Géraldine Maynaud

Céline Druilhe

Mylène Daumoin

Julie Jimenez

Dominique Patureau

Michel Torrijos

Anne-Marie Pourcher

Nathalie Wéry
email: nathalie.wery@inra.fr

Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter

Keywords: digestate, biodegradability, complexity, anaerobic digestion, respirometry Anaerobic digestion Organic fraction of municipal solid waste

The stability of digestate organic matter is a key parameter for its use in agriculture.

Here, the organic matter stability was compared between 14 post-treated digestates and the relationship between organic matter complexity and biodegradability was highlighted. Respirometric activity and CH 4 yields in batch tests showed a positive linear correlation between both types of biodegradability (R 2 =0.8). The accessibility and complexity of organic matter were assessed using chemical extractions combined with fluorescence spectroscopy, and biodegradability was mostly anti-correlated with complexity of organic matter. Post-treatments presented a significant effect on the biodegradability and complexity of organic matter. Biodegradability was low for composted digestates which comprised slowly accessible complex molecules. Inversely, solid fractions obtained after phase separation contained a substantial part of remaining biodegradable organic matter with a significant easily accessible fraction comprising

Introduction

Anaerobic digestion (AD) is a biological treatment that converts organic waste into methane (CH 4 ). It can thus produce energy, while the digestate is regarded as a potential organic fertilizer. The digestate is a fully fermented nutrient-rich material which can ideally replace inorganic fertilizer (Tambone et al., 2010). Many studies focus on the optimization of CH 4 production from organic waste but rarely on the assessment of the agronomic value of digestate and its valorisation through land application (Teglia et al., 2010). At times, when digestate is not fully stabilized, it can present residual biodegradability and contain complex organic elements such as lignocellulosic compounds which the digestion process cannot degrade (Bayard et al., 2015).

Consequently, post-treatments such as solid-liquid separation, drying or composting, are generally recommended before land application and the fertilizer produced should also fulfil current standards, i.e. efficiency and safety (Teglia et al., 2011).

To be used as an organic fertilizer, stability or biodegradability of a digestate has to be fully assessed prior to land application. The biodegradability of organic residues Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 4 can be evaluated using (i) biological methods including aerobic respirometric tests and anaerobic test methods (Binner and Zach, 1999;Wagland et al., 2009) and (ii) nonbiological methods including dry matter, organic matter (OM) and total organic carbon content (Godley et al., 2004). The use of the biomethane potential test (BMP) aims at assessing the biogas potential of both organic waste and digestate. It is the most common method applied for measuring the anaerobic biodegradability of organic residues [START_REF] Schievano | Predicting anaerobic biogasification potential of ingestates and digestates of a full-scale biogas plant using chemical and biological parameters[END_REF]. However, the BMP test protocol can vary from one laboratory to another [START_REF] Raposo | Biochemical methane potential (BMP) of solid organic substrates: Evaluation of anaerobic biodegradability using data from an international interlaboratory study[END_REF] thus leading to large discrepancies. It is also a time-consuming method (>30 days). For these reasons, alternative approaches such as respirometric tests have been considered (Cossu and Raga, 2008; [START_REF] Scaglia | Estimating biogas production of biologically treated municipal solid waste[END_REF].

These aerobic methods are based on the oxygen (O 2 ) measurement uptake or carbon dioxide (CO 2 ) production which are related to OM biodegradation under controlled conditions. As organic residues can either be directly applied on land or stored prior to their application, they can be exposed to both aerobic and anaerobic environments. An assessment of their biodegradability under aerobic and anaerobic conditions thus provides complementary information on their characteristics. Furthermore, correlations between aerobic and anaerobic tests have been reported by several authors, especially for organic waste before its biological treatment, such as AD processes or composting The objectives of this study are (i) to investigate the biodegradability under both anaerobic and aerobic conditions of 14 post-treated digestates (seven composted or dried solid fractions of digestate and seven solid fractions of digestate obtained after

Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 5 liquid-solid separation) and (ii) to characterize their OM in order to better assess the impact of post-treatments on their characteristics and stability. In this view, a recent method developed to characterize both the accessibility and complexity of organic wastes (Jimenez et al., 2015) has been applied. This method combines successive chemical fractionations to 3D fluorescence spectroscopy. The final goal was correlating biodegradability and OM characterization and describing the effect of post-treatment on the digestate stability. This could help to define better strategies for agricultural re-use of these organic residues.

Material and methods

Sampling of digestates

Fourteen digestates were sampled from 13 AD sites in France: 7 solid fractions of digestate (SL1S, SL2S, A1S, T1S, T2S, T3S, T4S) obtained following solid-liquid phase separation, 1 dried and composted solid fraction of digestate (A2D) and 6 composted solid fractions of digestate (A3C, A4C, SL1C, O1C, O2C, M1C). The AD sites treated various types of waste, including agricultural waste, sewage sludge from a wastewater treatment plant (WWTP), municipal solid waste (MSW), the organic fraction of MSW (OFMSW), food processing waste (FPW), all according to different digestion processes (wet or dry, mesophilic or thermophilic). The designation of the digestates and their origins are presented in Table 1. All of the samples collected (about 50 kg each) were homogenized for further analyses.

Physico-chemical characterisation of digestates

Residual biodegradability assays

Aerobic biodegradability assay (BDaero)

The aerobic biodegradability was measured on fresh digestates (between 2 and 4 kg according to the bulk density) using a dynamic respirometric method (Berthe et al.,

2007

). The respirometric device consisted of 10 L hermetically sealed stainless steel cells. These were filled with the studied substrates mixed with plastic Pall rings acting as inert bulking agent. Each cell was maintained at 40 °C by means of a water bath and was supplied with a continuous air flow rate of 70 L h -1 . The incoming air was preheated to the water bath temperature while the aeration system also included a rapid recirculation of part of the exhaust air (360 L h -1 ), thus ensuring homogeneous conditions throughout the substrate. In order to measure the O 2 uptake rate (OUR) during the respirometric experiment, the oxygen concentrations of the inlet and outlet gases were monitored every 2 min using a paramagnetic analyser (Magnos 206, ABB, Zurich, Switzerland). The respirometric test was finally halted when the O 2 uptake rate fell to a low and stable value. phase of the first, second and third peak, respectively. Following the previously described calculations, MBi and MHi were expressed in %BDaero.

Anaerobic biodegradability assay (BDana)

An anaerobic batch test was used to quantify the residual CH 4 production in digestate.

This was measured on fresh digestate without inoculation for 40 days using the following protocol: 200 g of fresh digestate were suspended in 200 mL of sodium bicarbonate solution (2.5 g L -1 ) in 500 mL flasks. To ensure anaerobic conditions, the solution in flask was flushed with N 2 for a few seconds. The flasks were sealed with gas-tight rubber stoppers and aluminium seals and incubated at 37 °C for 40 days in the dark and under a constant shaking. Each digestate was tested in duplicate. Biogas production and gas composition were estimated twice a week using a water displacement method and gas chromatography (Perkin Elmer Clarus®580), respectively. The CH 4 yield was calculated using the experimental results and expressed in NL CH 4 kg

Fluorescence spectroscopy analysis

Comment

ܸ ሺiሻሺUA mg COD L ିଵ ሻ = ሺ୧ሻ େୈ ౩ౣ౦ౢ × 1 ୗሺ୧ሻ ∑ ୗሺ୧ሻ ళ సభ ൘ (3) ܲ ሺiሻሺ%ሻ = ሺ୧ሻ ∑ ሺ୧ሻ ళ సభ × 100 (4)
Where:

V f (i) (U.A. mg COD L -1 ): the raw volume of the zone i, COD sample (mg L -1 ): the COD concentration of the sample, S(i) (nm²): the area of a zone i, P f (i) (%): the fluorescence proportion of a zone i.

Statistical analysis

Correlations between the CH 4 yield and each of the parameters obtained from respirometry (BOD, AT 4 and DRI 24 ), and between anaerobic (BDana) and aerobic (BDaero) biodegradabilities were investigated using linear regression evaluation. The analytical data were processed using principal component analysis (PCA) to identify multiple correlations between variables including BDana, BDaero, MBi and MHi (i=1,

Comment

Results and discussion

Characterization of digestates

Table 2 summarizes the physico-chemical and biological properties of digestates. Anaerobic and aerobic biodegradabilities were calculated with the CH 4 yield and BOD, respectively, and with respect to CODtot content of digestate.

