Romain Roulard

Michel Trentin

Valerie Lefebvre

Francoise Fournet

Ludivine Hocq

Jerome Pelloux

Eric Husson

Christophe Pineau

Loic Dupont

Arash Jamali
email: arash.jamali@u-picardie.fr

Valérie Lefebvre

Françoise Fournet

Jérôme Pelloux

Éric Husson

Loïc Dupont

In situ ESEM using 3-D printed and adapted accessories to observe living plantlets and their interaction with enzyme and fungus

Keywords: ESEM accessories, In Situ observation, Arabidopsis thaliana, Linum usitatissimum, Polygalacturonase, Verticillium dahliae

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction:

Many macroscopic properties and qualitative characteristics of plants and biomaterials originate from their cell wall composition and organization [START_REF] Holland | Plant cell walls: Impact on nutrient bioaccessibility and digestibility[END_REF]. Observation of cells, tissues and organs and subtle alterations of their morphology often requires highresolution imaging [START_REF] Talbot | Cell surface and cell outline imaging in plant tissues using the backscattered electron detector in a variable pressure scanning electron microscope[END_REF]. Therefore, specialized microscopy techniques have been developed to reveal structural features such as distribution of different cell types, and to characterize cell wall components at micron and submicron-scale [START_REF] Mayo | Micron-scale 3d imaging of wood and plant microstructure using high-resolution x-ray phase-contrast microtomography[END_REF][START_REF] Sozzani | Advanced imaging techniques for the study of plant growth and development[END_REF]. Application of high-resolution microscopy in plant studies, however, is not always straightforward. The crucial limitations can arise from the nature of the samples and difficulties in their preparation and conservation, as well as requirements imposed by the microscopy techniques, such as specific imaging environment and complex image reconstitution [START_REF] Costa | Plant cell wall, a challenge for its characterisation[END_REF][START_REF] Wu | Imaging techniques for elements and element species in plant science[END_REF]. Scanning electron microscopy (SEM), ever since the early days of its application by biologists, has been widely used to observe plant cells, tissues and their surface morphology [START_REF] Carr | Intercellular pectic strands in parenchyma: Studies of plant cell walls by scanning electron microscopy[END_REF][START_REF] Stant | The role of the scanning electron microscope in plant anatomy[END_REF]. SEM can provide high-resolution pseudo-three dimensional (3D) images of the surfaces at a broad magnification range. These images are acquired more rapidly and comprehended more easily than those obtained by more complex techniques used in two or threedimensional microscopic analyses of plant cells (Barbier de [START_REF] Barbier De Reuille | A protocol to analyse cellular dynamics during plant development[END_REF][START_REF] Costa | Plant cell wall, a challenge for its characterisation[END_REF]. Nevertheless, the application of conventional SEM to study the surface of hydrated tissues or living cells has some limitations. Hydrated samples can lose their water in an uncontrolled dehydration process when exposed to vacuum of the microscope chamber, often resulting in deformation or destruction of their native structure [START_REF] Bogner | A history of scanning electron microscopy developments: Towards "wet-stem[END_REF][START_REF] Bozzola | Electron microscopy: Principles and techniques for biologists[END_REF]. Plant materials have usually a very high water content and are strongly dependent on turgor pressure for their structural integrity [START_REF] Ensikat | Scanning electron microscopy of plant surfaces: Simple but sophisticated methods for preparation and examination[END_REF][START_REF] Sorbo | Applications of environmental scanning electron microscopy (ESEM) in botanical research[END_REF]. Therefore, sample preparation is crucial in order to conserve structural features of plants tissues and cells for observation under the high vacuum of an electron microscope [START_REF] Pathan | Sample preparation for SEM of plant surfaces[END_REF]. Accordingly, specialized sample preparation protocols have been developed to minimize the artefacts and artificial changes that may occur in the original structure of the hydrated samples during their conservation process [START_REF] Ensikat | Scanning electron microscopy of plant surfaces: Simple but sophisticated methods for preparation and examination[END_REF][START_REF] He | Highly efficient nanoscale analysis of plant stomata and cell surface using polyaddition silicone rubber[END_REF][START_REF] Kirk | Application of environmental scanning electron microscopy to determine biological surface structure[END_REF][START_REF] Popielarska-Konieczna | Extracellular matrix of plant callus tissue visualized by ESEM and SEM[END_REF]. These methods however, are not suitable for in situ observation and analyzing the live plant tissues in their native state [START_REF] Tihlaříková | In-situ preparation of plant samples in ESEM for energy dispersive x-ray microanalysis and repetitive observation in SEM and ESEM[END_REF]. Development of environmental SEM (ESEM), first commercialized by ElectroScan and later by Philips/FEI (Amsterdam, the Netherlands) in the 1980s, introduced the possibilities of imaging wet objects in a gaseous environment with no prior specimen preparation [START_REF] Donald | Fracture of plant tissues and walls as visualized by environmental scanning electron microscopy[END_REF][START_REF] Gilpin | Biological applications of environmental scanning electron microscopy[END_REF][START_REF] Muscariello | A critical overview of ESEM applications in the biological field[END_REF]. In an ESEM, the hydration state of the specimen is maintained by simultaneously controlling its temperature and the water vapor pressure in the chamber [START_REF] Donald | The use of environmental scanning electron microscopy for imaging wet and insulating materials[END_REF][START_REF] Stokes | Principles and practice of variable pressure/environmental scanning electron microscopy[END_REF]. The ESEM, therefore, has been widely used in biological research including plant science to study the ultrastructure and the morphology of hydrated samples [START_REF] Stabentheiner | Environmental scanning electron microscopy (ESEM)-a versatile tool in studying plants[END_REF], and the use of ESEM in comparative studies of static morphology of biological materials has received considerable attention. Nevertheless, the observation of dynamic processes in biology has been largely confined to the in situ mechanical testing of tissues or physical interactions with biological surfaces such as wetting behaviors [START_REF] Dauwe | Etching of the seed cuticle by cold plasma shortens imbibitional leakage in linum usitatissimum[END_REF][START_REF] Mcgregor | ESEM imaging of dynamic biological processes: The closure of stomatal pores[END_REF][START_REF] Stabentheiner | Environmental scanning electron microscopy (ESEM)-a versatile tool in studying plants[END_REF][START_REF] Wigzell | Smart water channelling through dual wettability by leaves of the bamboo phyllostachys aurea[END_REF]. Attempts have been made to test the viability of plant cells and view dynamic natural processes in living tissue using ESEM. [START_REF] Zheng | Investigation of viability of plant tissue in the environmental scanning electron microscopy[END_REF] could maintain the cells of the upper epidermal tissue of Allium cepa (onion) hydrated and viable inside the ESEM chamber. [START_REF] Mcgregor | ESEM imaging of dynamic biological processes: The closure of stomatal pores[END_REF] successfully recorded the stomatal movements in freshly-cut leaf tips of Tradescantia andersonia (Spiderwort) which are regarded as robust tissues with suitable stomatal movements for ESEM imaging. Such ESEM observations were realized by controlling the physiological temperature and adjusting the water vapor pressure with the thermodynamic constraints in the chamber and minimizing the electron beam damage [START_REF] Mcgregor | ESEM imaging of dynamic biological processes: The closure of stomatal pores[END_REF]. In fact, achieving a precise and careful control of chamber's conditions to maintain the sample in its natural state is complex and often counted as the main concern for performing in situ observation in the ESEM [START_REF] Kirk | Application of environmental scanning electron microscopy to determine biological surface structure[END_REF]. Furthermore, the optimal working conditions for biological applications are always sample-specific and determined empirically, which prevents the standardization of ESEM methods [START_REF] Tihlaříková | In-situ preparation of plant samples in ESEM for energy dispersive x-ray microanalysis and repetitive observation in SEM and ESEM[END_REF]. Consequently, despite the strong interests in using ESEM for studying the plants cells and tissues, application of ESEM by the plant science community has not yet reached its full capacity. For example, the use of ESEM for monitoring the time-course of changes in plant cell walls and tissues during growth has not been reported. Limitations in maintaining the plant sample viable during examination and lack of commercial accessories dedicated to such analyses have been considered as the major hindering factors for these applications [START_REF] Kwon | Development of SEM and stem-in-SEM grid holders for eds analysis and their applications to apatite phases[END_REF][START_REF] Zhang | Applications of ESEM on materials science: Recent updates and a look forward[END_REF]. In this study, we modified a commercially available sample holder of an ESEM and optimized the chamber condition and imaging parameters for in situ observation of living plant cells and tissues. We describe how we obtained electron microscopic images during early growth stages of living model plants i.e. Arabidopsis thaliana and flax (Linum usitatissimum). We also show the effect of polygalacturonase (PG), a biopolymer-modifying enzyme, on cell wall structure of Arabidopsis, and illustrate the fungal colonization of roots of flax seedlings and invasion of the epidermal cells by the hyphae on the living plant surfaces.

Materials and methods

Environmental scanning electron microscopy (ESEM) and accessories

ESEM and Low vacuum modes

We used a Quanta 200 field emission gun environmental scanning electron microscope (ESEM, FEI Company, USA). Environmental (ESEM) and low vacuum (LoVac) modes of this SEM allow the users to image samples in a gaseous environment using Pressure Limiting Apertures (PLA) and cones. The standard configuration of the environmental mode requires installing a gaseous secondary electron detector (GSED) and a thermoelectric cooling (Peltier) stage that can operate in a temperature range from -25°C to 55°C. The desired relative humidity (RH), when using water vapor, is obtained by controlling the pressure of the ESEM chamber from 10 Pa (0.075 Torr) to 2700 Pa (20 Torr) and the temperature of the sample by the thermoelectric controller of the Peltier stage (FEI Company, 2010). These adjustments are made using liquid/gas phase border according to the relative humidity isobar. The phase diagram of pure water and the isobar chart are presented in supplemental Figure S1. In the low vacuum mode a large field detector (LFD) is used and the pressure ranges from 10 Pa (0.075 Torr) to 130 Pa (0.97 Torr).

