A Martins

G Spengler

M Martins

L Rodrigues

M Viveiros

A Davin-Regli

J Chevalier

I Couto

J M Pagès

L Amaral
email: lamaral@ihmt.unl.pt

Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes

Keywords:

A c c e p t e d M a n u s c r i p t

ABSTRACT

Enterobacter aerogenes predominates among Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum -lactamases.

Although this mechanism of resistance to -lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Among these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium.

The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.

A c c e p t e d M a n u s c r i p t

Introduction

The advent of multidrug resistance in Gram-negative bacteria has increased the range of infections owing to the problematic nature of therapy. Consequently, infections by Enterobacter aerogenes that were once less frequent are now becoming increasingly more frequent in the hospital environment [START_REF] Deal | Predictors of in-hospital mortality for bloodstream infections caused by Enterobacter species or Citrobacter freundii[END_REF]. As an example, two clones of ceftazidime-resistant E. aerogenes have been spreading throughout Belgian hospitals to the point that they have become a serious healthcare issue both in hospitals and in the community [START_REF] Goossens | Antibiotic resistance and policy in Belgium[END_REF]. Moreover, strains of E. aerogenes now account for >50% of all Gram-negative organisms that produce extended-spectrum -lactamases (ESBLs) and, inasmuch as ESBLs are the major cause of treatment failure, the opportunity for the development of multidrug-resistant (MDR) phenotypes has resulted [START_REF] Giamarellou | Multidrug resistance in Gram-negative bacteria that produce extended-spectrum -lactamases (ESBLs)[END_REF]. Among the mechanisms that contribute to a MDR phenotype in E. aerogenes is overexpression of genes that regulate and code for efflux pumps which extrude unrelated antibiotics before they reach their intended targets [START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF]. Interestingly, whilst the appearance of multidrug resistance in this species has been essentially made possible by the production and release of type C -lactamases, the existence of an overexpressed efflux pump further contributes to the progressive evolution of multidrug resistance [START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF][START_REF] Martins | An AcrAB-mediated multidrug-resistant phenotype is maintained following restoration of wild-type activities by efflux pump genes and their regulators[END_REF][START_REF] Chevalier | Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and 2003[END_REF].

The MDR phenotype of E. aerogenes coupled with its ESBL-producing capacity has resulted in increased emergence of this pathogen in hospital units [START_REF] Vogelaers | ANTHICUS Study Investigators. Patterns of antimicrobial therapy in severe nosocomial infections: empiric choices, proportion of appropriate therapy, and adaptation rates-a multicentre, observational survey in critically ill patients[END_REF][START_REF] Chang | Clinical characteristics and predictors of mortality in patients with Enterobacter aerogenes bacteremia[END_REF][START_REF] Anderson | Molecular and descriptive epidemiology of multidrug-resistant Enterobacteriaceae in hospitalized infants[END_REF] and A c c e p t e d M a n u s c r i p t 5 in the description by the media as 'killer bugs' or 'superbugs'. Indeed, the infections they cause result in a high cost in terms of morbidity, treatment failures and subsequent mortality [START_REF] Goossens | Antibiotic resistance and policy in Belgium[END_REF]. Although at the time of this writing no agents are available for inhibition of type C -lactamases, a variety of agents have been identified as having the potential to inhibit overexpressed efflux pumps of Gramnegative bacteria [START_REF] Pagès | Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria[END_REF]. However, although some of these inhibitors of efflux pumps affect the function of the pump itself [efflux pump inhibitors (EPIs)] [START_REF] Lomovskaya | Efflux pumps: their role in antibacterial drug discovery[END_REF][START_REF] Lomovskaya | Vacuuming the periplasm[END_REF], there is now evidence that some EPIs may have an effect on regulatory genes involved in the control of efflux expression and genes that code for transporters [START_REF] Bailey | RamA confers multidrug resistance in Salmonella enterica via increased expression of acrB, which is inhibited by chlorpromazine[END_REF][START_REF] Klitgaard | Reversal of methicillin resistance in Staphylococcus aureus by thioridazine[END_REF]. Because of this, assessment of EPIs must also include their potential genetic actions. Nevertheless, although the genetic basis for an overexpressed efflux pump is essential, the actual function of an efflux pump need not be compatible with its level of genetic expression and regulation [START_REF] Viveiros | Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes[END_REF].

