub-types has been associated with technical purification difficulties and thus the ability to attribute particular functions to each subtype [START_REF] György | Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles[END_REF]. Nevertheless, there is accumulating evidence that EV carry diverse cargoes including proteins, RNA species such as mRNA and miRNA and lipids that can be transported and exchanged between cells, strongly suggesting that EV play key roles in cell-cell communication at both paracrine and systemic levels [START_REF] Colombo | Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles[END_REF][START_REF] Rajendran | Emerging roles of extracellular vesicles in the nervous system[END_REF].

MPs are defined as phospholipid microvesicles with a diameter ranging from 100 to 1,000 nm that are derived from the cytoplasmic membrane of cells submitted to stress conditions that result in apoptosis or activation. These conditions lead to local cytoskeletal rearrangements and membrane budding [START_REF] Beaudoin | Shedding of vesicular material from the cell surface of eukaryotic cells: different cellular phenomena[END_REF][START_REF] Freyssinet | Apoptosis in vascular disease[END_REF]. Indeed, the increase of intracellular Ca 2+ induced by these conditions affects the function of three enzymes, namely floppase, scrambase and flippase that are involved in the asymmetry of the cellular lipid bilayers, and leads to the externalization of phosphatidylserine (PS) [START_REF] Gilbert | Platelet-derived microparticles express high affinity receptors for factor VIII[END_REF][START_REF] Hoyer | Microparticles-messengers of biological information[END_REF]. PS exposure is believed to be a key event in MP formation. Indeed, Scott syndrome, characterized by impaired ability to externalize PS and linked to genetic defects of several enzymes involved in cell PS exposure, has been associated with a decrease of MP shedding and a bleeding disorder [START_REF] Suzuki | Calcium-dependent phospholipid scrambling by TMEM16F[END_REF][START_REF] Toti | Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications, is an inherited disorder[END_REF][START_REF] Castoldi | Compound heterozygosity for 2 novel TMEM16F mutations in a patient with Scott syndrome[END_REF]. Moreover, the rise of intracellular Ca 2+ activates proteases that cleave the cytoskeleton, weaken its interaction with the cytoplasmic membrane, thereby allowing the release of MPs [START_REF] Morel | Cellular mechanisms underlying the formation of circulating microparticles[END_REF].

Accordingly, MPs are usually described as exhibiting PS externalization, although MPs without externalized PS have also been described [START_REF] Connor | The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib[END_REF].

The protein composition of MPs reflects that of the cell from which they are derived, including cell-type specific antigens which allow the identification of their cellular origin. Moreover, MPs also contain functional molecules that could have been induced on the parental cell by the factor responsible for triggering MP release [START_REF] Combes | In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant[END_REF][START_REF] Jimenez | Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis[END_REF]. Thus, both cell origin and nature of trigger(s) impact the phenotype of MPs and their functional properties. It has been shown for example that the level of externalized PS and the type of membrane receptors exhibited by endothelial cell-derived MP vary according not only to the triggering factor, namely apoptotic stimuli or tumor necrosis factor, but also to the vascular origin of the endothelial cells [START_REF] Jimenez | Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis[END_REF].

MPs have been detected in multiple biological fluids including urine, broncho-alveolar lavage fluid, sputum, synovial fluid, ascites, saliva and plasma [START_REF] Burger | Microparticles: biomarkers and beyond[END_REF]. In physiological conditions, it has been shown that plasma samples contain platelet-, erythrocyte-, endothelial cell-and leukocyte-derived MPs [START_REF] Morel | Cellular mechanisms underlying the formation of circulating microparticles[END_REF].