Cooling stage

The Peltier cooling/heating stage in the ESEM is used to control the sample's temperature within 20 °C around the ambient, ranging from -25 °C to + 55 °C. This stage can be used to create water condensation on the sample surface and achieve 100% relative humidity (RH) with respect to the water vapor pressure of the chamber (FEI Company, 2010). The original cooling stage provides a support for a standard cylindrical ESEM sample holder with diameter of 10 mm (Figure 1a and1b). This emplacement is sufficient for holding small-sized samples such as Arabidopsis and flax seeds, however, it does not provide enough surface to accommodate growing seedlings of these species. Upon germination of the seeds, the seedlings grow rapidly and within a few days can reach the edge of the sample holder (Figure 1c). Therefore, we aimed to assemble a new module with increased surface area that could hold a bigger ESEM mount to accommodate the growing seedlings. We first dismounted the original cooling stage and specified the dimension and technical details of its components.

Based on the original design and according to the microscope manufacturer's requirements, new pieces were redesigned and fabricated. Autodesk Fusion 360 software (Autodesk, Inc., San Rafael, CA, USA) was used to design all the components and develop 3D models. The designed pieces were saved in stl. format and machined from stainless steel and aluminium or fabricated with PVA filament using a Prusa MK3 3D-printer (Prusa Research, Czech Republic). Electronic parts and wires were purchased from the local stores. Technical details of the original and new designed accessories are provided in supplementary materials (supplemental Figure S2).

Bio-transfer module

We designed a simple specimen holder (hereinafter referred to as "bio-transfer module") that could fit the modified cooling stage. Two prototypes of a bio-transfer module were assembled.

The first prototype included a base composed of a 3-dimensional printed plastic ring and an aluminium cylinder with a diameter of 25 mm that provided ≈5 cm 2 metal contact surface with the Peltier module (supplemental Figure S3). In this model, the plastic part can be replaced by other materials for specific applications, for example, glass or alternatively shatter-resistant acrylic glass to increase the exposed surface to light as required for germination of some plants seeds [START_REF] Toole | Interaction of temperature and light in germination of seeds[END_REF]. In the second prototype, the whole base of the bio-transfer module was machined from aluminium providing ≈10 cm 2 of metal surface. The larger metal surface in the latter model, which was then used throughout this study, provides a high electronic conductivity enabling the analyses of larger specimens and using higher accelerating voltages during observation. A transparent cover composed of polycarbonate permitted light transmission to the seedlings and their protection against contamination during their growth and transport (supplemental Figure S3).

Biological samples

Arabidopsis thaliana and Polygalacturonase enzyme

Arabidopsis thaliana (A. thaliana) is a model plant with a fully-sequenced genome and many interesting characteristics such as a short life cycle, prolific seed production, and numerous mutant lines (The Arabidopsis Genome Initiative, 2000). We used the ESEM to study the cells of elongated hypocotyls in etiolated seedlings and their modification after exogenous application of heterologously expressed enzymes. Pectin depolymerizing enzymes such as polygalacturonases (PG) can induce pectin remodeling and affect cell elongation and integrity (Hocq et al., 2020). Here we added an Arabidopsis polygalacturonase (AtPG (endo-PG, EC 3.2.1.15)) into the growth medium of Arabidopsis to observe the induced modifications on the cells on the hypocotyl surfaces. The active enzyme was expressed heterologously in the yeast Pichia pastoris X-33 strain and purified as previously described (Hocq et al., 2020). Arabidopsis seeds were surface sterilized, soaked in 1mL water and placed for 2 days in a cold chamber for stratification. Seeds were then placed to germinate in sterilized 24-microwell plate containing a ½MS semi-solid medium plus either active or inactivated (control) enzymz (8µg AtPG /600µL/well). Microplate containing the seeds were first exposed to 6 hours of flash light (120µmoles/m²/s), then positioned vertically in the dark for 48-72 hours to favor the straight elongation of the hypocotyls [START_REF] Desnos | Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark-and light-grown arabidopsis seedlings[END_REF]. The 2-days old seedlings were carefully transferred into the sample holder for ESEM observations.

Flaxseeds and seedlings

Flax (Linum usitatissimum L.) is an important agricultural plant with an increasing application in nutraceutical and bio-pharmaceutical industries, fibers, human food and animal feed. Many interesting characteristics of flax are influenced by its seeds germination and seedlings properties [START_REF] Dauwe | Etching of the seed cuticle by cold plasma shortens imbibitional leakage in linum usitatissimum[END_REF][START_REF] Jhala | Flax (linum usitatissimum L.): Current uses and future applications[END_REF][START_REF] Wang | Effect of germination on vitamin c, phenolic compounds and antioxidant activity in flaxseed (linum usitatissimum L.)[END_REF][START_REF] Wang | Effect of germination on lignan biosynthesis, and antioxidant and antiproliferative activities in flaxseed (linum usitatissimum L.)[END_REF].

The seeds of brown flax (cv. Baladin), kindly provided by the seed company Laboulet (Airaines, France), were visually screened and those with similar sizes and weights were selected as the experimental material. Flax seeds were germinated on wet filter paper in 90-mm-diameter Petri dishes as described previously [START_REF] Dauwe | Etching of the seed cuticle by cold plasma shortens imbibitional leakage in linum usitatissimum[END_REF] or directly in the bio-transfer module.

After 48h growth in the dark, they were then transferred to a phytotron chamber and grown under controlled conditions (at 23°C and 80% humidity) using a light-dark cycle (16h:8h) with a light intensity of 3350 lux to 3500 lux. These seedlings were then transferred into the ESEM using the bio-transfer module for microscopic observation.

Verticillium dahliae

Verticillium dahliae is a fungus that causes Verticillium wilt in many crops plant. This fungus predominantly infests the fiber flax cultures in northern France causing significant yield losses [START_REF] Blum | Verticillium wilt on fiber flax: Symptoms and pathogen development in planta[END_REF]. We have artificially infected the flax seedlings using an undisclosed protocol developed by the company "Linéa Semences" (Grandvilliers, France). Briefly, this protocol consists of inoculation of roots of two-week old flax plantlets grown under controlled conditions (as above) with a spore suspension of V. dahliae grown on a potato dextrose agar (PDA) medium for 7 days. Using ESEM, we observed the native fungal structure and morphology in their hydrated culture medium, and studied the interaction of fungus mycelium with plantlets by imaging the epidermal and cortical cells of artificially infested plants.

Results and discussion

Modification of accessories

In situ observation of samples such as hypocotyls of Arabidopsis and germinated flaxseeds and their growing plantlets necessitated modification of the standard thermoelectric cooling stage. The hydration of a specimen in the ESEM was controlled by the temperature and the pressure of water vapor in the microscope chamber. The pressure control of the ESEM chamber is achieved by the use of differential pumping system and pressure-limiting apertures (PLAs), and the temperature of the sample is adjusted by a cooling stage operating by Peltier effect [START_REF] Stokes | Principles and practice of variable pressure/environmental scanning electron microscopy[END_REF]. For an accurate control and reading of the sample's temperature, we aimed to maximize the contact of the samples with the holder by redesign of the cooling stage. The holder is in contact with a thermoelectric (Peltier) cooling device that is typically manufactured from two thin ceramic wafers which sandwich a series of p and n doped bismuth telluride (Bi2Te3) semiconductor materials. The ceramic material on both sides of the thermoelectric device adds rigidity and the necessary electrical insulation. When current passes through these elements, one side of the wafer heats and the opposite one cools, and reversing the polarity switches cold and hot sides (Supplemental Figure S2) (FEI Company, 2010;[START_REF] Shourideh | A comprehensive study of an atmospheric water generator using peltier effect[END_REF]. In our redesigned module, we have replaced the original Peltier device (10 x10 mm 2 ) with a larger one (30 x30 mm 2 ) that could consequently receive a larger mount recipient with the diameter of 25 mm. The mount recipient is a plate into which the sample holder is inserted. A thermistor (PT 100, RS components, UK) was placed inside of the new mount recipient to give an accurate temperature reading in close vicinity under the sample. The mount recipient was installed on the new Peltier using a thermal grease. The inner cable and water hoses were then connected to the base of the stage to pass the current and remove excess heat efficiently as required by the microscope manufacturer. The details on the configuration and dimensions of the new module are presented in supplementary data (Supplemental Figure S2). The functionality of the new assembly was confirmed by comparison of temperature and water condensation and evaporation rate of this module with that of the standard module in an identical condition. Figure 2 shows the original cooling stage module and the modified design allowing comparison of the surface areas of the two modules regarding their provided surface areas (Figure 2). The higher surface contact and temperature exchange area of the new cooling stage module, enabled us to assemble a compatible specimen bio-transfer mount to transfer the germinating seeds and growing seedlings to the SEM chamber with minimal change in their natural posture. In such way, samples in the biotransfer module could be transported into and out of the microscope with minimal dislodgement or loss of their contact with the holder. As discussed later in the following sections of this paper, such a configuration enabled us to maintain the sample in their native state during the ESEM observation and analyze the structure and organization of the cells and the effect of the enzyme and fungus on their morphological aspects during the early growth stages of the seedlings.