As an example, in Escherichia coli strains whereas transcription of the acrEF gene is 80-fold greater in a tetracycline-resistant strain than that of its parental tetracycline-susceptible strain, its level of resistance is equal to that of the tetracycline-resistant strain whose acrAB is transcribed only 6-fold above that of its parent [START_REF] Viveiros | Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes[END_REF].

With the exception of some early demonstrations of efflux pumps as well as a recent report [START_REF] Spengler | Characterisation of intrinsic efflux activity of Enterococcus faecalis ATCC29212 by a semi-automated ethidium bromide method[END_REF], assessment of the function of specific efflux pumps is essentially absent from the literature. Understanding that resistance-nodulationcell division (RND) tripartite efflux pumps derive their energy for function from the A c c e p t e d M a n u s c r i p t 6 membrane p (potential gradient, p =  + RT logpH, where  is the difference in electrochemical gradient across the membrane, RT is the amount of ATP harnessed at 37 C and pH is the difference in pH across the membrane) (proton-motive force across inner membrane) suggests that the efficacy of these pumps would be best at low pH and less so at pH >7 given the paucity of protons at the latter pH. At pH >7 one would suppose that ATP-binding cassette (ABC)type transporters would derive their energy from the hydrolysis of ATP, inasmuch as such hydrolysis would be favoured by the F 0 -F 1 energy system of the cell [START_REF] Zharova | Energy-linked binding of P i is required for continuous steady-state proton-translocating ATP hydrolysis catalyzed by F 0 •F 1 ATP synthase[END_REF]. Moreover, because the dissociation constant (K d ) for substrates bound by the AcrB transporter is low at high pH and high at low pH [START_REF] Su | Ligand-transporter interaction in the AcrB multidrug efflux pump determined by fluorescence polarization assay[END_REF], the dissociation of a substrate such as ethidium bromide (EB) from the AcrB transporter could be sufficiently slow at physiological pH 7 to render the AcrAB efflux pump somewhat limited if the organism is challenged with a noxious substrate that it must extrude for its survival [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF]. Given these considerations, the described study here has assessed the role of pH in the activity of the efflux pump system of the intrinsically susceptible E. aerogenes ATCC reference strain as well as two MDR phenotypic strains whose resistance is due to an overexpressed efflux pump system.

A c c e p t e d M a n u s c r i p t 7

Materials and methods

Materials

Mueller-Hinton (MH) in powder form was used for the preparation of MH broth and MH agar. Antibiotics in powder form were purchased from Difco Laboratories (Detroit, MI). EB, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Phe-Arg--naphthylamide (PAN) were purchased from Sigma-Aldrich Química SA (Madrid, Spain).

Bacteria

Antibiotic-susceptible E. aerogenes ATCC 13048 and MDR E. aerogenes EA27 and CM64 strains have been previously fully characterised [START_REF] Chevalier | Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and 2003[END_REF][START_REF] Tran | Occurrence of efflux mechanism and cephalosporinase variant in a population of Enterobacter aerogenes and Klebsiella pneumoniae isolates producing extended-spectrum -lactamases[END_REF][START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF]. The MDR strains are resistant to norfloxacin, chloramphenicol, tetracycline and other antibiotics and overexpress the AcrAB-TolC efflux pump [START_REF] Chevalier | Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and 2003[END_REF][START_REF] Tran | Occurrence of efflux mechanism and cephalosporinase variant in a population of Enterobacter aerogenes and Klebsiella pneumoniae isolates producing extended-spectrum -lactamases[END_REF][START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF].

Methods

Minimum inhibitory concentrations (MICs)

The MIC of antibiotics, CCCP and PAN against the strains employed in this study were determined in MH medium adjusted to pH 5, 7 and 8 by the microbroth dilution method according to Clinical and Laboratory Standards Institute guidelines [START_REF] Espinel-Ingroff | Quality control and reference guidelines for CLSI broth microdilution susceptibility method (M 38-A document) for amphotericin B, itraconazole, posaconazole, and voriconazole[END_REF].