Functional properties of MPs

A large number of functional properties have been associated with MPs. The first and probably best described property is the ability to promote coagulation [START_REF] Wolf | The nature and significance of platelet products in human plasma[END_REF][START_REF] Owens Ap 3rd | Microparticles in hemostasis and thrombosis[END_REF]. Two physical characteristics of MPs have been linked to their procoagulant activity. The externalization of PS results in a negatively charged surface, allowing assembly of coagulation factors and thrombin generation [START_REF] Pereira | Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state[END_REF]. MPs released from endothelial cells and monocytes may also display tissue factor (TF) on their surface and thus support coagulation via the factor VII (FVII)/TF-dependent pathway [START_REF] Combes | In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant[END_REF][START_REF] Kasthuri | PF4/heparin-antibody complex induces monocyte tissue factor expression and release of tissue factor positive microparticles by activation of FcγRI[END_REF]. Furthermore, it has been shown that MPs from leukocytes can transfer TF to platelets and contribute to the recruitment of cells and the accumulation of TF at sites of vascular injury [START_REF] Hrachovinová | Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A[END_REF]. Furthermore, tumor-derived MPs are known to exhibit procoagulant properties [START_REF] Bastida | Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis[END_REF]. In agreement with their described procoagulant properties, high MP concentrations have been detected in various clinical conditions associated with increased incidence of thrombosis including paroxysmal nocturnal hemoglobinuria [START_REF] Hugel | Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia[END_REF], cardiovascular disease [START_REF] Mallat | Elevated levels of shed membrane microparticles with procoagulant potential in the peripheral circulating blood of patients with acute coronary syndromes[END_REF] and deep venous thrombosis [START_REF] Ye | Circulating tissue factor positive microparticles in patients with acute recurrent deep venous thrombosis[END_REF].

Various blood cell-derived MPs have also been shown to regulate the production of reactive oxygen species (ROS) and thus oxidative stress level. MPs shed by endothelial cells [START_REF] Burger | Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts[END_REF], monocytes [START_REF] Essayagh | Microparticles from apoptotic monocytes induce transient platelet recruitment and tissue factor expression by cultured human vascular endothelial cells via a redox-sensitive mechanism[END_REF] and lymphocytes [START_REF] Yang | Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways[END_REF] promote endothelial O 2 -and H 2 O 2 production in cultured endothelial cells through processes involving different enzymatic systems, and thus may induce apoptosis [START_REF] Distler | Induction of apoptosis in circulating angiogenic cells by microparticles[END_REF].

Pro-inflammatory stimuli provoke the release of MPs, which in turn may directly contribute to the inflammatory response. For instance, MPs derived from polymorphonuclear leukocytes or from monocytes promote the production of IL-6/MCP in cultured endothelial cells [START_REF] Mesri | Leukocyte microparticles stimulate endothelial cell cytokine release and tissue factor induction in a JNK1 signaling pathway[END_REF] and IL-8/MCP in airway epithelial cells [START_REF] Cerri | Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells[END_REF], respectively. The effects of MPs seem to be cell type-dependent since it has been shown that macrophages are inhibited upon incubation with MPs released from polymorphonuclear leukocytes [START_REF] Gasser | Activated polymorphonuclear neutrophils disseminate antiinflammatory microparticles by ectocytosis[END_REF][START_REF] Eken | Ectosomes released by polymorphonuclear neutrophils induce a MerTK-dependent anti-inflammatory pathway in macrophages[END_REF] suggesting that some MPs may exhibit both pro-and anti-inflammatory properties.

MPs from various blood cell types may also induce a pro-adhesive phenotype to endothelial cells. In this respect, treatment of endothelial cells with platelet-and endothelial cell-derived MPs are associated with increased expression of cell adhesion molecules and monocyteendothelial cell interactions [START_REF] Burger | Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts[END_REF][START_REF] Barry | Modulation of monocyte-endothelial cell interactions by platelet microparticles[END_REF].

In addition to previously cited biological pathways, MPs have been implicated in the regulation of angiogenesis, vascular function and apoptosis [START_REF] Van Der Pol | Classification, functions, and clinical relevance of extracellular vesicles[END_REF][START_REF] Martínez | Shed membrane microparticles from circulating and vascular cells in regulating vascular function[END_REF].