The TS content ranged between 40.9-74.6 % for dried or composted solid digestate fractions, the highest value being observed for the dried sample A2D. In solid digestate fractions the TS content varied between 22.3-26.4 %. The VS ranged between 39.3-86.5

% TS and the CODtot between 1245-1696 gO 2 kg -1 VS in all the digestates, without any notable differences related to the applied post-treatment.

The calculated aerobic and anaerobic biodegradabilities (BDaero and BDana)

presented differences amongst digestates depending on post-treatment applied. Indeed,

composted

Correlation between both aerobic and anaerobic biodegradabilities

Fig. 1 illustrates linear positive correlations between anaerobic and aerobic biodegradabilities (BDana, BDaero) and between CH 4 yield and parameters obtained from respirometry data (BOD, AT 4 and DRI 24 ) for the 14 post-treated digestates.

Concerning the anaerobic biodegradability, the mean value of the CH 4 yield measured in duplicate was chosen for Fig. 1 because values indicated a good reproducibility (coefficient of variation <15 %, data not shown).

Significant positive correlations were obtained between BDana and BDaero and between CH 4 yield and BOD (R 2 =0.8), whereas the correlations between CH 4 yield and AT 4 and between CH 4 yield and DRI 24 were less significant (R 2 of 0.7 and 0.6, respectively). This was due to the underestimation of the aerobic biodegradability of one digestate, A1S (point surrounded on Fig. 1b and Fig. 1c), using AT 4 and DRI 24 . Indeed, this sample presented the slowest respirometric kinetic, with an oxygen uptake rate of about 40 mmO 2 h -1 kg -1 VS after 200 h, and three clear OUR peaks (Fig. 3b).

Short-term aerobic indices such as AT 4 and DRI 24 appeared to be insufficient to represent the overall biodegradability of this type of substrate still highly biodegradable and with a high activity level for a longer period of time. The biodegradability is, in this case, better estimated with the BOD value.

A significant positive correlation between both aerobic and anaerobic

Relation between biodegradability, accessibility and complexity of OM

The full dataset was analysed though PCA in order to highlight correlations between the aerobic and anaerobic biodegradabilities (BDana and BDaero), the accessibility of OM and its complexity, obtained by combining chemical extractions and fluorescence spectroscopy. The two major components, PCA1 and PCA2, were sufficient to describe 76.3 % of the data variability (Fig. 2). The PCA1 essentially anaerobic tests; it could therefore be applied to evaluate the biodegradability and thus the status of digestate as a relatively stabilized fertilizer.

Kinetics of respirometric activity

Only the respirometric kinetics have been taken into account in the results.

Indeed, the O 2 uptake rate was measured more frequently (every 2 min) than the CH 4 production rate (twice a week). During the respirometric experiments, the OUR kinetics showed various profiles according to the studied digestate (Fig. 3). Nine digestates, including all the dried and composted solid fractions of digestate, were characterized by a profile with only one peak of O 2 consumption, followed by a decrease of activity until endogenous respiration (Fig. 3a). These profiles were classical on solid organic wastes, such as municipal waste [START_REF] Adhikari | Biodegradability of municipal organic waste: a respirometric test[END_REF] or mixtures of sludge and bulking agent (Mohajer et al., 2009). At the opposite, five digestates showed atypical profiles with a second (SL1S, T1S, T2S, T3S and A1S) and a third OUR peak (T3S and A1S) before reaching the endogenous respiration (Fig. 3b). Atypical OUR kinetics could be explained by combining two phases of OM biodegradation according to (i) the OM accessibility and/or (ii) the released OM components, that both changed with time.

The applied AD process (e.g., temperature and retention time) and the type of treated waste could also impact the OM composition of the obtained digestate as well as its respirometric profile. Atypical kinetics with two OUR peaks have already been demonstrate that the type of digestion process and the nature of treated waste do not seem to explain the observed atypical kinetics of respirometric activity.

In order to understand the appearance of two or three OUR peaks, a PCA was carried out on the different parameters describing the OM accessibility, its complexity and the fractionation of BDaero into MBi and MHi (i=1, 2 or 3) according to the number of OUR peaks (calculations described in 2.3.1) (Fig. 4). Two components, PCA1 and PCA2, were sufficient to describe 67.4 % of the data variability. Variables describing the PCA1 axis were MB1 which characterized the easily biodegradable OM linked to the first OUR peak, and the fluorescence-related complexity of each accessible fraction (zones 1-7). As for the previous results, the variable describing the PCA2 axis was the chemical accessibility of OM. MB1 was positively correlated with the simpler molecules of OM fractions (Fig. 4a and R 2 of the correlation between MB1 and fluorescence zones 1-3 of SPOM+REOM+SEOM = 0.5-0.7, p-values<0.05, data not shown). Even with the data description using the other PCA axes, the other MBi and MHi axes were not significantly correlated with the accessibility and/or the complexity (data not shown). Moreover, axes of PCA led to the separation from other digestates of four out of the five digestates presenting atypical kinetics (Fig. 4b). The parameters then used to describe the OM in this study were not sufficient and/or relevant to explain the observed atypical OUR kinetics. Further work is still required for a better comprehension of the observed multiple peaks. For example, during the respirometric test, enzymatic activities and bacterial and fungal community composition (by highthroughput sequencing analysis) could also contribute to the evolution of the nature of the OM. Moreover, the aqueous compartment of OM could be characterized in terms of apparent molecular weight by size exclusion chromatography.

Conclusions

Solid fractions were found to comprise a significant part of remaining biodegradable OM despite the digestion process. Composted solid fractions were characterized by low biodegradable OM containing complex molecules, thus implying the presence of a more stable OM. The relation between complexity and biodegradability that has been demonstrated in this study has allowed for the effect of post-treatments on the stability of digestates to be better understood. Consequently, these data on OM stability should contribute to the improvement of agricultural re-use of digestates as fertilizers.

An example of decomposition of a spectra obtained by fluorescence spectroscopy analysis into seven zones of fluorescence is presented in the Supplementary Figure 1.

Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. • Biodegradability is mostly anti-correlated with the of organic matter

• Post-treatments have a significant effect on biodegradability and complexity

• Kinetics of respirometric activity present multi-peaks for certain digestates

(

  Cossu and Raga, 2008; Ponsá et al., 2008; Barrena et al., 2009; Bohm et al., 2010; Liu et al., 2015). However, information is scarce concerning such correlations on digestates.

7

  Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057The biological oxygen demand (BOD) of the digestate corresponded to the cumulative O 2 uptake during the test. Two classical aerobic indices were also calculated for each substrate: AT 4 related to the cumulative O 2 uptake in four days and DRI 24 related to the average OUR in the 24 h of maximum activity (Ponsa et al., 2010). Moreover, the aerobic biodegradability (BDaero) could be expressed as the ratio between BOD and CODtot, according to Equation 1(Liu et al., 2015).BDaero(%CODtot) = (BOD(gO 2 kg -1 VS) x 100) / CODtot(gO 2 kg -1 VS) (1)A conceptual approach of typical OUR profiles has been described by Trémier et al. (2005): firstly (i) a rise to a peak value, OURmax, associated with the biodegradation of the easily biodegradable OM (MB) and biomass growth, secondly (ii) a break in the exponential growth and OUR decrease assumed to correspond to the biodegradation of the slowly biodegradable OM (MH) that has to be be hydrolysed before biological consumption, and thirdly (iii) the stabilization of OUR at a low and constant value associated with the disappearance of all biodegradable OM and the endogenous respiration of the biomass. On the basis of these previous assumptions, a simplified estimation of MB and MH could be carried out: the MB value represented the cumulative O 2 consumption during the rising phase, until OURmax, while the MH value characterised the cumulative O 2 uptake during the decrease phase, from OURmax to endogenous respiration. During this study, different OUR profiles were obtained: typical profiles with a single OUR peak, atypical profiles with two or three O 2 uptake peaks. A simplified estimation of MB and MH was then performed for each separated peak: the MB1, MB2 and MB3 values represented the cumulative O 2 consumption during the rising phase of the first, second and third peak respectively, and the MHi values (i=1, 2 or 3) represented the cumulative O 2 consumption during the decreasing Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 8

  citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 10 Similar to the procedure developed by Jimenez et al. (2014 and 2015), 3D fluorescence spectroscopy was applied to the extracted fractions, from which OM complexity could be quantified. The fluorescence spectrometer used was a Perkin Elmer LS55. Excitation wavelengths varied from 200 to 600 nm with 10 nm increments. According to Jimenez et al. (2014) and Muller et al. (2014), the spectra were decomposed into seven zones corresponding to biochemical fluorescence family-types. The proportion of fluorescence in a given zone "i" P ሺiሻ was calculated from the fluorescence zone volumes V f (i) according to Equations 3 and 4. An example of spectra decomposed into seven fluorescence zones is presented in the Supplementary figure 1.

  citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 11 2 or 3), the complexity related to fluorescence and the %CODtot of extracted OM fractions and NEOM fraction. A Pearson correlation matrix was performed in order to test for correlations between anaerobic and aerobic biodegradabilities and %CODtot of each extracted fraction. Statistical analysis was performed using the Rcmdr package of R-Software (R Foundation for Statistical Computing, Vienna, Austria).

  Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 14

  biodegradabilities and biological indices has been observed in previous studies considering raw waste or organic residues during composting treatment (Binner and Zach, 1999; Cossu and Raga, 2008; Barrena et al., 2009; Bohm et al., 2010; Liu et al., 2015). Few studies have reported this correlation for digestates. Bayard et al. (2015) demonstrated a linear regression between both biodegradabilities for four substrates and four raw digestates from residual municipal solid waste and biowaste. Schievano et al. (2008) also reported a positive correlation between anaerobic biogasification potential and O 2 uptake rate on 46 samples including waste mixtures (agricultural, agro-industrial wastes and organic fraction of municipal solid waste) and their raw digestates. Finally, Ponsa et al. (2008) described a positive and linear correlation between aerobic and anaerobic stability indices for organic solid wastes during different stages of biodegradation including raw digestates and digestates post-treated by composting. Hence, the present study appears to be the first that has demonstrated a significant correlation between both aerobic and anaerobic indices for digestate samples posttreated by phase separation and composting. Interestingly, the slope of the linear correlation between BDaero and BDana (1.8, Fig. 1d) suggested that the OM contained in all digestates was less biodegradable Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 15 under anaerobic conditions than aerobic conditions. This result on digestate samples has already been obtained by Bayard et al. (2015) on raw digestates from AD of residual municipal solid waste and biowaste. According to Monlau et al. (2012), certain biodegradable parts are not accessible to microorganisms during AD, most likely due to the compositional and structural characteristics of ligno-cellulosic materials. Wagland et al. (2009) also reported that microbial growth efficiency under anaerobic conditions was lower than under aerobic conditions. Consequently the CH 4 yield and BOD constitute good indicators for describing the biodegradability and the stability of digestates. They can thus also represent useful tools for assessing the efficiency of biological treatment process (Cossu and Raga, 2008; Ponsá et al., 2008; Barrena et al., 2009; Bayard et al., 2015).

  describes the complexity of each accessible fraction (1-7 zones) and the aerobic and anaerobic biodegradabilities, while the PCA2 mainly describes the chemical accessibility of OM. The PCA correlation circles also illustrated that BDana and BDaero were particularly well correlated with the variables describing the first component, i. e. the OM complexity. The low correlation values between digestate biodegradability and OM accessibility was confirmed by a Pearson's correlation Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 16analysis for BDana, BDaero and %CODtot of each extracted fraction of OM (Table3).Indeed, this analysis demonstrated that the BDana and BDaero biodegradabilities only correlated with the most easily accessible fraction SPOM (R 2 =0.8 and 0.6, respectively), and not with the other accessible fractions REOM, SEOM and PEOM. Likewise, Bayard et al. (2015) studied the correlations between the BMP and different OM fractions. These authors pointed to a significant positive correlation between BMP and water soluble COD, and negative correlations between BMP and Van Soest residual fraction and humic substances, respectively. Here, the PCA correlation circle (Fig 2a) illustrated that the two biodegradability indicators were positively correlated with the simplest forms of molecules (zones 1-3 of complexity related to fluorescence) and were negatively correlated with complex molecules (zones 4-7 of complexity related to fluorescence) such as fulvic acid-, melanoidin-, lignocellulose-or humic acid-like molecules. The previous correlations between anaerobic biodegradability and OM complexity have already been reported in studies carried out on wastes intended for AD processes (Gunaseelan, 2007) or treated by AD (Jimenez et al., 2014). The PCA1 axis governed the classification of digestates into two distinct groups (Fig. 2b). Composted and dried solid fractions of digestate were clearly separated from solid fractions of digestate, revealing that the composting post-treatment step significantly affected the biological and biochemical properties of digestate. This has also been reported by Bayard et al. (2015) on substrates and digestates from biowaste and residual municipal solid waste. Composted and dried solid fractions of digestate were characterized by low aerobic and anaerobic biodegradabilities. They mostly contained the slowly accessible fraction SEOM associated to humic-like substances including complex molecules (zones 4-7 of complexity related to fluorescence) as Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 17 reported by Jimenez et al. (2015). Indeed, the complexity of composted solid fractions of digestate could be attributed to humic and fulvic acid-like and lignocellulosic-like compounds found in fluorescence zones 4-7 of the easily and slowly accessible fractions (SPOM+REOM+SEOM), which are known to be recalcitrant and slowly biodegradable. Inversely, solid fractions of digestate without a composting posttreatment were characterized by high amounts of aerobic and anaerobic biodegradability indicators and contained a large part of easily accessible fraction (SPOM) comprising simple forms of molecules. Solid fractions of digestate contained proteins molecules observed in fluorescence zones 1-3 of easily and slowly accessible fractions (SPOM+REOM+SEOM).These results suggest that OM contained in digestates present different levels of complexity according to the applied post-treatment. Composted or dried solid fractions of digestate characterized by low biodegradability and high complexity were found to be more stable than solid digestate fractions, thus allowing for their agricultural re-use by land spreading[START_REF] Ponsá | Comparison of aerobic and anaerobic stability indices through a MSW biological treatment process[END_REF] Teglia et al., 2011). On the contrary, the solid digestate fractions, which were characterized by a higher biodegradability and lower complexity, should most likely be subjected to a complete composting step or to a storage period in order to ensure their stabilization before they can be re-used for agricultural purposes. Although the impact of post-treatments on the biodegradability of organic matter in solid-fractions of digestate was here demonstrated, it must be pointed out that other factors such as types of feedstocks or digester operating conditions can also impact the organic matter properties of digestates. Finally, OM characterization using fluorescence spectroscopy is easier and faster to perform than aerobic and Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 18

  reported by Zeng et al. (2016) in a study on digestates obtained after thermophilic AD of municipal solid waste. Atypical kinetics were here observed in digestates from both mesophilic (T1S, T2S, T3S) and thermophilic (A1S and SL1S) AD processes, and with different treated wastes (agriculture waste, territorial waste, urban sludge). These results Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 19

  Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 20

Fig. 1 .

 1 Fig. 1. Correlations between BOD and CH 4 yield (a), AT 4 and CH 4 yield (b), DRI 24 and

Fig. 2 .Fig. 3 .

 23 Fig. 2. Principal Components Analysis on biodegradabilities and organic matter

Fig. 4 .

 4 Fig. 4. Principle Components Analysis on respirometric kinetics and organic matter

aA:FigFig. 2 .FigFigHighlights•

 2 Fig. 1.

  Representative aliquots of digestate (about 2 kg) were used to perform all the analytical tests. For each sample, the total solid (TS) content was measured by drying 100 g of sample at 105 °C for 48 h. The volatile solid (VS) content was measured after calcination of this dried sample at 550 °C for 3 h. Prior to total chemical oxygen demand (CODtot) measurements, the dried sample was ground to a particle size of 500 µm. Then, CODtot was determined by titration after digesting 1 g of TS with H 2 SO 4 and K 2 Cr 2 O 7 in accordance with the AFNOR standard NF T90-101. CODtot results were expressed in gO 2 kg -1 VS. All measurements were performed in triplicate.

Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 6

  or dried solid fractions of digestate were characterized by low CH 4 yields and BOD (0-28 NLCH 4 kg -1 VS and 39.8-254.4 gO 2 kg -1 VS) corresponding to BDana of

	treatment plant, mostly comprised slowly biodegradable OM. These authors
	biodegradability under both aerobic and anaerobic conditions (BDana<0.5 % and demonstrated that the easily biodegradable fraction of OM ranged between 2.7 and 6.3
	BDaero=2.8 % CODtot), thus suggesting that the OM of A4C was the most stable of all % COD with no observable distinction between raw digestates and composted
	studied samples. For solid fractions, A1S presented highest biodegradable COD under digestates. The non-extracted fraction (NEOM), calculated according to the extracted
	both conditions (BDana=18.6 % and BDaero=38 % CODtot). The total retention time of fractions, represented a large part of CODtot (between 15.3-70.8 %). For most of the
	the treatment line for A1S was short (between 14 and 20 days, table 1) when compared digestates (10 out of 14) the NEOM fraction represented more than 40 % CODtot.
	to the other digestates (between 15 and 95 days). This could explain the important part Jimenez et al. (2015) reported that the NEOM of sludge digestate ranged from 30.0 to
	43.1 % CODtot with more than 40 % CODtot for the digested sludge compost. In the
	present study, the composted or dried digestates, excepting two composted digestates
	(A3C and SL1C, <20 % CODtot), also presented a NEOM fraction greater than 40 %
	CODtot.
	(SEOM+PEOM=25-78 % CODtot). The most accessible fractions SPOM+REOM only
	<0.5-5.2 % CODtot and BDaero of 2.8-18.6 % CODtot. The solid fractions of digestate represented 2.7-10.6 % CODtot. In terms of accessibility, no difference was observed
	showed higher values (41.1-93.8 NLCH 4 kg -1 VS, BDana=7.2-18.6 % CODtot and between digestates. Teglia et al. (2011) reported similar results: the remaining OM of
	237.0-550.0 gO 2 kg -1 VS, BDaero=14.6-38.0 % CODtot). These results demonstrated digestate and composted digestate obtained after digestion of food processing waste,
	13

that the composting step of the solid fraction significantly reduce the biodegradable OM Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 12 content. Among the solid fraction composts, A4C was characterized by the lowest of residual biodegradability in this digestate. The values of biodegradability for the solid fractions were in the range of values previously obtained by Bayard et al. in 2005. These authors reported that the BDana of digestates from AD of residual municipal solid waste or biowaste ranged between 14.6 and 31.4 % COD and the BDaero ranged between 19.8 and 44.1 % COD. Consequently, despite the digestion process, certain digestates still contained a significant portion of residual biodegradable OM, as has been previously reported (Teglia et al., 2011; Bayard et al., 2015). Depending on the post-treatment conditions, this portion can be more or less important. In terms of biodegradability, the differences observed between digestates were probably due to the differences in the structure of OM. The characterization of OM by chemical sequential extractions describing the accessibility of OM (% CODtot for each extract) showed that the remaining OM in all digestates was essentially composed of less accessible fractions agricultural wastes, organic fraction of municipal solid waste and sludge of wastewater Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057

  of the organic matter in the mechanical-biological treated waste. Gunaseelan, V. N., 2007. Regression models of ultimate methane yields of fruits and vegetable solid wastes, sorghum and napiergrass on chemical composition. Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 24 21. Tambone, F., Scaglia, B., D'Imporzano, G., Schievano, A., Orzi, V., Salati, S., Adani, F., 2010. Assessing amendment and fertilizing properties of digestates from anaerobic digestion through a comparative study with digested sludge and compost.

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Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 21 biological stability and Solutions. Stratford-upon-Avon, UK, 28-30 September, 40-49. Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 22 9. doi:10.1016/j.watres.2013.10.048 12. Liu, X., Bayard, R., Benbelkacem, H., Buffiere, P., Gourdon, R., 2015. Evaluation of the correlations between biodegradability of lignocellulosic feedstocks in anaerobic digestion process and their biochemical characteristics. Biomass Bioenergy 81, 534-543. doi:10.1016/j.biombioe.2015.06.021 13. Mohajer, A., Trémier, A., Barrington, S., Martinez, J., Teglia, C., Carone, M. 2009.

Microbial oxygen uptake in sludge as influenced by compost physical parameters.

Table 1 .

 1 Digestate designation and origin. -: unknown, d RT: retention time including the time of post-digestion when applied (*), e sample results from the digestate mix originated from two reactors (one at 37°C and one at 55°C) operating in parallel.

	fraction of digestate, D: dried and composted solid fraction of digestate, C: composted solid fraction of digestate, b WWTP: wastewater treatment plant, FPW: food

a A: agricultural waste, SL: urban sludge, T: territorial waste, O: organic fraction of municipal solid waste (OFMSW), M: municipal solid waste (MSW), S: solid Comment citer ce document : Maynaud, G., Druilhe, C., Daumoin, M., Jimenez, J., Patureau, D., Torrijos, M., Pourcher, A.-M., Wéry, N. (Auteur de correspondance) (2017). Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter. Bioresource Technology, 213, 65-74. DOI : 10.1016/j.biortech.2017.01.057 processing waste, c

Table 2 .

 2 Physico-chemical and biological properties of digestates. Analysis of TS, VS, CODtot were performed in triplicate and CH 4 yield in duplicate. The mean and the standard deviation are given for these analyses.

	Digestate a TS (%)	VS (%TS)	CODtot (gO 2 kg -1 VS)	CH 4 yield (NL CH 4 kg -1 VS)	BOD (gO 2 kg -1 VS)	BDana (%CODtot)	BDaero (%CODtot)	Chemical sequential extraction (% CODtot) SPOM REOM SEOM PEOM NEOM
	SL1S	22.3 ± 0.2 70.4 ± 0.1 1696 ± 6	83.7 ± 3.8	550.0	14.1	32.4	5.3	3.2	18.6	49.5	23.4
	SL2S	22.4 ± 0.1 51.7 ± 0.5 1311 ± 44	73.2 ± 2.0	332.1	16.0	25.3	5.6	4.6	10.4	48.4	31.0
	A1S	25.6 ± 0.4 86.5 ± 0.4 1439 ± 13	93.8 ± 2.4	546.9	18.6	38.0	6.6	4.1	11.2	32.9	45.2
	T1S	23.9 ± 0.1 74.3 ± 0.1 1554 ± 12	66.5 ± 1.3	368.1	12.2	23.7	4.1	3.6	11.1	24.8	56.4
	T2S	24.9 ± 0.1 80.1 ± 2.2 1615 ± 14	55.1 ± 3.4	492.6	9.7	30.5	2.7	2.1	4.2	34.3	56.7
	T3S	26.4 ± 0.3 82.6 ± 0.6 1508 ± 13	48.0 ± 7.3	446.2	9.1	29.6	3.0	2.2	8.0	40.3	46.5
	T4S	22.8 ± 0	61.0 ± 0.5 1627 ± 44	41.1 ± 0.9	237.0	7.2	14.6	1.9	2.4	7.0	17.9	70.8
	A2D	74.6 ± 0.8 61.3 ± 0.1 1366 ± 10	19.1 ± 0.3	122.7	4.0	9.0	2.1	1.9	15.8	37.6	42.6
	A3C	43.9 ± 0.4 51.7 ± 0.4 1530 ± 35	17.5 ± 0.6	177.8	3.3	11.6	3.1	3.8	30.3	47.5	15.3
	A4C	40.9 ± 1.7 47.0 ± 2.4 1425 ± 138	<0.5	39.8	0.0	2.8	1.5	3.8	20.2	19.6	54.9
	SL2C	59.1 ± 0.8 50.4 ± 7.3 1440 ± 61	14.6 ± 0.2	93.0	2.9	6.5	5.0	4.8	24.9	46.0	19.3
	O1C	60.1 ± 1.1 46.6 ± 1.0 1245 ± 15	22.7 ± 0.2	152.7	5.2	12.3	3.7	1.5	7.5	42.9	44.4
	O2C	53.3 ± 1.2 39.3 ± 1.7 1367 ± 133	17.8 ± 0.6	254.4	3.7	18.6	1.6	1.1	4.1	34.0	59.2
	M1C	55.8 ± 0.3 42.1 ± 1.1 1541 ± 38	28.0 ± 0.4	176.1	5.2	11.4	2.1	2.2	6.8	29.7	59.2

Acknowledgement

This research was financially supported by the Agence de l'Environnement et de la Maitrise de l'Energie (ADEME) (grant number: 1306C0032).