A c c e p t e d M a n u s c r i p t 8

Assessment of putative efflux of ethidium bromide by the ethidium bromide agar method

Assessment of efflux by the EB agar method has been previously described in detail [START_REF] Viveiros | New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps[END_REF][START_REF] Martins | An instrument-free method for the demonstration of efflux pump activity of bacteria[END_REF]. Briefly, plates of MH agar of pH 5, 7 and 8 containing increasing concentrations of EB were swabbed with each bacterial strain as spokes of a wheel (cartwheel manner) and the plates were incubated for 18 h at 37 C, examined under ultraviolet light and photographed using a GEL DOC XR instrument (Bio-Rad Laboratories, Hercules, CA). The minimum concentration of EB that produces fluorescence indicates that the amount of EB that the cell can extrude has been exceeded and hence EB accumulates within the cell.

Assessment of efflux pump activity

Assessment of efflux pump activity was conducted by the automated EB method as described previously [START_REF] Viveiros | Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method[END_REF]. This method affords a real-time estimation of accumulation of EB and its efflux. Briefly, strains were cultured in MH medium until they reached an optical density at 600 nm (OD 600 ) of 0.6 and centrifuged at 13 000 rpm for 3 min. The pellets were re-suspended in saline buffer, the OD 600 adjusted to 0.6 and 1 mL aliquots were transferred to microtubes. The microtubes were centrifuged and the pellets were re-suspended in saline buffer at pH 5, 7 or 8. Aliquots of 0.045 mL were transferred to microtubes of 0.1 mL and then 0.045 mL of EB in glucose-free saline of pH 5, 7 and 8 was added to A c c e p t e d M a n u s c r i p t 9 the respective tubes of the same pH. Because metabolic energy is required for efflux of EB, the absence of glucose ensures sufficient accumulation of the agent. The final concentration of EB for all experiments was 1.0 mg/L. As concentrations of EB much greater than 1 mg/L exceed the ability of the cell to extrude the agent, the level of intracellular agent rapidly increases and results in its intercalation between the nucleic bases of DNA. EB when bound to DNA is no longer available for extrusion [START_REF] Sharples | Correlation of the base specificity of DNAintercalating ligands with their physico-chemical properties[END_REF]. The tubes were rapidly transferred to a Corbett 3000 thermocycler (Qiagen, Doncaster, Australia) programmed with cycles of 1 min at a constant temperature of 37 C. The total average time from addition of the EB-saline to as many as 12 individual tubes to the transfer of the tubes to the instrument and re-starting the instrument did not exceed 60 s.

Accumulation of EB in each tube was followed on a real-time basis by assessment of fluorescence emitted. Excitation and emission wavelengths were 535 nm and 585 nm, respectively. Whereas the medium containing 1.0 mg/L of EB does not appreciably fluoresce, as the concentration of EB builds up in the periplasm of the Gram-negative bacterium, fluorescence is readily detected by the instrument [START_REF] Viveiros | New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps[END_REF]. Accumulation of EB at various pH levels was followed for up to 25 min. Assessment of efflux of EB at pH 5, 7 and 8 was conducted as follows.

Accumulation of EB at pH 5, 7 and 8 as described above was first conducted for up to 25 min, after which time the instrument was stopped and 0.010 mL of saline at pH 5, 7 and 8 either lacking or containing glucose to yield a concentration of A c c e p t e d M a n u s c r i p t 10 0.4% was added to the respective tubes. The tubes were then transferred to the instrument and the instrument was re-started. The total amount of time for this addition did not exceed 2.5 min. Fluorescence was followed for a minimum period of 10 min.

The effects of varying concentrations of CCCP and PAN on efflux of EB was carried out as follows. Accumulation of EB in glucose-free saline of pH 5, 7 and 8 was conducted as described and when the plateau of accumulation was reached the instrument was stopped and 0.045 mL of glucose-saline of pH 5, 7 and 8 containing varying containing CCCP and PAN was added to the salinecontaining tubes at the respective pH. The tubes were placed into the instrument, the instrument was re-started and fluorescence was followed for up to 15 min. The total time for these additions did not exceed 2.5 min. For some experiments, addition of CCCP at varying concentrations was in matched-pH glucose-free medium. This component of the experiment afforded an additional control that would define any role of metabolic energy in conjunction with any effect produced by given concentrations of CCCP on efflux of EB and modulation of efflux at a given pH. The concentrations of CCCP and PAN were <0.25 MIC and had no effect on the viability of the E. aerogenes strains employed in this study.