However, it should be noted that most of the previously cited studies addressing these mechanisms have been performed with MPs generated in vitro. Since the biological content and the properties are related to the triggers leading to their release, the functional properties of circulating MPs produced in vivo need to be confirmed.

Mechanisms of MP-mediated biological effects

In contrast to coagulation, the molecular mechanisms involved in the other biological effects mediated by MPs remain elusive and alternative putative processes have been proposed.

Several studies have documented direct physical interactions of MPs with their target cells [START_REF] Burger | Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts[END_REF][START_REF] Terrisse | Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow[END_REF][START_REF] Faille | Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells[END_REF]. Triggering of signaling pathways may result from binding of MP surface antigens to their specific counter-receptor. For example, the induction of monocytic cells adhesive receptor expression by endothelial-derived MPs could be inhibited by treatment with antibodies directed against ICAM-1, an adhesive receptor expressed by MP-derived endothelial cells, and its counter receptor, beta2 integrin expressed by monocytes [START_REF] Sabatier | Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor-dependent procoagulant activity[END_REF].

Complementary experiments strongly suggested that the induced pro-adhesive phenotype of monocytic cells did not involve membrane fusion, but rather receptor binding [START_REF] Sabatier | Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor-dependent procoagulant activity[END_REF].

Alternatively, the binding of MPs to target cells may change the panel of exposed antigens and thus modify functional properties of these cells. For instance, such a mechanism has been proposed for the increase of leukocyte-leukocyte interactions mediated by platelet-derived

MPs [START_REF] Forlow | Leukocyte-leukocyte interactions mediated by platelet microparticles under flow[END_REF]. The fusion of MPs with cells and the subsequent transfer of their content has also been proposed to be involved in the cellular relocation of functional membrane receptors [START_REF] Rozmyslowicz | Platelet-and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV[END_REF] and bioactive substances such as lipids [START_REF] Barry | Modulation of monocyte-endothelial cell interactions by platelet microparticles[END_REF][START_REF] Barry | Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles[END_REF] shown to mediate several MP functional properties.

MPs may contain enzymes with reactive oxygen species (ROS)-generating capacity [START_REF] Brodsky | Endothelium-derived microparticles impair endothelial function in vitro[END_REF][START_REF] Mostefai | Phosphatidylinositol 3-kinase and xanthine oxidase regulate nitric oxide and reactive oxygen species productions by apoptotic lymphocyte microparticles in endothelial cells[END_REF].

Thus, they have the potential to directly produce ROS known to be involved in several pathways mediated by MPs such as apoptosis, inflammation and endothelial dysfunction [START_REF] Touyz | NOX isoforms and reactive oxygen species in vascular health[END_REF].

However, the de novo production of ROS by MPs remains to be proven. In contrast, it has been unambiguously shown that MPs derived from erythrocytes may scavenge nitric oxide (NO) almost as efficiently as plasma hemoglobin because of their hemoglobin high content [START_REF] Donadee | Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion[END_REF][START_REF] Liu | Nitric oxide scavenging by red cell microparticles[END_REF].

In aggregate, these studies strongly suggest that the diverse biological effects induced by MPs are mediated by several distinct mechanisms.

Characterization of MPs

A large number of approaches have been used for MP characterization and detection including flow cytometry, solid-phase methods followed by enzyme-linked immunosorbent assay or functional assay, electron microscopy, atomic force microscopy, nanoparticle tracking analysis, dynamic light scattering and tunable resistive pulse sensing [START_REF] Burger | Microparticles: biomarkers and beyond[END_REF][START_REF] Lacroix | Overcoming limitations of microparticle measurement by flow cytometry[END_REF][START_REF] Thulin | Circulating cell-derived microparticles as biomarkers in cardiovascular disease[END_REF].