Observation of live plantlets

Optimization and imaging

We first optimized the imaging parameters and the chamber condition of the microscope to observe the native structure of very small seedlings of Arabidopsis. In dicotyledonous seedlings, the hypocotyl is part of the stem between the root and the cotyledons and is widely used as a model for the physiological study of the mechanism of cell elongation and variety of other biological events [START_REF] Gendreau | Cellular basis of hypocotyl growth in arabidopsis thaliana[END_REF]. The young etiolated hypocotyls with a diameter smaller than 250 µm and a length varying between 2 to 20 mm are very fragile and known to be highly susceptible to changes of relative humidity [START_REF] Burgert | Plant micro-and nanomechanics: Experimental techniques for plant cell-wall analysis[END_REF].

As a reference, we therefore began by analyzing young hydrated etiolated Arabidopsis seedlings (2-days old dark-grown) to obtain images of their surfaces and cellular structure.

Theoretically, hydration of the samples in the ESEM is achieved by setting the microscope to a specific water vapor pressure and cooling the specimen to the relevant temperature indicated by the phase diagram of water (as described above, supplemental Figure S1). In practice, however, the young plantlets were greatly impacted before reaching the desired relative humidity, because of dehydration during the pump-down and evacuation of the air from the chamber. This resulted in deformation of cells in hypocotyls and other organs such as roots and testa (Figure 3a-d). We could mitigate this issue by cooling the sample down to the target temperature and adding several drops of ultrapure water to the stage base before closing and pumping down of the chamber. Furthermore, purging water vapor into the specimen chamber (five cycles) during pump-down could improve saturating the sample's surroundings to prevent dehydration before reaching the designated chamber pressure.

These steps have previously shown to be effective in preventing dehydration of desiccationsensitive samples in the ESEM [START_REF] Jenkins | Observing fibers swelling in water with an environmental scanning electron microscope[END_REF]. We observed that the overall structure of the live seedlings was preserved in the ESEM chamber when the relative humidity was maintained around 85% (Figure 3e), conforming to their structural characteristics previously revealed in the fixed samples [START_REF] Gendreau | Cellular basis of hypocotyl growth in arabidopsis thaliana[END_REF]. At lower RHs the hypocotyl structure was extensively deformed (Figure 3f). We could achieve high levels of RH, by maintaining the temperature at 20 C° and adjusting the pressure to 14 torr. Higher vapor pressures could reduce the charging effect and proved to be essential for preserving the tissues of the fragile seedlings, however, further increase in the relative humidity compromised the sharpness of the image with significant loss of resolution. Moreover, condensation of vapor formed droplets and films of water on the surface of the specimens (Figure 3g), which obscured the underlying structure and reduced contrast as previously reported for ESEM observations [START_REF] Jansson | Novel method for controlled wetting of materials in the environmental scanning electron microscope[END_REF][START_REF] Jenkins | Observing fibers swelling in water with an environmental scanning electron microscope[END_REF]. Details on optimal image acquisition parameters of Arabidopsis seedlings are summarized in Table 1.

Optimization of imaging parameters for ESEM analyses of the sensitive hypocotyl cells provided reference points to observe other cells and tissues of the seeds and small seedlings of Arabidopsis. Figure 4 shows examples of ESEM images of diverse organs and cells including germinating seed surfaces and collumella of their mucilage secreting cells and cotyledons, root and shoot apical meristems, trichomes, stomata, rhizoderm cells and extremely delicate root hairs in the growing seedlings. Theses ESEM images of Arabidopsis organs and cells conform to the previously reported SEM images that were obtained from the chemically-fixed tissues [START_REF] Byrne | Shoot meristem function and leaf polarity: The role of class iii hd-zip genes[END_REF][START_REF] Pillitteri | Stomatal development in arabidopsis[END_REF][START_REF] Tsukaya | Leaf development[END_REF]. Our ESEM results not only show the disposing of sample preparation steps that are required for classical SEM observations, but also the ability of this approach to accurately study the surface morphology and anatomical development of the cells in their native state in the live growing seedlings.

Time course in situ ESEM and repetitive observation of growing plantlets

To observe the early stages of seedling growth and development on a daily basis by in situ ESEM, we combined our home-designed cooling stage and bio-transfer module. The flax seeds germinated readily in the bio-transfer module in a phytotron. The module containing germinated seeds could be easily transferred to the ESEM chamber and fitted on the newly designed cooling stage. We could subsequently image diverse cells and tissues and follow their time-course of modification in the germinating seeds and seedlings (Figure 5), then return the module back to the phytotron after observation. Similar to our ESEM observation of Arabidospis seedlings, imaging of flax seedlings also required an adjustment of the acquisition parameters for different tissues and cells. The combination of the optimal parameters used for ESEM imaging of different parts of the living seedlings of Arabidopsis and flax are outlined in Tables 1. Natural resistance of plant tissues to dehydration and beam damage varies by their types and upon their growth [START_REF] Stabentheiner | Environmental scanning electron microscopy (ESEM)-a versatile tool in studying plants[END_REF]. Structures such as seeds or pollens are usually tolerant to desiccation in most plants, however, there is a great variation in the resistance and deformation of vegetative tissues upon dehydration [START_REF] Oliver | Desiccation tolerance in vegetative plant cells[END_REF][START_REF] Wakui | Scanning electron microscopy of desiccationtolerant and-sensitive microspore-derived embryos of brassica napus L[END_REF]. We found that, the Arabidopsis hypocotyls were more resistant to partial vacuum in older plantlets and could be observed under low vacuum (pressure not exceeding ∼1 torr) after a week of germination without showing significant structural deformation. The increase of the cell wall thickness following the expansion of the thin primary cell walls in growing plants, could play a role in the resistance of the older plant to the vacuum and dehydration [START_REF] Kumar | Secondary cell walls: Biosynthesis and manipulation[END_REF][START_REF] Vicré | Insights into the cellular mechanisms of desiccation tolerance among angiosperm resurrection plant species[END_REF][START_REF] Vogler | Measuring the mechanical properties of plant cell walls[END_REF], and therefore to the partial vacuum or oscillation of the humidity in the microscope chamber during analyses [START_REF] Burgert | Plant micro-and nanomechanics: Experimental techniques for plant cell-wall analysis[END_REF][START_REF] Treitel | Elasticity, plasticity, water content, and hardness of plant tissues of different ages[END_REF]. Furthermore, cuticle that is a complex structure mainly composed of a lipid polymer, i.e., cutin and waxes, coats the outermost layer of periclinal cell walls of all aerial organ epidermal cells. The cuticle layer provides a barrier to prevent non-stomatal water-loss, and plays various roles in response to environmental stresses [START_REF] Jacq | The arabidopsis lipid transfer protein 2 (atltp2) is involved in cuticle-cell wall interface integrity and in etiolated hypocotyl permeability[END_REF][START_REF] Yeats | The formation and function of plant cuticles[END_REF]. This could also explain why it is possible to observe older plant tissues and cells by SEM at lower pressure i.e. low vacuum mode. We observed that the roots, even in older seedlings, suffered more easily from dehydration caused by the partial vacuum in the microscope. We could prevent this dehydration by wrapping the roots with blotting paper soaked in water. The lack of a strong diffusion barrier, such as cuticle, at the surfaces of epidermis cells in the primary growth stage could explain the higher sensitivity of the roots to the variation of humidity of the ESEM chamber compared to aerial plant organs [START_REF] Berhin | The root cap cuticle: A cell wall structure for seedling establishment and lateral root formation[END_REF]. Beside appropriate operation parameters to maintain the tissue fully hydrated, the viability of cells depend on the beam intensity, particularly in relation to the beam accelerating voltage and the total electron dose [START_REF] Zheng | Investigation of viability of plant tissue in the environmental scanning electron microscopy[END_REF]. During the imaging process, the image acquisition parameters such as working distance, acceleration voltage, scan speed and spot size were optimized to minimize beam damage to the living cells (Table T1). The images were generally recorded at acceleration voltages between 5 and 20 kv and a working distance between 5 to 10 mm. The beam was blanked during imaging intervals to reduce the beam damage to the samples and visible artifacts that could occur during ESEM observation as previously reported [START_REF] Dobberstein | Beam blanking studies in environmental scanning electron microscopy[END_REF]. Our post-ESEM surveying of the seedlings that underwent such repetitive exposure to the vacuum and electron beam showed that they maintained their vitality and vigor and continued their growth and development (Supplemental Figure S4). Nevertheless, the growth rate of these plantlets was found to be slightly lower compared to those that had not been observed in the ESEM. Furthermore, during our repetitive observations, we occasionally noticed the occurrence of epidermal meristems on the hypocotyls (Figure 5, D9 b). The presence of such meristems has been previously attributed to the exposure of seedlings to drought or radiation stresses (Tafforeau et al., 2004). These findings could indicate the presence of stresses in the underlying cells that remain undetectable at the surface. Such damage can occur, for example by an increase in the reactive oxygen species (ROS) or the rupture of plasma membranes (Bandurska et al., 2012;[START_REF] Muscariello | A critical overview of ESEM applications in the biological field[END_REF][START_REF] Zheng | Investigation of viability of plant tissue in the environmental scanning electron microscopy[END_REF], which may be triggered in the ESEM chamber due to the formation of free radicals through electron beam interactions or pressure oscillation.

Faster image acquisition could prevent possible beam and thermal damage to the samples, however, it compromised the signal to noise ratio and lowered the quality of the images. It should be noted that the insertion of pressure limiting aperture (PLA, 500 µm) on the pole piece, essential for ESEM operation, limited the field of view and restricted the lowest magnification to around 200x. Increasing the working distance and subsequently acceleration voltage to compensate the beam scattering and the weak signals, could provide a larger view, albeit at the expense of optimization time for imaging in the wet conditions of the chamber [START_REF] Muscariello | A critical overview of ESEM applications in the biological field[END_REF].