A c c e p t e d M a n u s c r i p t

Results

The MICs of various antibiotics against the E. aerogenes strains used in this study are presented in Table 1. As noted in the table, the CM64 and EA27 strains that overexpress the AcrAB efflux pump exhibit a MDR phenotype. Evaluation of the effect of agents for inhibitory activity against efflux requires that the agent be used at a concentration that has no significant effect on the viability and growth rate of the bacterium [START_REF] Viveiros | Isoniazid-induced transient high-level resistance in Mycobacterium tuberculosis[END_REF]. Consequently, the MICs of EB, CCCP and PAN against each of the E. aerogenes strains were also determined in MH broth at pH 5 and 8, thus allowing the selection of concentrations that had no effect on the viability of the strains employed in this study (data not shown).

The effect of pH on retention of EB by the E. aerogenes strains is illustrated in Fig. 1. Whereas at pH 8 fluorescence is detected for all of the strains at 0.5 mg/L EB, at pH 5 the CM64 and EA27 strains do not fluoresce even with a concentration as high as 2.5 mg/L EB. Unlike these latter MDR strains, the ATCC 13048 strain begins to fluoresces with 0.2 mg/L EB at pH 5 (data not shown).

These results collectively suggest that: (i) the viability of the organism is not affected by concentrations as high as 600 mg/L EB; (ii) permeability of the strains to EB at pH 8 is sufficient with a concentration of EB as low as 0.5 mg/L; (iii) the strains are able to extrude EB given the difference observed between ATCC and resistant strains; and (iv) the MDR strains EA27 and CM64 have an A c c e p t e d M a n u s c r i p t 12 overexpressed efflux pump system that is readily evident at pH 5 compared with that present in the ATCC 13048 strain [START_REF] Bolla | Membrane permeability and regulation of drug 'influx and efflux' in enterobacterial pathogens[END_REF][START_REF] Martins | An AcrAB-mediated multidrug-resistant phenotype is maintained following restoration of wild-type activities by efflux pump genes and their regulators[END_REF][START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF].

The effect of pH and glucose on the accumulation of EB by the strains was evaluated by the automated EB method [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF]. As shown in Fig. 2, accumulation of EB by the ATCC 13048 strain is not affected by pH unless glucose is absent from the medium, and only at pH 8. Similar results were obtained with the MDR strains EA27 and CM64 (data not shown). These results suggest that at pH 8 extrusion of EB is markedly affected by the absence of glucose, as would be expected if the efflux pump system at this pH was dependent on the membrane energy conferred by glucose metabolism (hydrolysis of ATP). Because no accumulation per unit period of time takes place at pH 5 where the pH is strong [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF], intracellular concentration of EB is dependent only on the availability of protons and is independent of metabolic energy (glucose). The accumulation assays without glucose at pH 5 and 8 were repeated and after 25 min the instrument was put into its pause mode and pH-matched saline containing glucose to yield a final concentration of 0.4% was added and the instrument re-started. As shown in Fig. 3, addition of glucose at pH 8 to the ATTC 13048-and CM64-containing tubes immediately causes a drop in fluorescence from 10 to 25 fluorescence units compared with the control without glucose. This drop in fluorescence is maintained for the duration of the assay. At pH 5, glucose has no effect. These results demonstrate that at pH 8 efflux is dependent upon a source of metabolic energy (glucose).