Each of these approaches has inherent advantages and limitations; however, since this topic was recently reviewed, it will not be discussed further in the present report [START_REF] Pasalic | Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis[END_REF][START_REF] Erdbrügger | Analytical challenges of extracellular vesicle detection: A comparison of different techniques[END_REF][START_REF] Revenfeld | Diagnostic and prognostic potential of extracellular vesicles in peripheral blood[END_REF]. Up to now, the most widespread technique used to analyze MPs is flow cytometry. This approach allows rapid determination of cellular origins and enumeration of MPs, and is available at most research facilities. However, flow cytometry has its own limitations, in particular for the detection of small vesicles [START_REF] Lacroix | Overcoming limitations of microparticle measurement by flow cytometry[END_REF], although recent generation flow cytometers are capable of detecting MPs as small as approximately 0.2 m in diameter [START_REF] Arraud | Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry[END_REF]. In addition to differences of flow cytometer characteristics, it has been clearly established that analytical and preanalytical procedures such as sample preparation are important sources of variability [START_REF] Erdbrügger | Analytical challenges of extracellular vesicle detection: A comparison of different techniques[END_REF].

Indeed, the lack of consensus in these variables contributes to the difficulty in drawing firm conclusions about MP characteristics and roles in clinical conditions such as SCD [START_REF] Hebbel | Microparticles in sickle cell anaemia: promise and pitfalls[END_REF].

Quantitative and qualitative microparticles pattern in sickle cell disease

Compared to healthy individuals, a 3-to 4-fold increase of plasma MPs has been repeatedly reported in SCD patients at steady-state condition, i.e. remote from acute complications, by multiple groups [START_REF] Hebbel | Microparticles in sickle cell anaemia: promise and pitfalls[END_REF] using either flow cytometry [64 -71] or solid-phase methods [START_REF] Van Tits | Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis[END_REF]. In these studies, MPs were found to derive mainly from platelets and RBCs; those originating from other blood-cell types such as endothelial cells, monocytes or granulocytes were either detected at variable levels or were undetectable [START_REF] Van Beers | Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease[END_REF]. Surprisingly, one group reported lower concentrations of MPs in SCD patients compared to controls [START_REF] Piccin | Circulating microparticles, protein C, free protein S and endothelial vascular markers in children with sickle cell anaemia[END_REF].

Multiple reasons could account for these discordant results. Since several of these studies were performed in SCD patients with a variety of sickle cell syndromes, i.e. SCA, HbSC disease or S/-thal [64 -66,71,73], it is tempting to speculate that these discrepancies could be related to the proportion of patients with each syndrome. However, a recent study performed on 180 SCD children --84 with HbSC disease and 96 with SCA --showed that although the former group exhibited lower blood MP concentrations, resulting mainly from a decrease of MPs originating from RBC and to a lesser extent from platelets, no significant difference in the numbers of MPs derived from other blood cells between the two groups was detected [START_REF] Garnier | Differences of microparticle patterns between sickle cell anemia and hemoglobin SC patients[END_REF]. To our knowledge, no study dedicated to patients with S/-thal has been conducted. Therefore, the variability of the cellular origins of MPs in SCD patients reported in the literature is most likely the consequence of non-standardized pre-analytical and analytical procedures, known to be critical factors impacting MP pattern, both quantitatively and qualitatively [START_REF] Hebbel | Microparticles in sickle cell anaemia: promise and pitfalls[END_REF].

The clinical course of the disease may also affect MP concentration. So far, conflicting results have been obtained on the impact of SCD complications on circulating MP concentrations.