C Kizlik-Masson

C Vayne

S E Mckenzie

Anne Poupon

Y Zhou

G Champier

C Pouplard

Yves Gruel
email: gruel@med.univ-tours.fr

J Rollin

5B9, a monoclonal anti-platelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies

Keywords: heparin, thrombocytopenia, monoclonal antibody, platelet activation

    Copyright 5B9, a monoclonal anti-platelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies

INTRODUCTION

Heparin-induced thrombocytopenia (HIT) is a frequent drug-adverse event caused in most patients by platelet-activating antibodies (Abs) directed against complexes of heparin (H) bound to platelet factor 4 (PF4) [START_REF] Kelton | Heparin-induced thrombocytopenia: a historical perspective[END_REF][START_REF] Greinacher | Heparin-Induced Thrombocytopenia[END_REF]. Most HIT Abs are IgG, which are potentially pathogenic because they can activate platelets with heparin via FcγRIIA receptors. The diagnosis of HIT is based on clinical and pathological criteria. The first category of laboratory HIT tests commonly used is immunologic and detects the binding of Abs to PF4 immobilized with heparin or polyvinyl sulfonate. The second category of assays is functional, more specific for pathogenic IgG Abs and therefore is necessary for confirming the diagnosis of HIT in many patients.

Nevertheless, these functional assays require strict quality controls, (i.e., testing a weak positive HIT serum and using an appropriate negative control, as well as checking that platelet activation is inhibited by a high heparin concentration) [START_REF] Warkentin | scientific, standardization committee of the international society on t, haemostasis. Laboratory testing for heparin-induced thrombocytopenia: a conceptual framework and implications for diagnosis[END_REF]. Functional assays performed with platelet-rich plasma (PRP) or whole blood are less sensitive than methods with washed platelets such as serotonin release assay, with high frequency of false-negative results [START_REF] Pouplard | Antibodies to platelet factor 4-heparin after cardiopulmonary bypass in patients anticoagulated with unfractionated heparin or a low-molecular-weight heparin : clinical implications for heparininduced thrombocytopenia[END_REF][START_REF] Warkentin | Polymorphisms of protein tyrosine phosphatase CD148 influence FcgammaRIIA-dependent platelet activation and the risk of heparin-induced thrombocytopenia[END_REF]. In addition, despite improvements in HIT laboratory assays, a standard is still lacking for both immunological and functional assays.

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In addition, platelets from healthy donors also show wide variability in response to HIT antibodies, and HIT does not develop in many patients with significant levels of antibodies against PF4/H. The mechanisms that regulate the pathogenicity of HIT antibodies have not been fully defined, although recent studies found that variations in CD148, FCGR2A and UBASH3B genes might have a significant effect on platelet activation induced by HIT antibodies [6][START_REF] Rollin | Increased risk of thrombosis in FcgammaRIIA 131RR patients with HIT due to defective control of platelet activation by plasma IgG2[END_REF][START_REF] Zhou | Anti-miR-148a regulates platelet FcgammaRIIA signaling and decreases thrombosis in vivo in mice[END_REF].

Several studies of the pathophysiology of HIT involved KKO, a murine monoclonal anti-PF4/H antibody that activates platelets and monocytes by cross-linking FcγRIIA and behaves like human HIT IgG [START_REF] Reilly | Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through FcgammaRIIA[END_REF][START_REF] Rauova | Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications[END_REF][START_REF] Kasthuri | PF4/heparin-antibody complex induces monocyte tissue factor expression and release of tissue factor positive microparticles by activation of FcgammaRI[END_REF][12]. However, KKO is an IgG2b antibody, a subclass absent in humans [START_REF] Bruhns | Mouse and human FcR effector functions[END_REF].

Moreover, IgG anti-PF4/H antibodies present in HIT plasma samples are predominantly IgG1.

Also, human and mouse Fcγ receptors (FcγRs) have shown large differences in binding abilities [START_REF] Bruhns | Mouse and human FcR effector functions[END_REF]. Indeed, mouse FcγRs efficiently bind human IgG subclasses, whereas human FcγRs do not or poorly bind mouse IgG subclasses. FcγR-IgG interactions are highly critical in the pathogenesis of HIT [START_REF] Rollin | Risk factors for heparin-induced thrombocytopenia: Focus on Fcgamma receptors[END_REF], so we aimed to develop a monoclonal anti-PF4/H antibody with a human Fc fragment by using transgenic mice homozygous for the Cγ gene of a G-class human immunoglobulin that directly produce chimeric IgG1 antibodies. We then studied the characteristics of the obtained monoclonal antibody, 5B9, and demonstrated that it fully mimicked the cellular effects of human HIT antibodies.

MATERIALS AND METHODS

Development of monoclonal antibodies to PF4/heparin complex

To develop an anti-PF4/H antibody with a human Fc fragment, we used transgenic Balb/c mice homozygous for the Cγ gene of a G-class human immunoglobulin that produce chimeric IgG1 antibodies composed of murine light chains and chimeric heavy chains with a murine variable region and a human constant region (GammaPrim

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purified human PF4 (50 μg, Hyphen BioMed, Neuville-sur-Oise, France), unfractionated heparin (UFH 2 IU; Heparin Choay; Sanofi), and 25μL Freund's complete adjuvant [START_REF] Arepally | Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies[END_REF]. Then, the same preparation containing preformed PF4/heparin complexes in PBS with Freund's complete adjuvant was injected intraperitoneally or through the tail vein in mice on days 15 and 30. Finally, 10 days after the later injection, a boost of human PF4/heparin complexes in PBS with Freund's incomplete adjuvant was injected intravenously in all mice 3 days before they were sacrificed. The synthesis of anti-PF4/heparin antibodies was monitored using a homemade ELISA, adapted from Arepally et al [START_REF] Arepally | Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies[END_REF] with microplates coated with purified human PF4 (2.5 µg/mL, Hyphen Biomed) with or without 0.05 IU/mL of heparin. Three mice positive for anti-PF4/H antibodies were killed and their spleens were removed for fusion procedures. Hybridomas were then obtained from chemical fusion of B cells with SP2/0 AG14 myeloma cells using a method adapted from Kölher and Milstein [START_REF] Kohler | Continuous cultures of fused cells secreting antibody of predefined specificity[END_REF]. Hybridomas were cultured for 13 days, and supernatants screened by ELISA to detect antibodies to PF4/H and PF4. Wells considered positive were then sub-cloned by limiting dilutions, and positive ELISA results were confirmed for 8 clones. Finally, 4 clones were isolated: 3 produced antibodies that equally bound PF4/H complex and PF4 alone on ELISA (1C12, 1E12, 2E1; supplemental Figure S1). The other clone produced an IgG1 named 5B9 that was mainly specific to PF4/H and was isolated from ascitic fluid by using affinity columns.

The reactivity of 5B9 was evaluated with

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Platelet activation and aggregation tests

Whole blood from healthy donors was collected after informed consent in acid-citrate-dextrose supplemented with prostaglandin E1 (0.1mM, Sigma-Aldrich), according to the Helsinki Declaration principles and International Society on Thrombosis and Haemostasis recommendations [START_REF] Cattaneo | Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH[END_REF]. Platelets were washed and suspended at a final count adjusted to 350 G/L. In addition, whole blood from healthy donors was collected on 0.129 M sodium citrate and PRP was isolated. Platelet aggregation tests (PAT) with PRP and washed platelets were performed in an APACT 4 aggregometer (ELITech Group, Puteaux, France) with different concentrations of 5B9 (50, 20 and 10 µg/mL) and heparin (0, 0.5 and 50 IU/mL).

Serotonin release assays (SRA) were performed [START_REF] Pouplard | Differences in specificity of heparin-dependent antibodies developed in heparin-induced thrombocytopenia and consequences on cross-reactivity with danaparoid sodium[END_REF], with the same concentrations of 5B9, with or without addition of exogenous PF4 (10 µg/mL).