A c c e p t e d M a n u s c r i p t 13

To determine that the degree of fluorescence reflective of accumulation of EB is indeed due to an efflux pump system, the EB accumulation assay was repeated with glucose-free saline at pH 5 and 8 for each of the E. aerogenes strains. After the instrument was put into pause mode, varying concentrations of CCCP, an uncoupler of membrane energy, in glucose-containing saline of pH 5 and 8 were added, the instrument was re-started and the amount of relative fluorescence was followed for an additional 10 min. Addition of pH-matched saline with and without glucose served as controls. The data presented in Fig. 4 are for addition of 5 mg/L CCCP. As is evident from Fig. 4, addition of CCCP at pH 5 causes an increase in the amount of fluorescence due to EB retained by the ATCC 13048 strain and a considerably lower increase for the two MDR strains EA27 and CM64. At pH 8, addition of CCCP markedly increases accumulation of EB in the ATCC 13048 and EA27 strains to the same level but does not affect the accumulation of EB by the CM64 strain, suggesting that at pH 8 the extrusion of EB is taking place by an ABC transporter that receives its energy directly from the hydrolysis of ATP [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF].

The smaller effect of CCCP on the accumulation of EB at pH 5 noted for the ATCC and EA27 strains is interpreted to be due to the large concentration of surface protons [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF][START_REF] Mulkidjanian | Protons @ interfaces: implications for biological energy conversion[END_REF] that exceed the binding capacity of CCCP. At pH 8, the proton concentration is rapidly bound such that the antiport proton-dependent efflux pump system of the ATCC 13048 and EA27 strains is denied the required PAN has been repeatedly described as an EPI [START_REF] Kadlec | Efflux-mediated resistance to florfenicol and/or chloramphenicol in Bordetella bronchiseptica: identification of a novel chloramphenicol exporter[END_REF][START_REF] Begic | Fluoroquinolone resistance of Serratia marcescens: sucrose, salicylate, temperature, and pH induction of phenotypic resistance[END_REF]. However, our recent results suggest that PAN has no effect on efflux but has an effect on accumulation. The effect of PAN on intracellular concentration is not due to direct inhibition of efflux pump activity but rather to competition with EB transport [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF][START_REF] Martins | An instrument-free method for the demonstration of efflux pump activity of bacteria[END_REF][START_REF] Viveiros | Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method[END_REF]. Nevertheless, we first assessed PAN for any effect on efflux, and hence accumulation, of EB by each of the strains in glucose-free medium at pH 5 and 8; after 25 min, 20 mg/L and 40 mg/L PAN in glucose-containing medium was added. As shown in Fig. 5, addition of PAN in glucose-containing saline at pH 5 and 8 has no effect on accumulation of EB by any of the E. aerogenes strains, even with a concentration as high as 40 mg/L. The additions contained glucose to ensure that the metabolic energy required for efflux at pH 8 was readily available.

Discussion

The two overexpressing strains studied in this work differ in terms of overexpression of efflux pumps, mutations and porin expressions [START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF][START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF].

According to Ghisalberti et al. [START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF], strain CM64, which became resistant A c c e p t e d M a n u s c r i p t 15 following prolonged exposure to increasing concentrations of chloramphenicol, presents overexpression of the AcrAB-TolC efflux pump. However, strain EA27, a clinical isolate also resistant to chloramphenicol, was reported as having a deletion in the acrR gene that induces upregulation of the acrAB efflux pump operon as well as exhibiting lower expression of Omp36, the major non-specific porin of E. aerogenes [START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF]. In contrast to EA27, the opposite is true for the CM64 and the parental strains [START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF][START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF]. These differences in resistance mechanisms adopted by the two MDR strains were also visible in our work. CM64 shows resistance mechanisms dependent on pH and glucose availability. On the other hand, these effects are not evident for the EA27 and ATCC strains. Because the mechanisms of resistance of the CM64 strain are based on overexpression of the AcrAB efflux pump, extrusion of EB is mediated by proton changes obtained directly from the transmembrane proton gradient. At pH 8, owing to slow dissociation of the EB substrate from the AcrB transporter [START_REF] Su | Ligand-transporter interaction in the AcrB multidrug efflux pump determined by fluorescence polarization assay[END_REF], efflux by the RND AcrAB efflux pump may be limited. It is at this pH 8 where the need for metabolic energy (glucose) is evident, suggesting the involvement of a putative ABC transporter [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF].