Six studies described increased numbers of MPs during sickle cell crises compared to steadystate disease [START_REF] Shet | Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes[END_REF][START_REF] Tantawy | Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications[END_REF][START_REF] Kasar | Clinical significance of circulating blood and endothelial cell microparticles in sickle-cell disease[END_REF][START_REF] Van Tits | Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis[END_REF][START_REF] Piccin | Circulating microparticles, protein C, free protein S and endothelial vascular markers in children with sickle cell anaemia[END_REF][START_REF] Hierso | Exacerbation of oxidative stress during sickle vaso-occlusive crisis is associated with decreased anti-band 3 autoantibodies rate and increased red blood cell-derived microparticle level: a prospective study[END_REF], while van Beers EJ et al detected similar MP concentrations in both conditions [START_REF] Van Beers | Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease[END_REF]. In addition, the cellular origins of MPs for which an increased concentration was observed varied between studies. Several important parameters differed between these studies, including cross-sectional or longitudinal design, clinical definition of sickle crisis or delay between blood sampling and hospital admission, among others. The largest longitudinal survey published so far, with 32 SCA patients, showed a 2-fold increase in the concentration of RBC-derived MPs during painful vaso-occlusive crisis [START_REF] Hierso | Exacerbation of oxidative stress during sickle vaso-occlusive crisis is associated with decreased anti-band 3 autoantibodies rate and increased red blood cell-derived microparticle level: a prospective study[END_REF]. Such an increase in MPs originating from RBC has also been reported by three other groups [START_REF] Tantawy | Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications[END_REF][START_REF] Kasar | Clinical significance of circulating blood and endothelial cell microparticles in sickle-cell disease[END_REF][START_REF] Piccin | Circulating microparticles, protein C, free protein S and endothelial vascular markers in children with sickle cell anaemia[END_REF]. However, further studies are warranted to better document the quantitative and qualitative changes in MPs during SCD-related complications.

High MP concentrations were reported in SCD patients exhibiting a severe vaso-occlusive phenotype [START_REF] Tantawy | Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications[END_REF][START_REF] Nébor | Fetal hemoglobin and hydroxycarbamide moduate both plasma concentration and cellular origin of circulating microparticles in sickle cell anemia children[END_REF] and a past history of complications such as acute chest syndrome, pulmonary hypertension [START_REF] Tantawy | Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications[END_REF] or osteonecrosis of the femoral head [START_REF] Marsh | Microparticles as biomarkers of osteonecrosis of the hip in sickle cell disease[END_REF]. Nevertheless, such associations with previous vaso-occlusive complications, acute chest syndrome or pulmonary hypertension have not been reproduced in other studies [START_REF] Garnier | Differences of microparticle patterns between sickle cell anemia and hemoglobin SC patients[END_REF], while the usefulness of MPs as biomarkers of osteonecrosis need to be confirmed.

Hydroxyurea (HU) effect on MP profiles

The effect of HU therapy on circulating MPs remains unclear. Indeed, HU treatment has been associated with either reduced plasma concentration of MPs, mainly those originated from RBC and platelets [START_REF] Tantawy | Circulating platelet and erythrocyte microparticles in young children and adolescents with sickle cell disease: Relation to cardiovascular complications[END_REF][START_REF] Nébor | Fetal hemoglobin and hydroxycarbamide moduate both plasma concentration and cellular origin of circulating microparticles in sickle cell anemia children[END_REF][START_REF] Westerman | Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy[END_REF], increased levels of MPs [START_REF] Piccin | Circulating microparticles, protein C, free protein S and endothelial vascular markers in children with sickle cell anaemia[END_REF][START_REF] Brunetta | Hydroxyurea increases plasma concentrations of microparticles and reduces coagulation activation and fibrinolysis in patients with sickle cell anemia[END_REF] or no change in MP concentration [START_REF] Kasar | Clinical significance of circulating blood and endothelial cell microparticles in sickle-cell disease[END_REF]. Since MP concentrations exhibit high inter-individual variability, the cross-sectional design of the previously cited studies may account partly for these inconsistent results. Currently, only one longitudinal study of patients treated with HU has been performed demonstrating similar MP concentrations before and after 2 years of treatment [START_REF] Garnier | Decrease of externalized phosphatidylserine density on red blood cell-derived microparticles in SCA patients treated with hydroxycarbamide[END_REF].