Assays of tissue factor mRNA levels after stimulation by 5B9 antibody

Monocytes were isolated from whole blood collected in healthy donors on 0.129 M sodium citrate with the Monocyte Isolation Kit II (Miltenyi Biotec, Paris, France) [START_REF] Kasthuri | PF4/heparin-antibody complex induces monocyte tissue factor expression and release of tissue factor positive microparticles by activation of FcgammaRI[END_REF]. P24 plates were seeded with 5x10 5 cells/well and various concentrations of purified human PF4 and heparin were then added, followed by the addition of 5B9 (20 µg/mL). After incubation at 37°C for 2 hours, total RNA was isolated using the RNeasy Mini kit (Qiagen, Courtaboeuf, France) and mRNA levels specific for tissue factor (TF) and CD14 genes were measured by qPCR as described [START_REF] Rollin | Increased risk of thrombosis in FcgammaRIIA 131RR patients with HIT due to defective control of platelet activation by plasma IgG2[END_REF].

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Human neutrophil degranulation assay

Blood neutrophils were isolated as described [START_REF] Korkmaz | Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates[END_REF]. After priming with cytochalasin B (5 μg/mL) and human tumour necrosis factor α (10 ng/ml), neutrophils (10 6 cells for each assay)

were stimulated with various concentrations of heparin, human PF4 (5 μg/mL), and 5B9 (20 μg/mL), or phorbol myristate acetate (100 ng/mL, Sigma-Aldrich) as positive control. After incubation for 90 min, supernatants were collected and neutrophil activation was assessed by using a fluorogenic substrate of neutrophil proteases (Abz-HPV-PVYAFSPQ-Yno2, 25μM, GeneCust Europe, Ellange, Luxembourg) [START_REF] Korkmaz | Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates[END_REF]. For each condition, maximal hydrolysis speed of the substrate (Vmax) was measured and ratio of values under stimulated and basal conditions was calculated.

Stimulation of human whole blood with 5B9 antibody

Whole blood from healthy donors on 0.129 M sodium citrate was incubated 60 min with 5B9 (20µg/mL), with and without heparin (0.5 IU/mL) at 37°C. After centrifugation, plasma was collected and clotting time was measured with the Procoag-PPL assay (Stago), which is sensitive to the presence of phospholipid microparticles [START_REF] Rollin | Increased risk of thrombosis in FcgammaRIIA 131RR patients with HIT due to defective control of platelet activation by plasma IgG2[END_REF]. Total RNA was isolated from cells using the Qiamp RNA blood Mini kit (Qiagen) and mRNA levels from TF and CD14 genes were measured by qPCR.

ELISA competition assays

The ability of 5B9 and KKO to bind PF4/H-coated wells in the presence of plasma from patients with anti-PF4/H antibodies was evaluated using the Asserachrom HPIA ® assay (Stago) with modification. Diluted plasmas were first incubated for 50 min at room temperature (RT) in wells coated with PF4/H complex. Then, 5B9 or KKO were added to wells without removing diluted plasma samples and incubated for 10 min at RT. The concentrations of 5B9 and KKO

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used (10 and 0.7 µg/mL, respectively) were defined in specific ELISA experiments and provided 75% of the maximal binding of each antibody to PF4/H complexes. After washings, alkalinephosphatase-conjugated goat anti-murine κ chain (1/500; Jackson ImmunoResearch, Suffolk, UK), was added and incubated for 1h at RT. After washings, pNPP substrate was added, and the reaction was stopped with NaOH 3N before absorbances were read at 405 nm. The reference value (OD ref) was measured after incubation of 5B9 or KKO without plasma, and the percentage binding inhibition was obtained as: [(ODref -ODtest) / ODref] x 100.

For 5B9/KKO competitive ELISA, 5B9 was first incubated 10 minutes at 2.5µg/ml in wells with PF4/H, and then KKO was added at different concentrations (0, 5, 10, 15 and 20 µg/mL). Finally, 5B9 binding was revealed using an anti-human Fc antibody.

In vivo effects of 5B9 in HIT mouse model

All studies in mice were conducted in accordance with the guidelines and approval of the

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Docking analysis of 5B9-PF4 complex

5B9 cDNA was sequenced using the Sanger method, and a model of the 3D structure of 5B9 antibody was then built by using MODELLER [START_REF] Webb | Comparative Protein Structure Modeling Using MODELLER[END_REF]. The structures used as templates for homology modelling were PDB:1F58 for the variable heavy (VH) chain and constant domains and PDB:5B9L for the variable light (VL) chain. The resulting 3D model was docked on PF4 complexed with fondaparinux (PDB:4R9W) [START_REF] Cai | Atomic description of the immune complex involved in heparin-induced thrombocytopenia[END_REF], using the method PRIOR optimized for antibody/antigen complexes [START_REF] Bourquard | Unraveling the molecular architecture of a G protein-coupled receptor/beta-arrestin/Erk module complex[END_REF]. The models for the assembly between heparin, three tetramers of PF4 and 3 antibodies were built manually, with the structure of heparin heptasaccharide (PDB:1SR5) used as a guide. Structures were imaged using the PyMOL Graphics System (v1.8).

Statistical analysis

Student t-test was used to compare data obtained after stimulating monocytes, neutrophils, or whole blood with 5B9 and during studies in vivo. P < 0.05 was considered statistically significant.

RESULTS

5B9 specifically binds to PF4/H complexes

Homemade ELISA revealed that 5B9 mostly bound to the PF4/H complex with no significant interaction with PF4 alone (Figure 1A). Also, the binding of 5B9 to PF4/H complex was optimal with concentrations of UFH and PF4 of 0.1 IU/mL and 5 µg/mL, respectively, (Figure 1B) and was inhibited with higher concentrations of heparin (5 and 25 IU/mL). The binding of 5B9 to modified PF4 was evaluated with 3 commercial IgG specific ELISA (i.e.

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with modified PF4. The 3 assays produced excellent correlation (R 2 > 0.98), but the binding of 5B9 to modified PF4 seemed lower with the Zymutest HIA ® than the 2 other immunoassays.

Therefore, the mean absorbance measured with 5 µg/mL of 5B9 was 1.3 with the Zymutest HIA ® , and above 2.0 with HPIA ® and Lifecodes PF4 ® (Figure 1C).

5B9 induces heparin-dependent platelet activation and aggregation

5B9 induced heparin-dependent platelet activation with maximal release of 14 C serotonin achieved with 0.1 IU/mL UFH, which was similar to the pattern observed with most of HIT plasma samples (Figure 2A). As expected, platelets were not activated by 5B9 when heparin was absent or was present at a high concentration (10 IU/mL). With exogenous PF4 (10 µg/mL) in the reaction mixture, 5B9 induced strong platelet activation without requiring heparin, but with complete inhibition with 10 IU/mL UFH (Figure 2B). The release of serotonin was always inhibited by the monoclonal IV.3 antibody whatever the conditions, with or without exogenous PF4. 5B9 also induced heparin-dependent platelet aggregation with PRP and washed platelets from healthy donors. This response (Figure 2C andD) was observed with 50 or 20 µg/mL 5B9 and 0.5 IU/mL UFH, with no aggregation obtained with 10µg/mL. Similar results were obtained with 5B9 tested in the presence of enoxaparin, a low-molecular-weight heparin (data not shown).

5B9 induces monocyte and neutrophil activation

5B9 with exogenous PF4 (5 µg/mL) increased tissue factor mRNA synthesis from isolated monocytes, effect which was potentiated by a low concentration of UFH (0.1 IU/mL) and fully inhibited by 10 IU/mL UFH (Figure 3A). In addition, after incubation of whole blood from healthy donors with 5B9 and UFH (0.5 IU/mL), TF mRNA level was 3.2 fold increased than without heparin (P<0.05), and this effect was inhibited by a high concentration of heparin (50

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IU/mL) (Figure 3B). This TF gene induction was associated with procoagulant activity because plasma clotting time was shortened after incubation of whole blood with 5B9 and UFH (39 vs. 60 sec without UFH; Figure 3C). Also, 5B9 incubated with PF4 and without UFH led to strong release of protease activity from isolated neutrophils (with a 5.9 fold increase in maximal hydrolysis speed compared to the non-stimulated condition) (Figure 3D). But, this effect was potentiated (8.9-fold) by a low UFH concentration (0.1 IU/mL) and inhibited with 10 IU/mL UFH.