Effect of enzyme application on hypocotyl structure

ESEM permitted us to observe the overall structure of hypocotyls and, in greater detail, identify the individual cells and their boundaries in the live seedlings without any sample preparation (Figure 4). The etiolated (dark-grown) seedlings of living Arabidopsis (wild type Col-0) featured typical morphological phenotypes such as apical hook, hypocotyls, unexpanded cotyledons and short root (figure 4d-f) in accord with the results of previous SEM studies on cryogenically-prepared samples [START_REF] Desnos | Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark-and light-grown arabidopsis seedlings[END_REF][START_REF] Gendreau | Cellular basis of hypocotyl growth in arabidopsis thaliana[END_REF]. The hypocotyls' cells in etiolated seedlings of Arabidopsis are known to have a simple anatomy and a postembryonic growth that is exclusively the result of a controlled series of cellelongation events [START_REF] Desnos | Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark-and light-grown arabidopsis seedlings[END_REF][START_REF] Gendreau | Cellular basis of hypocotyl growth in arabidopsis thaliana[END_REF]. Such characteristics could make this organ an apt model to study the effects of diverse growth factors on cell elongation. We used heterologously expressed AtPG, a PG (EC 3.2.1.15) that induces pectin remodeling (Hocq et al. 2020) to illustrate structural modification of hypocotyls cells. Our ESEM analyses revealed that the presence of active AtPG, modified the integrity of the hypocotyls by impairing the longitudinal and ultimately transversal adhesion of cell walls in treated seedlings (Figure 6b andc), more apparently in epidermis cells and occasionally in their underlying cortex cells (Figure 6 c). The hypocotyls of the control seedling grown with inactivated AtPG (denaturated protein) did not show any separation of the adjacent cells (Figure 6 c). These ESEM observations are comparable to the observation we made on cryo-prepared samples using low-vacuum SEM and light microscopy as shown in Figure 6 (d-i) and accord with the results of prior studies on the relation between the role of polygalacturonases in modification of cell wall rigidity and elongation [START_REF] Sénéchal | Homogalacturonan-modifying enzymes: Structure, expression, and roles in plants[END_REF]. Polygalacturonase is a pectin lytic enzyme implicated in cell expansion and elongation by degrading pectin in plants cell walls. This enzyme hydrolyses the α-1,4-linked-D-galacturonic acid units which compose the homogalacturonans (HG), the major polymer of pectins [START_REF] Babu | Suspensor length determines developmental progression of the embryo in arabidopsis[END_REF]Hadfield and Bennett, 1998;Xiao et al., 2014). Our finding on separation of the cells could be attributed to the softening and decomposition of pectin in HG-rich middle lamella of the cells by PGs, however, the exact function of these enzymes during cell elongation is not known [START_REF] Zhang | Photo-sensitive leaf rolling 1 encodes a polygalacturonase that modifies cell wall structure and drought tolerance in rice[END_REF]. During our observation, we noted that the AtPG-treated hypocotyls were more sensitive to the vacuum of the ESEM compared to the untreated ones, probably because of the enzyme's effect on the loss of integrity of the epidermal tissue and weakening of their mechanical properties. The vulnerability of such organs in the ESEM environment, could be mitigated by increasing purge cycles during the pump-down of the chamber and faster image acquisition during observations as described above. Our method of observation shows promise as a means of analyzing and illustrating the structural modification of living cell walls of plantlets upon the application of biopolymer-modifying enzymes and this may lead to broader use of this technique with the increasing interest in the in situ enzymatic treatment of plant cells.

In situ plant-fungus interaction

We could observe a laboratory culture of Verticillium dahliae mycelium in its native state in the ESEM. In situ observation of the structure of mycelium networks of this pathogen in the culture medium and then its identification during its development in infected plantlets (Figure 7a-i) was performed by optimization of imaging parameters at a relative humidity of 75% (Table 1). V. dahliae is a soil borne pathogen and belongs to the fungal class Deuteromycetes (Fungi Imperfecti), a group of fungi with an unknown sexual stage. We imaged the vegetative part of this fungus, by which it spreads in plants, and could identify different hyphal structures such as conidium, conidiophore and phialide in the fungal colony in the medium (Figure 7b andc). The structural and anatomical information on the fresh fungus provided by the ESEM observation in their native state accord with those previously reported on the chemically-fixed samples from isolated V. dahliae obtained by optical or scanning electron microscopy [START_REF] Babu | Suspensor length determines developmental progression of the embryo in arabidopsis[END_REF][START_REF] Inderbitzin | Phylogenetics and taxonomy of the fungal vascular wilt pathogen verticillium, with the descriptions of five new species[END_REF]. ESEM therefore could avoid the time-consuming sample preparation and thus had advantages over other imaging techniques for in situ studies.

Attention should be paid, however, to the high sensitivity of fungal cells to dehydration and their low intrinsic contrast during their ESEM analyses [START_REF] Kaminskyj | High spatial resolution surface imaging and analysis of fungal cells using SEM and AFM[END_REF]. Our observation of the infected seedlings showed that the fungus surrounded the roots with a thick mantle of closely interlaced hyphae (Figure 7d-f). The hyphae expanded to the higher parts of the plantlets and colonized stem (hypocotyl) and leaves (cotyledon) three days after infection covering the epidermal cells in these zones (Figure 7g-i). To observe the presence of mycelium underneath the epidermis, we transversally cut the roots and stems using a sharp single-edged razor blade and immediately observed the freshly cut surface using the ESEM.

The external structures of the transversally-cut roots, including cortex and epidermis cells did not withstand the vacuum and collapsed, with the exception of the central cylinder retaining its structure (supplemental Figure S5). To overcome the deformation of the freshly-cut sections and observe the sub-epidermal tissues and the fungus hyphae, the infected seedlings were plunge-frozen in liquid nitrogen, and then fractured in the root and hypocotyl regions as previously described [START_REF] Wakui | Scanning electron microscopy of desiccationtolerant and-sensitive microspore-derived embryos of brassica napus L[END_REF]. The fractured surfaces of roots and hypocotyls then were instantly observed in low vacuum mode at ≈1 torr to elucidate the presence of fungus in the internal structure of plantlets. Preparation of these tissues by freeze-fracturing could preserve their overall structure and components such as the epidermal and cortex cells and vascular cylinder, and allowed detection of fungal mycelium in the internal surfaces of roots (Figure 7j-l). These images showed that the hyphae could penetrate into the cortical cells, that could later colonize the vascular tissue of the plants as previously shown by bright-field and fluorescent images by [START_REF] Blum | Verticillium wilt on fiber flax: Symptoms and pathogen development in planta[END_REF]. Obviously, freeze-fracturing and low vacuum observation of the plant samples, did not preserve their vitality, but it was unavoidable to detect details of internal structure of tissues and to complement the ESEM observation of the external surfaces. These results, indeed, can be always supplemented by the conventional SEM analyses following classical sample preparations.

Conclusion

In this work, we modified a commercially available ESEM sample holder to observe and image live growing plantlets in situ. The use of newly designed and 3-D printed accessories in combination with optimized control of the relative humidity and tuning of imaging parameters permitted maintaining the plant seedlings viable during observation and acquiring quality ESEM images. This approach allowed us to observe structural modification of cells in living plantlets upon the application of a biopolymer-modifying enzyme or interaction with a fungus. These novel in situ ESEM observations are largely comparable to those made on cryo-prepared samples using low-vacuum SEM and light microscopy. Therefore, we conclude that our adopted approach in using ESEM for in situ analysis of the surfaces of living plantlets is a reliable means of assessing their evolution and growth, modification by enzymes, or degradation by fungi at cellular levels. There is clearly considerable scope to use this method to examine the interaction of various physical, chemical and biological agencies with surfaces of living plants or other biological samples. However, maintaining the humidity and viability of samples during the ESEM observations, imposed some limitations such as smaller field of view and shorter observation timespan compared to conventional SEM. Further research will address the technical limitations of this approach by employing technologies currently commercially available, such as micromanipulators and in-chamber optical cameras for faster sample navigation or novel detection systems, and developing more sample-specific accessories to expand the application of in situ ESEM into the relevant fields. (arrowed in h) and remaining of endosperm layer 3days after infection (i); freeze-fracturing of a 21 days-old flax plantlet root, 3days after their artificial infection by V. dahliae (j-I), low vacuum SEM revealing the presence of fungi mycelium on the external surfaces (j and k) and its penetration into the sub-epidermal cells (arrowed in l)

Figure and Table captions

Figure 1 :Figure 2 :

 12 Figure 1: Standard cooling stage module: (a) cooling stage (Peltier/Seebeck) assembly containing a standard ESEM sample holder, installed on the stage of the Quanta 200 ESEM chamber, (b) the image and dimensions of a standard ESEM sample holder, and (c) a 3-days old Arabidopsis plantlet (arrowed in upper image) and a flaxseed (arrowed in lower image) showing their size relative to the standard holder's surfaces. The yellow arrows in b and c indicate the width of the edge of the holder

Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :

 34567 Figure 3: The effect of chamber pump-down and relative humidity on the structure of a very young (2-days old) hydrated Arabisopsis seedling: (a) a low magnification image of an entire seedling taken at a long working distance showing the dehydrated cells, (b-d) higher magnification images of different organs of the same plant, showing the deformation of the cell walls in testa, root and hypocotyl, (e-g) images of the hypocotyls showing the effect of chamber's relative humidity in conservation of their structure during the ESEM observation;note the structural deformation at RH lower than 75% (f) and water film formation and submersion of the tissue at 100 % RH (g) (images were acquired at 5Torr and between 4 to 6°C)

Table 1 :

 1 Summary of the optimal parameters for Low vacuum (LoVac) and Environmental SEM (ESEM) observations of tissues of living plantlets and fungus

The authors thank "Région Hauts-de-France" for its financial support. Arash Jamali and Romain Roulard are grateful to Dr. Sylvain Lecomte at the ""Linéa Semences" for his helpful advice in the application of fungi for artificial contamination of flax plantlets. Arash Jamali would like to thank Dr. Emmanuelle Etienne from "Direction de la Recherche " at Université de Picardie Jules Verne for her administrative assistance and advice in this research project.