Interestingly, modification of two quantitative flow cytometry parameters of MPs shed by RBC, namely forward scatter (FS) and mean fluorescence intensity (MFI) of Annexin V (related to MP size and density of phosphatidylserine at the membrane surface, respectively), were detected in this study. HU-treated patients exhibited significantly higher FS indices and lower MFI values compared to values before treatment. Moreover, the relationship between these two parameters and HU dose strengthens the hypothesis of an HU-mediated effect.

Further studies are needed to confirm these observations and, more importantly, to analyze their potential functional consequences.

Triggering factors of MP release in sickle cell disease

Several factors responsible for the increased release of MPs have been identified in SCD. The best characterized are those targeting RBCs and platelets, while those affecting the other blood cell types have been less studied.

Sickle RBCs were the first pathologic cell type to be identified as a source of MPs [START_REF] Allan | Release of spectrin-free spicules on reoxygenation of sickled erythrocytes[END_REF].

Indeed, it has been established that repeated RBC sickling/unsickling induce the shedding of MPs due to uncoupling between the membrane skeleton and the lipid bilayer, resulting partly from RBC membrane protein oxidation [81 -83]. These oxidative processes, as well as sickling of RBC, are the main driving forces of intravascular hemolysis. Therefore, it is not surprising that several studies have detected relationships between RBC-derived MP concentration and Hb, as well as expression of hemolytic parameters [START_REF] Van Beers | Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease[END_REF][START_REF] Nébor | Fetal hemoglobin and hydroxycarbamide moduate both plasma concentration and cellular origin of circulating microparticles in sickle cell anemia children[END_REF][START_REF] Westerman | Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy[END_REF][START_REF] Nouraie | The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe[END_REF].

Another mechanism induced by alteration in the RBC membrane is involved in the generation of sickle RBC-derived MPs. Mechanical stress in RBC, exacerbated in SCD, activates sphingomyelinase, an enzyme implicated in membrane stability, vesiculation and MP formation [START_REF] Cuschieri | Acid sphingomyelinase is required for lipid Raft TLR4 complex formation[END_REF][START_REF] Dinkla | Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure[END_REF]. A recent study showed that the abnormal activation of acid sphingomyelinase in sickle RBCs enhanced the shedding of MPs by these cells [START_REF] Awojoodu | Acid sphingomyelinase is activated in sickle cell erythrocytes and contributes to inflammatory microparticle generation in SCD[END_REF].

In addition, it has been shown that thrombospondin-1, a major platelet protein detected at high level in sickle plasma [START_REF] Browne | Disturbance of plasma and platelet thrombospondin levels in sickle cell disease[END_REF], triggers in vitro erythrocyte conversion into spicule-covered echinocytes and induces RBC-derived MP shedding [START_REF] Camus | Erythrocyte microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease[END_REF].

Several mechanisms responsible for enhanced MP release by platelets in sickle patients have been characterized. Free hemoglobin, detected at high levels in SCD patient blood as a consequence of intravascular hemolysis, is clearly a major factor contributing to platelet activation in SCD by limiting the bioavailability of NO, a molecule known to inhibit platelet activation [START_REF] Rother | The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease[END_REF][START_REF] Villagra | Platelet activation in patients with sickle disease, hemolysis-associated pulmonary hypertension, and nitric oxide scavenging by cell-free hemoglobin[END_REF]. More recently, it has been shown that free HbS may directly participate in platelet activation. Indeed, its binding to GP1b on the platelet surface induced activation via the Lyn, PI3K, Akt and ERK pathway with associated shedding of MPs [START_REF] Annarapu | HbS Binding to GP1bα Activates Platelets in Sickle Cell Disease[END_REF]. Lastly, the release of intra-erythrocyte ADP by intravascular hemolysis may also lead to platelet activation [START_REF] Helms | Mechanisms of hemolysis-associated platelet activation[END_REF].