5B9 induces thrombocytopenia in the hFcγRIIA/hPF4 transgenic mouse model

In vivo, 5B9 injected with UFH at 15.8 µg/g in transgenic mice expressing human FcγRIIA and PF4 induced thrombocytopenia with a 38% to 40% decrease in platelet count as compared to the basal value (Figure 4A). This effect of 5B9 was similar to that obtained with KKO injected at 30 µg/g (decrease in platelet count between 29% and 35%). Plasma level of TAT was measured at times that coincided with platelet nadirs in each experimental group. TAT plasma levels significantly increased after injection of UFH in all treated mice, but this effect was more pronounced on day 1 with 5B9 (mean 47 vs. 5 ng/mL at day 0) than with KKO (33 vs. 11 ng/mL).

This difference was no longer observed 2 days after injection of UFH, but TAT levels remained high in all mice studied (36 and 33 ng/mL with 5B9 and KKO, respectively).

5B9 binds an epitope close to that recognized by human HIT antibodies

Cross-competition experiments were performed to determine whether 5B9 and pathogenic HIT antibodies could recognize overlapping epitopes in the PF4/H complex. The binding of 5B9 to PF4/H complexes was inhibited (i.e. inhibition > 20%) by 60% of HIT plasma samples tested (15/25) and only 12% of plasma samples containing non-pathogenic anti-PF4/H antibodies (3/25)(Figure 5A). Noticeably, there was no significant difference in the main clinical

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HIT patients with inhibitory and non-inhibitory antibodies (supplementary table). Similar cross-

competition experiments were performed with KKO: its binding to the PF4/H complex was inhibited by 32% of HIT plasma samples (8/25), and 0% of non-HIT samples. Levels of 5B9 and KKO binding inhibition to PF4/H by HIT plasma samples were also highly correlated (R 2 = 0.85), which suggests that epitopes recognized by both antibodies are similar. In addition, competitive assays with KKO and 5B9 showed that KKO inhibited in a concentration-dependent manner the binding of 5B9 to the PF4/H complex, with absorbance varying from 2.05 without KKO to 0.55 with 20 µg/mL KKO (Figure 5C).

Modelling the 5B9 scFv/PF4 complex by docking

After 5B9 cDNA was sequenced, we defined a docking model based on the VH and VL sequences of 5B9 (scFv) obtained, and with recently described data for the crystal structure of PF4 tetramer modified by fondaparinux [START_REF] Cai | Atomic description of the immune complex involved in heparin-induced thrombocytopenia[END_REF]. According to this model, 5B9 appeared to bind B and D monomers of PF4 (Figure 6A) and this interaction could stabilize the PF4 tetramer and then macromolecular complexes composed of several PF4 tetramers bound to extended glycosaminoglycan chains (Figure 6B). The epitope of 5B9 was predicted to involve 24 aminoacids, 12 in monomer B and 12 in monomer D. Comparatively, KKO mostly interacts with monomer B, with a binding site involving 22 residues including Gln9 and Val13-Thr15 (VKT) that are also recognized by 5B9 (Figure 6C andD). Comparatively, KKO bound only 7 amino acids of monomer D, but 3 of these (i.e., Gln9-Cys10 and Thr38) also appeared to contribute to the epitope of 5B9. Five other residues, (i.e., Ala32-Pro34 or AGP and Cys36-Pro37) either in monomer B or monomer D, were also showed to interact with KKO and 5B9, respectively (Figure 6D).

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DISCUSSION

In this study, we characterized the first chimeric monoclonal HIT antibody that contains a human Fc fragment, and showed that it behaves similarly to human IgG anti-PF4/H antibodies.

5B9 was obtained after injecting purified hPF4/H complexes into a transgenic mouse able to produce IgG antibodies with a human constant region. Among the clones obtained, only 5B9

produced an IgG1 antibody specific to the PF4/H complex with very low reactivity against PF4 alone.

Two other HIT activating antibodies have previously been developed. The first is KKO, a monoclonal antibody that also mainly binds PF4/H complexes and weakly PF4 alone [START_REF] Arepally | Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies[END_REF].

However, KKO is a murine IgG2k antibody, a subclass absent in humans, and therefore does not interact with FcγRs similar to HIT human IgG antibodies. More recently, Asada et al. developed a monoclonal murine IgG1 antibody, but it bound PF4/H complex and PF4 alone similarly and induced platelet activation without heparin [START_REF] Asada | Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia[END_REF].

Importantly, 5B9 was shown to induce heparin-and FcγRIIA-dependent platelet activation similar to that observed with plasma samples from HIT patients, whether tested with PRP or washed platelets. We recently demonstrated that the platelet response to 5B9 widely varied among donors, particularly when testing PRP [START_REF] Rollin | Increased risk of thrombosis in FcgammaRIIA 131RR patients with HIT due to defective control of platelet activation by plasma IgG2[END_REF]. This variability was similar to that observed with human HIT plasma samples and likely explains the relatively low sensitivity of platelet aggregation tests for the diagnosis of HIT [START_REF] Warkentin | Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia[END_REF]. Hence, 5B9 may be used as a positive control in functional assays to determine whether healthy platelet donors are good or poor responders to HIT antibodies. In addition to activating platelets, 5B9 can also activate monocytes inducing TF mRNA synthesis even in the absence of exogenous heparin as previously reported [START_REF] Arepally | Antibodies from patients with heparin-induced thrombocytopenia stimulate monocytic cells to express tissue factor and secrete interleukin-8[END_REF][START_REF] Pouplard | Induction of monocyte tissue factor expression by antibodies to heparin-platelet factor 4 complexes developed in heparin-induced thrombocytopenia[END_REF], and procoagulant activity, which in vivo likely contributes to the risk of thrombosis [START_REF] Kasthuri | PF4/heparin-antibody complex induces monocyte tissue factor expression and release of tissue factor positive microparticles by activation of FcgammaRI[END_REF]28]. 5B9 also activates neutrophils with a significant release of a protease activity, which confirms previous data obtained with HIT plasma samples [START_REF] Xiao | Immune complexes formed following the binding of anti-platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion[END_REF], but the pathophysiological significance of this effect remains undefined.

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When 5B9 was tested in ELISA, the concentration allowing about 75% of maximal binding was 10 µg/mL, although less than 1 µg/mL KKO was enough to achieve a similar binding. This finding suggests a lower affinity of 5B9 for PF4/H complex than KKO. However in functional assays, 20 µg/mL 5B9 were sufficient to induce platelet activation with heparin, but without addition of exogenous PF4. Comparatively, 80 µg/mL KKO were used to induce heparindependent platelet activation with 10 µg/mL PF4 [START_REF] Arepally | Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies[END_REF][START_REF] Reilly | PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model[END_REF]. Our in vivo experiments also demonstrated that 5B9 induced thrombocytopenia after intravenous injection with heparin in transgenic mice expressing human FcγRIIA and PF4, an effect similar to that obtained with a higher dose of KKO. In addition, thrombin-antithrombin level, validated as a surrogate marker of thrombosis in patients with HIT [START_REF] Warkentin | The pathogenesis of venous limb gangrene associated with heparin-induced thrombocytopenia[END_REF], and of thrombosis in mouse models [START_REF] Lay | Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities[END_REF][START_REF] Reilly | Prothrombotic factors enhance heparin-induced thrombocytopenia and thrombosis in vivo in a mouse model[END_REF], was significantly higher in mice having received 5B9 than in those injected with KKO.