At neutral pH, the energy derived from glucose or other sources is used to export cytoplasmic H + to the periplasmic space where it is used by the AcrB pump for drug antiport activity. Addition of CCCP collapses the energy of proton transport to the periplasm, reducing the H + concentration within the periplasm and consequently altering AcrB function. At acidic pH, the proton periplasmic PAN is again shown not to have any effect on EB efflux by Gram-negative bacteria [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF][START_REF] Viveiros | New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps[END_REF][START_REF] Viveiros | Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method[END_REF], confirming that the mechanism by which PAN reduces efflux-mediated resistance to a given antibiotic is due to competition between PAN and EB [START_REF] Lomovskaya | Waltzing transporters and 'the dance macabre' between humans and bacteria[END_REF]. The degree of competition has recently been shown to be in favour of extrusion of PAN, resulting in a Henri-Michaelis-Menten constant (K M ) of 4.21 mg/L versus 1 mg/L EB by the efflux machinery of E. coli [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF]. Lastly, because dissociation of an AcrB substrate is very fast at low pH [START_REF] Su | Ligand-transporter interaction in the AcrB multidrug efflux pump determined by fluorescence polarization assay[END_REF], if a true inhibitor of an efflux pump is to be designed its activity must be assessed at pH 5 to ensure that it truly binds and inhibits the RND efflux pump of Gram-negative bacteria.

It is also interesting to mention that PAN is able to increase significantly, at neutral pH, the intracellular accumulation of chloramphenicol in the E. aerogenes strains tested [START_REF] Chevalier | Quinazoline derivatives are efficient chemosensitizers of antibiotic activity in Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa resistant strains[END_REF]. This increase suggests that PAN and chloramphenicol may use the same affinity sites in the AcrB cavity, in contrast to EB. Consequently, PAN may successfully compete for extrusion with chloramphenicol as previously reported and partially restore chloramphenicol susceptibility in different efflux-mediated resistant strains [START_REF] Martins | pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: protection during its passage and eventual colonization of the colon[END_REF]. Alternatively, EB may use a that is open to the TolC exit canal [START_REF] Tal | A coordinated network of transporters with overlapping specificities provides a robust survival strategy[END_REF].

At this moment, it is important to note that E. aerogenes, like E. coli, is a commensal bacterium of the digestive tract where it faces an acidic pH that consequently may provide sufficient periplasmic proton concentration to drive the proton antiport necessary for drug extrusion. a CM 64 strain overexpresses the AcrAB-TolC efflux pump [START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF][START_REF] Ghisalberti | Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes[END_REF].

b EA27, a clinical isolate, is resistant to chloramphenicol and has a deletion in the acrR gene that induces upregulation of the acrAB efflux pump operon as well as exhibiting reduced expression of Omp36, the major non-specific porin of E.

aerogenes [START_REF] Ghisalberti | Chloroquinolines block antibiotic efflux pumps in antibiotic-resistant Enterobacter aerogenes isolates[END_REF].

Edited Table 1 A c c e p t e d

A

  c c e p t e d M a n u s c r i p t 14 energy for its action. At pH 8 any contribution made by the presence of metabolic energy is masked by the acute accumulation of EB produced by CCCP. Unlike the effects of CCCP on the ATCC 13048 and EA27 strains, addition of CCCP has no effect on the accumulation of EB by the CM64 strain.

A

  c c e p t e d M a n u s c r i p t 16 concentration is high and CCCP cannot downregulate the activity of the AcrB pump.

A

  c c e p t e d M a n u s c r i p t parallel efflux pump during the fist step before reaching the central AcrB cavity

Fig. 1 .

 1 Fig. 1. Effect of pH on the accumulation of ethidium bromide (EB) by strains of

Fig. 2 .

 2 Fig. 2. Effects of pH and glucose (glu) on the accumulation of ethidium bromide

Fig. 3 .

 3 Fig. 3. Effect of glucose (glu) on efflux of ethidium bromide at pH 5 and 8 by (A)

Fig. 4 .

 4 Fig. 4. Effect of 5 mg/L carbonyl cyanide m-chlorophenylhydrazone (CCCP) on

Fig. 5 .