MP formation from other blood cell types is presumably the consequence of several pathophysiological processes such as hypoxia/reperfusion, inflammation, and oxidative stress, among others.

Microparticles in sickle pathophysiology

The involvement of MPs in pathophysiological processes of SCD including coagulation, inflammation and abnormal cellular adherence has been proposed in several studies.

Different cell type-specific MPs may trigger coagulation through TF-independent or TFdependent mechanisms. The former mechanism may involve MPs originating from RBCs and platelets, the two most abundant circulating MPs in SCD that do not express TF but exhibit docking sites for activated clotting factors; indeed, an accelerating effect on thrombin generation was observed with RBC-derived MPs in in vitro assays [START_REF] Gerotziafas | The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles[END_REF][START_REF] Noubouossie | Evaluation of the procoagulant activity of endogenous phospholipids in the platelet-free plasma of children with sickle cell disease using functional assays[END_REF]. Accordingly, positive correlations between RBC-derived MP concentrations and expression of coagulation markers such as prothrombin fragment F1+2 and D-dimer as well as acceleration in the propagation phase of thrombin generation have been observed [START_REF] Van Beers | Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease[END_REF][START_REF] Gerotziafas | The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles[END_REF]. Interestingly, a recent study presented evidence that the density of externalized PS, assessed by the mean fluorescent intensity of Annexin V, is higher for RBC-derived MPs compared to platelet-derived MPs [START_REF] Garnier | Differences of microparticle patterns between sickle cell anemia and hemoglobin SC patients[END_REF]. On the other hand, initiation of coagulation by TF-bearing MPs was supported by the inhibition of clotting activity of sickle MPs in normal plasma using a neutralizing antibody to TF, as well as the correlations between TF positive MP numbers and levels of circulating coagulation markers [START_REF] Shet | Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes[END_REF]. Despite these discrepancies, these data strongly suggest that MPs of various cellular origins in SCD are bio-effectors involved in the hypercoagulation state and thrombosis, both considered a leading cause of death and significant contributors to vessel occlusion in SCD patients [95 -97].

Other functional properties for sickle RBC-derived MPs have been documented. Injection of RBC-derived MPs, produced in vitro from sickle RBCs, in SCD mice induced renal vasoocclusion [START_REF] Camus | Erythrocyte microparticles can induce kidney vaso-occlusions in a murine model of sickle cell disease[END_REF]. In vitro, these MPs induced ROS production by endothelial cells, endothelial apoptosis and a pro-adhesive phenotype. A subsequent study by the same group demonstrated heme docked by externalized PS in MPs could be the dominant driving factor of these biological effects, which also included compromised microvascular dilation [START_REF] Camus | Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease[END_REF]. In addition, these MPs could be internalized by monocytes, where they promoted the production and secretion of some proinflammatory cytokines and enhanced monocyte adhesion to endothelial cells [START_REF] Awojoodu | Acid sphingomyelinase is activated in sickle cell erythrocytes and contributes to inflammatory microparticle generation in SCD[END_REF]. Although these data strongly suggest functional links between RBC-derived MPs with pathologic processes in SCD, it is worthwhile to note that these studies have been performed with MPs generated in vitro and thus which may not fully recapitulate the properties of circulating MPs.

Conclusion

This overview describes the current state of knowledge regarding MP formation, structural characteristics, biological impacts and factors which affect genesis and function of MPs derived from blood cells in SCD. Despite a significant volume of studies, uncertainties remain including the precise quantitative and qualitative pattern of circulating MPs in SCD, at steady state, during acute SCD complications and in SCD patients treated with HU. These unsolved points illustrate the need for standardized procedures and improved protocols for MP purification and analysis. Carefully designed studies based on large and well-characterized SCD cohorts are currently needed to clarify the biomarker status of MPs in SCD.

Furthermore, the functional properties of circulating MPs, in addition to those generated in vitro, remain to be resolved.