Funding

This research was funded by the Région Hauts-de-France, through the ENSEMBLE project (120-2016 RDIPROJINV-000083 RDIPROJFT-000192).

Samples


Claire Bal

Dit Sollier

Natacha Berge

Sara Hamadouche

Caren Brumpt

Alain Stepanian

Patrick Henry

Virginie Siguret

Ludovic Drouet

Jean-Guillaume Dillinger
email: jean-guillaume.dillinger@aphp.fr

Claire Bal Dit Sollier

Is platelet function testing at the acute phase under P2Y12 inhibitors helpful in predicting bleeding in real-life patients with acute coronary syndrome? The AVALANCHE study

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

internationales actuelles préconisent l'emploi de nouveaux inhibiteurs P2Y12 comme traitement de première intention malgréun risque de saignement plus élevé par rapport au clopidogrel.

Objectif. -L'étude AVALANCHE avait pour objectif de tester si les fonctions plaquettaires (VASP, agrégation à l'ADP) à la phase aiguë chez les patients avec SCA traités par P2Y12 pouvaient prédire le saignement (classification BARC 2-5) durant le suivi.

Méthodes. -Dans cette étude rétrospective, tous les patients adultes consécutifs admis au CHU Lariboisière pour un SCA, quel que soit l'inhibiteur P2Y12 et ayant bénéficié d'un test de la fonction plaquettaire (VASP et agrégométrie optique) au cours de l'hospitalisation initiale, ont été inclus. Le suivi a été réalisé afin dede recueillir les événements hémorragiques selon la classification BARC.

Résultats. -364 patients ont été inclus, dont 123 traités avec ticagrelor, 105 par le prasugrel et 136 par le clopidogrel. Parmi eux, 42,3 % après un STEMI, 27,1 % après un NSTEMI et 30,6 % présentant un angor instable. L'âge moyen était de 64 ± 11 ans. L'indice VASP médian était significativement plus bas avec les nouveaux inhibiteurs de P2Y12 (14 % sous ticagrelor, 14 % sous prasugrel) par rapport à 42 % sous clopidogrel. Malgré ces différences du degré d'inhibition plaquettaire, la fréquence des saignements (BARC 2-5) au cours du suivi était de 7,7 % au total et était similaire pour tous les inhibiteurs de P2Y12 (8,9 % sous ticagrelor, 6,6 % sous prasugrel et 7,4 % sous clopidogrel). En effectuant les courbes ROC, il n'a pas été possible pour chacun des inhibiteurs P2Y12 de déterminer un seuil d'indice VASP prédictif du risque hémorragique durant la première année de traitement. De même, l'agrégation plaquettaire induite par l'ADP était plus profondément inhibée (chez les patients sous ticagrelor ou prasugrel que sous clopidogrel sans qu'il soit possible de déterminer un seuil pour prédire le risque hémorragique pendant le suivi.

Conclusions. -Malgré la survenue d'un nombre significatif de saignements pendant le suivi, l'évaluation des fonctions plaquettaires (indice VASP et agrégation à l'ADP) mesurées à la phase aiguë n'est pas prédictive des saignements. L'intérêt de l'évaluation des fonctions plaquettaires après la phase aigüe pour prédire le risque hémorragique au cours du suivi reste à démontrer.

KEYWORDS

Background

Antiplatelet drugs are a cornerstone therapy for the prevention or treatment of arterial thrombosis, an acute complication of atherosclerosis. After an acute coronary syndrome (ACS), all patients systematically receive dual antiplatelet therapy (DAPT), consisting of a low dose of aspirin combined with an oral inhibitor of the platelet P2Y12 receptor. The only matters for discussion are DAPT duration and the type of P2Y12 inhibitor. Current international guidelines recommend more constant, more potent and more predictable P2Y12 inhibitors (i.e. prasugrel, a new-generation thienopyridine; or ticagrelor, a cyclopentyl-triazolopyrimidine reversible P2Y12 antagonist) as first-line treatment, because they provide faster and stronger platelet inhibition compared with clopidogrel [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF][START_REF] Levine | ACC/AHA/SCAI Focused Update on Primary Percutaneous Coronary Intervention for Patients With ST-Elevation Myocardial Infarction: An Update of the 2011 ACCF/AHA/SCAI Guideline for Percutaneous Coronary Intervention and the 2013 ACCF/AHA Guideline for the Management of ST-Elevation Myocardial Infarction: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Society for Cardiovascular Angiography and Interventions[END_REF]. However, the benefit of these drugs is counterbalanced by a greater bleeding risk than clopidogrel [START_REF] Wallentin | Ticagrelor versus clopidogrel in patients with acute coronary syndromes[END_REF][START_REF] Wiviott | Prasugrel versus clopidogrel in patients with acute coronary syndromes[END_REF]. Thus, clopidogrel is now recommended for patients who cannot receive ticagrelor or prasugrel or have an increased bleeding risk, such as those requiring oral anticoagulation. Consequently, clopidogrel is still widely used in current practice [START_REF] Khayata | Comparison of Clopidogrel With Prasugrel and Ticagrelor in Patients With Acute Coronary Syndrome: Clinical Outcomes From the National Cardiovascular Database ACTION Registry[END_REF][START_REF] Zeymer | P2Y12 receptor inhibitors in patients with non-STelevation acute coronary syndrome in the real world: use, patient selection, and outcomes from contemporary European registries[END_REF].

The clinical relevance of platelet function testing in the acute phase of ACS under aspirin or P2Y12 inhibitors to identify patients at high risk of ischaemic events has been demonstrated in various clinical settings [START_REF] Dillinger | High platelet reactivity on aspirin in patients with acute ST elevation myocardial infarction[END_REF][START_REF] Gum | A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease[END_REF][START_REF] Tantry | Consensus and update on the definition of on-treatment platelet reactivity to adenosine diphosphate associated with ischemia and bleeding[END_REF]. A positive predictive value for high on-treatment platelet reactivity and occurrence of ischaemic events has been largely explored. Conversely, but more recently, clinical studies have shown an association between low on-treatment platelet reactivity with clopidogrel and prasugrel and the occurrence of bleeding complications (low on-treatment platelet reactivity being defined mostly using the vasodilator-stimulated phosphoprotein phosphorylation [VASP] index, and different values of the VASP index have been proven predictive, such as VASP < 16%) [START_REF] Tantry | Consensus and update on the definition of on-treatment platelet reactivity to adenosine diphosphate associated with ischemia and bleeding[END_REF]. Among the P2Y12 antagonists, clopidogrel and prasugrel decrease the VASP index only by inhibition of the P2Y12 receptor pathway. Consequently, for these thienopyridines, there is a direct correlation between the degree of inhibition of the P2Y12 receptor pathway and the decrease in the VASP index. Besides, ticagrelor potentially decreases the VASP index by two different pathways: one is certain (i.e. inhibition of the P2Y12 pathway similarly to thienopyridines), but the other (through stimulation by adenosine of the platelet A2b pathway) is under discussion [START_REF] Parker | Equilibrative nucleoside transporter 1 gene polymorphisms and clinical outcomes following acute coronary syndromes: findings from the PLATelet inhibition and patient Outcomes (PLATO) study[END_REF]. The increase in plasma adenosine level is caused by the inhibition by ticagrelor of the "erythrocyte equilibrative nucleoside transporter", the active transporter of adenosine incorporation in red blood cells [START_REF] Bonello | Ticagrelor increases adenosine plasma concentration in patients with an acute coronary syndrome[END_REF]. This could elevate local adenosine levels at sites of ischaemia and tissue damage, although this latter effect has been challenged recently; it is unclear whether ticagrelor via this mechanism can impact the ex vivo platelet functions (VASP index, adenosine diphosphate [ADP]-induced platelet aggregation). As adenosine has a very short half-life in whole blood (a few seconds), because of its very rapid cell uptake and metabolism, adenosine present in vivo is not likely to be present ex vivo during the tests, but platelets challenged in vivo by the increased adenosine plasma level could produce the effect. Therefore, if besides P2Y12 receptor inhibition, ticagrelor induced additional adenosine-related VASP phosphorylation, a similar decrease in the VASP index should not confer the same clinical haemorrhagic risk when it is achieved by a thienopyridine or by ticagrelor. Several studies have shown that ticagrelor is associated with higher platelet inhibition and a higher incidence of low on-treatment platelet reactivity than prasugrel after ACS, without an increased incidence of bleeding [START_REF] Dillinger | Ticagrelor effectiveness overestimated by VASP index: platelet inhibition by ticagrelor versus prasugrel in acute coronary syndrome patients according to platelet function tests[END_REF][START_REF] Kerneis | Platelet effect of prasugrel and ticagrelor in patients with STsegment elevation myocardial infarction[END_REF][START_REF] Laine | COMparison of Platelet reactivity following prAsugrel and ticagrelor loading dose in ST-Segment elevation myocardial infarctION patients: The COMPASSION study[END_REF]. These results suggest that the VASP index should not have the same predictive value for haemorrhagic risk in patients treated with a thienopyridine compared with ticagrelor.