The class of 5B9 and KKO and the origin of their Fc portion are not the same, these variations probably explaining differences in platelet reactivity to these IgG antibodies in vitro and in vivo. 5B9 is indeed composed of murine light chains and chimeric heavy chains with a murine variable region and a human IgG1 constant region, and IgG1 is the most frequently represented IgG subclass among human HIT antibodies [START_REF] Pouplard | Antibodies to platelet factor 4-heparin after cardiopulmonary bypass in patients anticoagulated with unfractionated heparin or a low-molecular-weight heparin : clinical implications for heparininduced thrombocytopenia[END_REF][START_REF] Arepally | Fc gamma RIIA H/R 131 polymorphism, subclass-specific IgG anti-heparin/platelet factor 4 antibodies and clinical course in patients with heparin-induced thrombocytopenia and thrombosis[END_REF]. In contrast, KKO is a full murine IgG2bk, a class absent in our species, and weakly binds human FcγRIIA. Indeed, the affinity of murine IgG2b for human FcγRIIA receptor is very low (association constants or KA = 1.2 and 0.9

x 10 5 M -1 for 131H and 131R isoforms, respectively) as compared with those of human IgG1 (5.2 and 3.5 x 10 5 M -1 ) [35]. Moreover, Fc receptors other than FcγRIIA likely contribute to the pathogenic effects of HIT antibodies [START_REF] Rollin | Risk factors for heparin-induced thrombocytopenia: Focus on Fcgamma receptors[END_REF], particularly FcγRIIIA, which does not bind murine IgG2b [36]. In contrast, human IgG1 subclass shows relatively high affinity (KA > 10 6 ) for this receptor [START_REF] Bruhns | Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses[END_REF] and an association between FcγRIIIA V158F polymorphism and the risk of HIT was previously demonstrated in patients with high titre of anti-PF4/H antibodies [START_REF] Gruel | The homozygous FcgammaRIIIa-158V genotype is a risk factor for heparin-induced thrombocytopenia in patients with antibodies to heparin-platelet factor 4 complexes[END_REF].

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In clinical practice, anti-PF4 antibodies in HIT patients are detected by different immunoassays based on variable PF4 complexes. 5B9 appeared to react similarly with PF4 bound to heparin (Stago assay) or polyvinyl sulfonate, a non-heparin polyanion (Immucor GTI assay), but its binding to modified PF4 seemed lower when evaluated by the Hyphen BioMed assay. In this assay, PF4 (and likely other heparin-binding antigenic proteins) is provided by a platelet lysate, and binds to heparin immobilized by protamine during the incubation step.

Therefore, 5B9 (as well as human HIT antibodies) likely interacts differently with PF4 modified by protamine-bound heparin (i.e., in the Hyphen BioMed assay) and PF4/H or PF4/PVS complexes used as antigens in the other methods. We also showed that 5B9 mainly bound to PF4 modified by heparin, and this binding was strongly reduced by a high concentration of heparin, similar to findings with KKO [START_REF] Arepally | Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies[END_REF] or most HIT human antibodies [START_REF] Sheridan | A diagnostic test for heparin-induced thrombocytopenia[END_REF].

Importantly, 5B9 binding to PF4/H complexes was decreased by human antibodies present in 15/25 HIT plasma samples, but this inhibitory effect was obtained with only 3 of 25 samples containing non-activating human anti-PF4/H antibodies. Hence, human HIT pathogenic antibodies may bind PF4 epitopes that are close to those recognized by 5B9, but different from those targeted by non-platelet-activating antibodies. In similar experiments with KKO, we found no significant inhibition of this antibody binding to PF4/H in the presence of non-pathogenic anti-PF4/H antibodies, but a significant inhibitory effect with 8 of 25 HIT plasma samples. These results agree with those from Cuker et al [START_REF] Cuker | Novel diagnostic assays for heparin-induced thrombocytopenia[END_REF], and confirm that the KKO binding site on modified PF4 likely overlaps epitopes recognized by some of the pathogenic antibodies in HIT patients.

Several HIT human antibodies also decreased significantly the binding of both 5B9 and KKO to PF4/H complex. Moreover, high concentrations of KKO also strongly inhibited 5B9/PF4 interaction. Accordingly, our predictive docking model suggested that the epitopes of 5B9 and KKO largely overlap. However, differences in these epitopes, notably because 5B9 seems to bind to both B and D monomers whereas KKO mostly interacts with the B monomer [START_REF] Cai | Atomic description of the immune complex involved in heparin-induced thrombocytopenia[END_REF], could explain some of the variation observed in ELISA competitive assays performed with both antibodies and HIT plasma samples. Several of these residues, including Pro34, Pro37 and the

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sequence Asp7-Gln9 were previously found critical in the epitopes recognized by human HIT antibodies [START_REF] Ziporen | Defining an antigenic epitope on platelet factor 4 associated with heparininduced thrombocytopenia[END_REF] and KKO [START_REF] Li | Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody[END_REF]. Also, the amino-acid region that binds both KKO and 5B9 is likely important to promote heparin-dependent platelet activation and thrombocytopenia. As recently demonstrated, KKO stabilizes the ternary complex KKO/PF4/H [START_REF] Cai | Atomic description of the immune complex involved in heparin-induced thrombocytopenia[END_REF], and initiates the oligomerization of PF4 tetramers [43]. Our functional studies also strongly suggest that like KKO, 5B9 probably also favours the formation of ultra-large immune complexes on cell surfaces, a prerequisite for potent FcγRIIA-dependent cell activation.

Our docking model was developed based on data obtained after crystallization of KKO Fab with PF4 and fondaparinux, which is a homogeneous molecule. We therefore cannot exclude the possibility that therapeutic glycosaminoglycans other than fondaparinux, are inducing other changes in PF4 antigenicity [44].

In conclusion, 5B9 is the first monoclonal antibody specific to PF4-heparin complex with a human Fc fragment, and could be used as a positive control and standard reagent for laboratory HIT assays. Moreover 5B9 fully mimics the effects of human antibodies and hence could be a useful specific tool when studying FcγR-dependent cell activation in the

Legend to figures

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  Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

  Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

  Asserachrom HPIA ® IgG, Zymutest HIA ® IgG and Lifecodes PF4 ® IgG) testing different complexes Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

  citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

(

  4T's, thrombosis, treatment) and biological (A450 value, maximal serotonin release) features in Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

  Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

  Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

  citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

Figure 1 :

 1 Figure 1: Binding of 5B9 to PF4/heparin complex. Panel A: Absorbances obtained with different concentrations of 5B9 added to wells coated with purified PF4 alone or PF4/heparin complex. Panel B: Absorbances obtained in ELISA with 5B9 (10 µg/mL) incubated in wells coated with PF4 (5µg/mL) and various concentrations of UFH. Panel C: Binding of 5B9 to modified PF4 evaluated by Asserachrom HPIA IgG, Zymutest HIA IgG and Lifecodes PF4 IgG (n=5). Data are mean ± SEM.

Figure 2 :

 2 Figure 2: Platelet activation and aggregation induced by 5B9. Panel A: Serotonin release from washed platelets (n = 10 donors) after incubation with 5B9 or HIT plasma samples and different concentrations of UFH. Panel B: Serotonin release induced by 5B9 (20µg/mL) with and without exogenous PF4 (10µg/mL) and IV.3 (10 µg/mL), a monoclonal antibody inhibiting FcγRIIA. Panel C: Representative platelet aggregation profile after addition of 5B9 (50, 20 and 10 µg/mL) and heparin (0.5 IU/mL) to platelet-rich plasma (PRP) from one healthy donor. Panel D: Representative platelet aggregation induced by 5B9 (20 µg/mL) in the absence or presence of heparin (0.5 or 10 IU/mL) and inhibitory effect of monoclonal IV.3 antibody (10 µg/mL). Data are mean ± SEM.

Figure 3 :Figure 4 :Figure 5 :

 345 Figure 3: Activation of blood cells with 5B9.

  Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

  pathophysiology of HIT. Sachais BS, Litvinov RI, Yarovoi SV, Rauova L, Hinds JL, Rux AH, Arepally GM, Poncz M, Cuker A, Weisel JW, Cines DB. Dynamic antibody-binding properties in the pathogenesis of HIT.

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ACKNOWLEDGMENTS

This study was supported by the Institut pour la Recherche sur la Thrombose et l'Hémostase (IRTH), and by the program "Investissements d'avenir Grant Agreement LabEx MAbImprove: ANR-10-LABX-53". Version postprint Comment citer ce document : Kizlik-Masson, C., Vayne, C., McKenzie, S., Poupon, A., Zhou, Y., Champier, G., Pouplard, C., Gruel, Y., Rollin, J. (2017). 5B9, a monoclonal antiplatelet factor 4 /heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. Journal of Thrombosis and Haemostasis, 15 (10), 2065-2075. DOI : 10.1111/jth.13786

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DISCLOSURE OF CONFLICTS OF INTERESTS

G. Champier is an employee of B Cell Design. Other authors have nothing to disclose.

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