Considering results of preliminary trials, such as TOPIC [START_REF] Cuisset | Benefit of switching dual antiplatelet therapy after acute coronary syndrome: the TOPIC (timing of platelet inhibition after acute coronary syndrome) randomized study[END_REF] and TROPICAL-ACS [START_REF] Sibbing | Guided de-escalation of antiplatelet treatment in patients with acute coronary syndrome undergoing percutaneous coronary intervention (TROPICAL-ACS): a randomised, open-label, multicentre trial[END_REF], a new concept of de-escalation of P2Y12 inhibitor treatment has been introduced and implemented in the latest European Society of Cardiology recommendations: based on platelet function testing in the chronic phase, it proposes a switch from prasugrel or ticagrelor to clopidogrel for patients in whom low on-treatment platelet reactivity under a newer P2Y12 inhibitor indicates a higher haemorrhagic risk and, consequently, to consider clopidogrel as an alternative strategy, especially for patients with ACS deemed unsuitable for 12 months of potent platelet inhibition [START_REF] Neumann | ESC/EACTS Guidelines on myocardial revascularization[END_REF].

The aim of the AVALANCHE (Analysis of VAsp index after ticagrelor or thienopyridines Loading And mainteNanCe doses to predict HEmorhages) study was to determine whether the VASP index or platelet aggregation results measured at the acute phase could predict bleeding in patients with ACS according to P2Y12 inhibitor.

Methods

Study design

This was a retrospective study on outcome events in patients with ACS treated with DAPT (aspirin and a P2Y12 inhibitor). All consecutive adult patients admitted to the Lariboisière University Hospital (AP-HP, Paris, France) from January 2012 to December 2016 for ST-segment elevation or non-ST-segment elevation myocardial infarction, and who had platelet aggregation tests because of at least one additional atherothrombotic risk factor (diabetes, smoking, age ≥ 65 years, chronic renal failure or multivessel coronary disease) were selected for the study. Patients were included if they were followed by our structure and they (or their relatives) had agreed to participate in a telephone interview. The exclusion criteria were cardiogenic shock, concomitant anticoagulation therapy, use of fibrinolytic drug or glycoprotein IIb/IIIa inhibitors, known serious haemostatic disorder, neoplasia, pregnancy or lactation, participation in another investigational drug study within 30 days, patient under curatorship and patient without social security affiliation.

All patients' clinical and biological baseline characteristics were obtained from the hospital database.

Follow-up was performed using the hospital database (computerized medical file [MiddleCare®.

Evolucare, Villers Bretonneux, France] along with telephone interview of the patient and their referring doctor if necessary. In case of a significant event during follow-up, all medical records available (hospitalization report, biological data, electrocardiogram, etc.) were collected and reviewed by two blinded investigators to determine the classification of the event. Bleeding events were categorized according to the Bleeding Academic Research Consortium (BARC) classification [START_REF] Mehran | Standardized bleeding definitions for cardiovascular clinical trials: a consensus report from the Bleeding Academic Research Consortium[END_REF] and also the Thrombolysis in Myocardial Infarction (TIMI) [START_REF] Rao | Thrombolysis in Myocardial Infarction (TIMI) Trial--phase I: hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase[END_REF] and PLATelet inhibition and patient Outcomes (PLATO) [START_REF] Becker | Bleeding complications with the P2Y12 receptor antagonists clopidogrel and ticagrelor in the PLATelet inhibition and patient Outcomes (PLATO) trial[END_REF]

scales (see Table A.1, Table A.2 and Table A.3). If DAPT was modified or interrupted during follow-up

without any event, the patient's follow-up was considered completed and events were not recorded after DAPT modification. The study was approved by the institutional review board in accordance with national guidelines (IDRCB 2017-A01159-44). All patients provided informed consent.

Antiplatelet drugs

The patients were treated initially with an aspirin loading dose of 250-500 mg intravenously, followed by a daily morning dose of 75 mg of non-enteric coated lysine acetylsalicylate (Kardegic®; Sanofi-Aventis, Paris, France). A loading dose of P2Y12 inhibitor (600 mg clopidogrel, 60 mg prasugrel or 180 mg ticagrelor) was given in the ambulance or just after coronary angiography. A maintenance dose was administered in the morning for clopidogrel (75 mg) or prasugrel (10 mg) and twice a day for ticagrelor (180 mg/day). DAPT was planned for 1 year according to European Society of Cardiology guidelines [START_REF] Ibanez | ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC)[END_REF][START_REF] Roffi | ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC)[END_REF]. The choice of P2Y12 inhibitor and the duration of DAPT was left to the discretion of the practitioner in charge of the patient, and was independent of the present study, which was retrospective.

Platelet function testing

Platelet function assays were assessed during the hospitalization for ACS before discharge as previously described (i.e. between day 2 and day 4) [START_REF] Dillinger | High platelet reactivity on aspirin in patients with acute ST elevation myocardial infarction[END_REF][START_REF] Roffi | ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC)[END_REF]. Compliance was systematically assessed before each blood sampling. All blood samples were taken under fasting conditions, and were performed in the morning at trough level (between 8:00 am and 9:00 am) before ingestion of the morning dose of aspirin (i.e. 24 hours after the last aspirin intake) and P2Y12 inhibitor (i.e. 24 hours after the last intake of clopidogrel or prasugrel, and 12 hours after the last intake of ticagrelor). Blood samples were delivered to the hospital's clinical laboratory within 1 hour.

Flow cytometry for VASP index and platelet activation characterization [22]

Samples of peripheral blood were collected in citrated anticoagulants tubes and processed within the 4 hours after sampling. Flow cytometry measurements were performed on a FACSCanto II flow cytometer, using FACSDiva software (Becton Dickinson, San Jose, CA, USA) for data acquisition and analysis.

VASP phosphorylation was measured by quantitative flow cytometry using a commercially available kit PLT VASP/P2Y12 (ByoCytex, Marseille, France). The VASP index was calculated from the median fluorescence intensity (MFI) of samples incubated with prostaglandin E1 (PGE1) and ADP, according to the formula: VASP index = [(MFI(PGE1)-MFI(PGE1+ADP))/MFI(PGE1)]×100

In addition, platelet-rich plasma diluted 1:40 in phosphate-buffered saline was incubated with anti-Pselectin phycoerythrin-conjugated monoclonal antibody (Clone AC1.2; Becton Dickinson, San Jose, CA, USA). Platelet activation markers were measured in unstimulated platelets and after stimulation of platelets with ADP (50 µmol/L final concentration) and thrombin receptor-activating peptide (TRAP; (70 µmol/L final concentration).

Light transmission aggregometry

Briefly, platelet-rich plasma was obtained by centrifugation of citrated whole blood at 160 g for 12 minutes at 20 °C, and platelet-poor plasma was obtained by further centrifugation at 2300 g for 10 minutes at 20 °C. Ex vivo platelet aggregation was measured at 37 °C within 1 hour following sampling, using an aggregometer (Model 490-4D; CHRONO-LOG Corporation, Kordia, the Netherlands) following the optical aggregometry method of Born [START_REF] Born | Aggregation of blood platelets by adenosine diphosphate and its reversal[END_REF]. To assess the biological response of the P2Y12 inhibitor, light transmission aggregometry (LTA) was induced by the addition of ADP at final concentrations of 5 µM, 10 µM (not shown) and 20 µM. Aggregation was expressed as the maximum aggregation intensity as well as the final aggregation extent (measured 6 minutes after ADP activation) using the autologous platelet-poor plasma as a reference [START_REF] Dillinger | High platelet reactivity on aspirin in patients with acute ST elevation myocardial infarction[END_REF][START_REF] Dillinger | Monitoring persistent platelet reactivity in patients with unprotected left main stenting[END_REF].

To assess the biological response to aspirin, platelet-rich plasma LTA triggered by arachidonic acid (AA) at concentrations of 0.8 µM (0.25 mg/mL) and 1.6 µM (0.5 mg/mL) was performed [START_REF] Dillinger | Biological efficacy of twice daily aspirin in type 2 diabetic patients with coronary artery disease[END_REF].

Follow-up of bleeding and ischaemic outcomes

Bleeding events were recorded chronologically starting from the ACS hospitalization, and followed throughout the time that the patient was on the DAPT regimen under which the platelet function studies were performed. Bleeding events were classified according to BARC, TIMI and PLATO scales [START_REF] Mehran | Standardized bleeding definitions for cardiovascular clinical trials: a consensus report from the Bleeding Academic Research Consortium[END_REF][START_REF] Rao | Thrombolysis in Myocardial Infarction (TIMI) Trial--phase I: hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase[END_REF][START_REF] Becker | Bleeding complications with the P2Y12 receptor antagonists clopidogrel and ticagrelor in the PLATelet inhibition and patient Outcomes (PLATO) trial[END_REF].

Statistical methods

The aim of the study was to determine for each P2Y12 inhibitor the optimal VASP index cut-off to predict bleeding (BARC score 2-5) in patients with ACS, using a receiver operating characteristic (ROC) curve model and calculation of Youden's J statistic [START_REF] Hajian-Tilaki | Receiver Operating Characteristic (ROC) Curve Analysis for Medical Diagnostic Test Evaluation[END_REF]. The optimal cut-off level was calculated by determining the smallest distance between the ROC curve and the upper left corner of the graph. ROC curves were also performed for other bleeding scales (PLATO, TIMI). Continuous variables were analysed for normal

Results

Cohort characteristics

The present study included 364 patients with ACS from January 2012 to December 2016. Among them, 123 were treated with ticagrelor, 105 with prasugrel and 136 with clopidogrel. An invasive approach was used in all patients. Patients with an ST-segment elevation myocardial infarction were admitted directly to the catheterization laboratory, and all patients with a non-ST-segment elevation myocardial infarction underwent a coronary angiography within 48 hours and most often within 24 hours after admission.

Platelet function testing (VASP index and aggregation tests) was performed on day 3 ± 1 day. The median follow up was 2.6 years (IQR 1.9-3.3 years). The clinical characteristics are presented in Table 1. Briefly, 297 patients (81.6%) were male, the mean age was 64 ± 11 years and 40.9% had diabetes. Seventeen patients were excluded because DAPT was modified during follow-up: 11 were no longer on clopidogrel at the time of the event (four were on aspirin only, three switched to prasugrel and four switched to ticagrelor); four patients on ticagrelor were switched to another treatment (one to clopidogrel, two to anticoagulant and one to aspirin only); finally, two patients on prasugrel were switched to another treatment (one to clopidogrel and one to aspirin only).

Platelet function tests

Spontaneous platelet activation

Spontaneous platelet activation measured by platelet P-selectin expression was similar in the three groups: 6% (IQR 4-9%) in the clopidogrel group; 5% (IQR 3-8%) in the prasugrel group; and 5% (IQR 3-7%) in the ticagrelor group (P = 0.38).

VASP index

Overall, the median VASP index was 22% (IQR 10.0-39.4%). The VASP index was lower in patients on ticagrelor (14.0%, IQR 7.8-23.0%) or prasugrel (13.9%, IQR 7.0-25.0%) than in those on clopidogrel (42.0%, IQR 25.0-64.0%) (P < 0.001) (Table 2 and Fig. 1). Of note, a greater interindividual variability of response was observed in patients on clopidogrel compared with in those on prasugrel or ticagrelor.

Platelet aggregation measured by LTA induced by ADP

Overall, the median maximal intensity of platelet aggregation measured by LTA induced by ADP 20 µM was 50% (IQR 34-70%); it was lower in patients on ticagrelor (37%, IQR 29-56%) or prasugrel (45%, IQR 30-60%) than in those on clopidogrel (69%, IQR 55-81%) (P < 0.001). The median residual intensity of platelet aggregation induced by ADP 20 µM at 6 minutes was 39% (IQR 15-55%); it was lower in patients on ticagrelor (20%, IQR 10-44%) or prasugrel (25%, IQR 5-47%) than in those on clopidogrel (50%, 36-65%) (P < 0.001). Consistent results were obtained for LTA induced by ADP 5 µM (Table 2).

Platelet aggregation measured by LTA induced by AA

No significant differences were observed between the biological responses to aspirin in the three subgroups, whatever the AA concentration used to induce aggregation. For LTA triggered by AA 0.8 µM tests, the median maximal intensity of platelet aggregation was 5% (IQR 0-10%) on ticagrelor, 5% (IQR 0-7%) on prasugrel and 4% (IQR 0-8%) on clopidogrel. For LTA triggered by AA 1.6 µM, the median maximal intensity of platelet aggregation was 12% (IQR 5-16%) on ticagrelor, 9% (IQR 6-16%) on prasugrel and 10% (IQR 4-17%) on clopidogrel (Table 2).

Follow-up

Seventy-seven haemorrhagic events were recorded: 28 concerning patients on ticagrelor, 18 on prasugrel and 31 on clopidogrel (Table 3). On the BARC scale, 36.3% of these events were rated equal to or greater than 2 out of 5, which corresponds to 7.7% of the total population. The delay between platelet function testing and the occurrence of the event was 10 months on average.

Platelet function tests and bleeding

Whatever the definition of bleeding that we explored (BARC score ≥ 2, TIMI and PLATO scales), there was no significant relationship between inhibition of platelet function (VASP index and ADP-induced platelet aggregation) and the subsequent occurrence of bleeding events (Fig. 2).

Discussion

Baseline cohort characteristics were similar to those in previous ACS studies [START_REF] Wallentin | Ticagrelor versus clopidogrel in patients with acute coronary syndromes[END_REF][START_REF] Wiviott | Prasugrel versus clopidogrel in patients with acute coronary syndromes[END_REF]. It should be noted that the proportion of patients with diabetes was relatively high (40.9 %). VASP index and LTA triggered by ADP 20 µM have been widely used by our team and others for platelet function testing under P2Y12 inhibitors [START_REF] Dillinger | High platelet reactivity on aspirin in patients with acute ST elevation myocardial infarction[END_REF][START_REF] Kerneis | Platelet effect of prasugrel and ticagrelor in patients with STsegment elevation myocardial infarction[END_REF][START_REF] Lordkipanidze | Comparison of four tests to assess inhibition of platelet function by clopidogrel in stable coronary artery disease patients[END_REF]. Median VASP indexes as well as ADP-induced maximal and residual intensities of platelet aggregation were significantly decreased in patients on ticagrelor or prasugrel compared with in those on clopidogrel, and the median values were in accordance with results from previous studies [START_REF] Bonello | Ticagrelor increases adenosine plasma concentration in patients with an acute coronary syndrome[END_REF][START_REF] Kerneis | Platelet effect of prasugrel and ticagrelor in patients with STsegment elevation myocardial infarction[END_REF][START_REF] Laine | COMparison of Platelet reactivity following prAsugrel and ticagrelor loading dose in ST-Segment elevation myocardial infarctION patients: The COMPASSION study[END_REF].

During the follow-up, the occurrence of bleeding was frequent: approximately 20% of patients reported haemorrhage on DAPT; overall, the occurrence of BARC 2-5 bleeding was 7.7%. Using TIMI major or minor score, the bleeding rate in our study lies between those in the TRITON and PLATO studies [START_REF] Wallentin | Ticagrelor versus clopidogrel in patients with acute coronary syndromes[END_REF][START_REF] Wiviott | Prasugrel versus clopidogrel in patients with acute coronary syndromes[END_REF]. So, despite the retrospective study design, the rate of events collected during the follow-up was in accordance with those in the literature.

Using ROC curves, the accuracy of the VASP index or platelet aggregation intensities at the acute phase to predict bleeding was poor, whatever the P2Y12 inhibitor used. These results conflict with previous studies showing that the lower the VASP index, the more bleeding occurs [START_REF] Tantry | Consensus and update on the definition of on-treatment platelet reactivity to adenosine diphosphate associated with ischemia and bleeding[END_REF]. Bonello et al. demonstrated that, in patients treated with thienopyridines, the prevalence of bleeding was 15% in those with VASP ≤ 15% vs 3-4% in patients with VASP > 15% [START_REF] Bonello | Relationship between post-treatment platelet reactivity and ischemic and bleeding events at 1-year follow-up in patients receiving prasugrel[END_REF]. According to Cuisset et al., the prevalence of bleeding in patients on thienopyridines was 33% when VASP was ≤ 10% vs 8% when VASP was > 10% [START_REF] Cuisset | Clinical implications of very low on-treatment platelet reactivity in patients treated with thienopyridine: the POBA study (predictor of bleedings with antiplatelet drugs)[END_REF]. Thus, the bleeding odds ratio was multiplied by 4.7 (95% confidence interval: 2.7-8.3) when the VASP index showed low on-treatment platelet reactivity. However, adaptation of DAPT according to aggregation test results does not modify patient prognosis. In the GRAVITAS study, bad responders had a worse prognosis than those who had an adapted pharmacodynamic response to clopidogrel. Nonetheless, the use of high-dose clopidogrel among patients with high on-treatment platelet reactivity after percutaneous coronary intervention (PCI) did not reduce the incidence of death from cardiovascular causes, non-fatal myocardial infarction or stent thrombosis [START_REF] Price | Standard-vs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial[END_REF]. Similarly, the ANTARCTIC study did not demonstrate that adjusting antiplatelet therapy according to platelet function monitoring improved clinical outcomes in patients undergoing PCI [START_REF] Cayla | Platelet function monitoring to adjust antiplatelet therapy in elderly patients stented for an acute coronary syndrome (ANTARCTIC): an open-label, blindedendpoint, randomised controlled superiority trial[END_REF]. In the TRIGGER PCI trial, switching from clopidogrel to prasugrel in stable patients with high on-treatment platelet reactivity afforded effective platelet inhibition without a difference in the outcomes rate. However, the study was stopped prematurely for futility because of the low rate of adverse ischaemic events after PCI with contemporary drug-eluting stents in stable coronary artery disease [START_REF] Trenk | A randomized trial of prasugrel versus clopidogrel in patients with high platelet reactivity on clopidogrel after elective percutaneous coronary intervention with implantation of drug-eluting stents: results of the TRIGGER-PCI (Testing Platelet Reactivity In Patients Undergoing Elective Stent Placement on Clopidogrel to Guide Alternative Therapy With Prasugrel) study[END_REF]. Other studies have aimed to adjust the therapeutics to the genetic profile of patients or to perform de-escalation of antiplatelet therapy, without proving any benefit [START_REF] Collet | Genetic and platelet function testing of antiplatelet therapy for percutaneous coronary intervention: the ARCTIC-GENE study[END_REF].

In the present retrospective study, we sought to determine a VASP index cut-off that could predict increased bleeding risk in patients treated with a P2Y12 inhibitor and, more specifically, to establish whether the threshold was similar for ticagrelor compared with thienopyridines. The rationale is that ticagrelor is known to induce a more profound decrease in the VASP index than thienopyridines, even a more profound decrease than prasugrel, without evidence of comparatively increased bleeding risk. The two main conclusions of this retrospective study are: (1) that the bleeding rate was similar with the three P2Y12 inhibitors, even though the VASP index was lower with prasugrel compared with clopidogrel and lower with ticagrelor compared with prasugrel; and (2) that in the overall cohort, the occurrence of bleeding during DAPT was not significantly related to the VASP index measured at the initial phase.

Moreover, for each of P2Y12 inhibitor individually, no relationship between ex vivo platelet inhibition and subsequent bleeding incidence was observed.

To explain the first point, we can hypothesize that patients treated with clopidogrel are those at higher bleeding risk; so, they are more likely to bleed with less platelet inhibition. Additionally, we put forward the hypothesis that ticagrelor could decrease the VASP index by two alternative pathways: the VASP index of patients treated with ticagrelor is lower without meaning more profound platelet reactivity inhibition, explaining why patients treated with ticagrelor do not experience more bleeding complications than patients treated with prasugrel, even if their VASP index is lower, but we admit that this hypothesis is controversial [START_REF] Bonello | Ticagrelor increases adenosine plasma concentration in patients with an acute coronary syndrome[END_REF].

To explain the absence of association between the VASP index and bleeding complications, even for P2Y12 inhibitors taken individually, the mean delay in occurrence of bleeding was 10 months in the overall cohort, which is a very late occurrence. The VASP index was assessed in the ACS acute phase. At this time, the degree of platelet inhibition could be more related to the efficacy of the platelet inhibitor (prevention of ischaemic events), and the platelet reactivity is modified both by platelet stimulation as a result of the clinical situation in the acute phase and by the effect of the antiplatelet treatment. Later in the evolution, the patient's platelet reactivity returns to baseline, and the effect of the inhibitor is probably more related to haemorrhagic risk. As in many other trials evaluating the association between platelet function monitoring and clinical events, we did not pay enough attention to this point, and our lack of association between clinical and laboratory data probably reflects this major point. We should consider early platelet testing as a test for efficacy and late platelet testing (e.g. at 1 month) as a test for safety. At present, platelet function tests cannot be proposed routinely as they are not cost-effective, even using the tests that are considered the gold standard, which are time consuming.

Study limitations

Our study has several limitations. First, the main limitation is the retrospective design. Secondly, it is possible that we were faced with a lack of statistical power, especially in the clopidogrel group. However, the bleeding rate during follow-up appeared to be what we expected. Further studies are needed to definitively determine VASP index cut-off values to predict bleeding in patients with coronary artery disease, including both early and late evaluations of platelet function.

Conclusions

This study shows that platelet function testing in the acute phase failed to predict bleeding events in patients with ACS and treated with P2Y12 inhibitors, whatever the drug. We could consider early platelet function testing as a test for efficacy and late platelet function testing as a test for safety. Data are expressed as number (%). VASP: vasodilator-stimulated phosphoprotein phosphorylation.

Table A.3

Thrombolysis in Myocardial Infarction (TIMI) bleeding scale [START_REF] Rao | Thrombolysis in Myocardial Infarction (TIMI) Trial--phase I: hemorrhagic manifestations and changes in plasma fibrinogen and the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase[END_REF].

Major

Any intracranial bleeding (excluding microhaemorrhages < 10 mm evident only on gradient-echo MRI)

Clinically overt signs of haemorrhage associated with a drop in haemoglobin of ≥ 5 g/dL or a ≥ 15% absolute decrease in haematocrit Fatal bleeding (bleeding that directly results in death within 7 days)

Minor Clinically overt (including imaging), resulting in haemoglobin drop of 3 to < 5 g/dL or a ≥ 10% decrease in haematocrit

No observed blood loss: ≥ 4 g/dL decrease in the haemoglobin concentration or ≥ 12% decrease in haematocrit Any overt sign of haemorrhage that meets one of the following criteria and does not meet criteria for a major or minor bleeding event, as defined above: Minimal Any overt bleeding event that does not meet the criteria above Any clinically overt sign of haemorrhage (including imaging) associated with a < 3 g/dL decrease in haemoglobin concentration or < 9% decrease in haematocrit MRI: magnetic resonance imaging.

  Abbreviations: AA, arachidonic acid; ACS, acute coronary syndrome; ADP, adenosine diphosphate; BARC, Bleeding Academic Research Consortium; DAPT, dual antiplatelet therapy; IQR, interquartile range; LTA, light transmission aggregometry; MFI, median fluorescence intensity; PCI, percutaneous coronary intervention; PGE1, prostaglandin E1; PLATO, PLATelet inhibition and patient Outcomes; ROC, receiver operating characteristic; TIMI, Thrombolysis in Myocardial Infarction; VASP, vasodilatorstimulated phosphoprotein phosphorylation.

  distribution with the D'Agostino-Pearson test. Continuous variables are presented as means and standard deviations if normally distributed and were compared with Student's unpaired t test; if not normally distributed, they are presented as median (interquartile range [IQR]), and were compared with the Mann-Whitney rank-sum test. Categorical variables are presented as counts and percentages and were compared by means of the χ 2 test or Fisher's exact test. Comparison of continuous variables between P2Y12 inhibitor groups was performed with a one-way analysis of variance. A P value < 0.05 was considered statistically significant. Statistical analyses were performed with the use of MedCalc Statistical Software (MedCalc Software bv, Ostend, Belgium).

Figure 1 .

 1 Figure 1. Vasodilator-stimulated phosphoprotein (VASP) index (%) distribution according to P2Y12 inhibitor.

Figure 2 .

 2 Figure 2. Receiver operating characteristic curves to predict bleeding (PLATelet inhibition and patient Outcomes [PLATO] minor/major score) according to the vasodilator-stimulated phosphoprotein (VASP) index. AUC: area under the curve.

Figure 2

 2 Figure 2.

  a. Requiring intervention (medical practitioner-guided medical or surgical treatment to stop or treat bleeding, including temporarily or permanently discontinuing or changing the dose of a medication or study drug) b. Leading to or prolonging hospitalization c. Prompting evaluation (leading to an unscheduled visit to a healthcare professional and diagnostic testing, either laboratory or imaging)

Table 1

 1 Baseline clinical characteristics of patients, overall and according to P2Y12 inhibitor.

	All patients	Ticagrelor	Prasugrel	Clopidogrel	P
	(n = 364)	(n = 123)	(n = 105)	(n = 136)

a 96.7%.

Table 2

 2 Baseline laboratory data of patients, overall and according to P2Y12 inhibitor.

	All patients	Ticagrelor	Prasugrel	Clopidogrel	P
	(n = 364)	(n = 123)	(n = 105)	(n = 136)

Table 3

 3 Bleeding events according to the Bleeding Academic Research Consortium (BARC), PLATelet inhibition and patient Outcomes (PLATO) and Thrombolysis in

	Myocardial Infarction (TIMI) scales.
		Overall			Ticagrelor			Prasugrel			Clopidogrel
		Total	VASP <	VASP ≥	Total	VASP <	VASP ≥	Total	VASP <	VASP ≥	Total	VASP <	VASP ≥
			16%	16%		16%	16%		16%	16%		16%	16%
		(n = 364) (n = 150)	(n = 214)	(n = 123) (n = 76)	(n = 47)	(n = 105) (n = 58)	(n = 47)	(n = 136) (n = 16)	(n = 120)
	BARC ≥ 1	77 (20.1)	23 (15.3)	54 (25.2)	28 (22.8) 10 (13.2)	18 (38.3)	18 (17.1)	10 (17.2)	8 (17.0)	31 (22.8) 3 (18.8)	28 (23.3)
	BARC ≥ 2	28 (7.7)	7 (4.7)	21 (9.8)	11 (8.9)	3 (3.9)	8 (17.0)	7 (6.7)	3 (5.2)	4 (8.5)	10 (7.4)	1 (6.3)	9 (7.5)
	BARC ≥ 3	16 (4.4)	3 (2.0)	13 (6.1)	7 (5.7)	0 (0.0)	7 (14.9)	2 (1.9)	2 (3.4)	0 (0.0)	7 (5.1)	1 (6.3)	6 (5.0)
	BARC ≥ 4	2 (0.05)	-	-	2 (1.6)	-	-	0 (0)	-	-	0 (0)	-	-
	BARC = 5	1 (0.02)	-	-	1 (0.8)	-	-	0 (0)	-	-	0 (0)	-	-
	PLATO	77 (20.1)	23 (15.3)	54 (25.2)	28 (22.8) 10 (13.2)	18 (38.3)	18 (17.1)	10 (17.2)	8 (17.0)	31 (22.8) 3 (18.8)	28 (23.3)
	Minor or major	27 (7.4)	7 (4.7)	20 (9.3)	10 (8.1)	3 (3.9)	7 (14.9)	7 (6.7)	3 (5.2)	4 (8.5)	10 (7.4)	1 (6.3)	9 (7.5)
	PLATO
	TIMI	77 (20.1)	23 (15.3)	54 (25.2)	28 (22.8) 10 (13.2)	18 (38.3)	18 (17.1)	10 (17.2)	8 (17.0)	31 (22.8) 3 (18.8)	28 (23.3)
	Minor or major	27 (7.4)	7 (4.7)	20 (9.3)	10 (8.1)	3 (3.9)	7 (14.9)	7 (6.7)	3 (5.2)	4 (8.5)	10 (7.4)	1 (6.3)	9 (7.5)
	TIMI

Sources of funding

This study was supported by AstraZeneca, France.

Disclosure of interest