 5 Fig. 5. Effect of 40 mg/L Phe-Arg--naphthylamide (PAN) on efflux of ethidium

Table 1

 1 Antimicrobial susceptibilities of the Enterobacter aerogenes strains studied

	[27,28]
		MIC (mg/L)
		ATCC 13048 CM 64 a EA 27 b
	Cefepime	0.125	0.5	64/128
	Imipenem	0.25	0.25	8
	Ceftazidime	0.5	1	512
	Chloramphenicol 8	512	512/1024
	Tetracycline	2	16	16
	Nalidixic acid	8	128	512
	Norfloxacin	0.5	4	256
	Ciprofloxacin	0.125	1	32
	Ofloxacin	0.5	N/D	>128
	Sparfloxacin	0.125	1	256
	Erythromycin	128	512	N/D
	Gentamicin	1	1	2
	MIC, minimum inhibitory concentration; N/D, not determined.

Funding

None.

Competing interests

None declared.

Ethical approval

Not required.


M Barry

Malcolm R Kell
email: malcolm.kell@nbsp.ie

RADIOTHERAPY AND BREAST RECONSTRUCTION: A META-ANALYSIS

Keywords: Postmastectomy radiotherapy, immediate and delayed breast reconstruction, prosthetic breast reconstruction, autologous breast reconstruction, postoperative morbidity

Introduction:

The optimum sequencing of breast reconstruction (BR) in patients receiving postmastectomy radiation therapy (PMRT) is controversial.

Methods

: A comprehensive search of published studies that examined postoperative morbidity following immediate or delayed breast reconstruction with combined radiotherapy was performed. Medical (MEDLINE & EMBASE)

databases were searched and cross-referenced for appropriate studies where morbidity following BR was the primary outcome measured.

. Results: 1,105 patients were identified from 11 appropriately selected studies.

Patients undergoing PMRT and BR are more likely to suffer morbidity compared to patients not receiving PMRT ((OR) = 4.2; 95% CI, 2.4-7.2(no PMRT vs.

PMRT)). Reconstruction technique was also examined with outcome when

PMRT was delivered after BR and this demonstrated that autologous reconstruction is associated with less morbidity in this setting ((OR) = 0.21; (95% CI, 0.1-0.4 (autologous vs. implant based)). Delaying BR until after PMRT had no significant effect on outcome ((OR) =0.87; 95% CI, 0.47-1.62 (delayed vs.

immediate)).

Conclusions: PMRT has a detrimental effect on BR outcome. These results suggest that where immediate reconstruction is undertaken with the necessity of PMRT, an autologous flap results in less morbidity when compared to implant based reconstruction.

INTRODUCTION:

Breast conservation surgery provides excellent locoregional control with improved quality of life when compared with mastectomy [START_REF] Fisher | Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF]. However many patients still require mastectomy as the optimum therapeutic cancer procedure, and many opt to undergo immediate breast reconstruction [START_REF] Chevray | Timing of breast reconstruction: immediate versus delayed[END_REF]. It is therefore essential that an appropriate strategy be proposed regarding the timing of reconstruction and postoperative radiotherapy. The enthusiasm for postmastectomy breast reconstruction aims to achieve good functional aesthetic outcome and maintain their quality of life, without negatively affecting the prognosis or detection of cancer recurrence [START_REF] Meretoja | Local recurrence of stage 1 and 2 breast cancer after skin sparing mastectomy and immediate breast reconstruction in a 15 year series[END_REF].

The Danish Breast Cancer Cooperative Group, 82b and 82c, trials in conjunction with findings from the Canadian trial, collectively demonstrate that patients randomised to receive post-mastectomy radiation have a lower 10-year rate of local regional recurrence and an additional survival advantage associated with post-mastectomy radiotherapy [START_REF] Overgaard | Postoperative radiotherapy in high risk premenopausal women with breast cancer who receive adjuvant chemotherapy[END_REF][START_REF] Ragaz | Adjuvant radiotherapy and chemotherapy in node positive premenopausal women with breast cancer[END_REF]. Based on these, and similar studies, there are now clear guidelines regarding the indications for postmastectomy radiation which are; large tumour size, direct involvement of the skin, and 4 or more metastatic axillary lymph nodes [START_REF] Recht | Postmastectomy radiotherapy: clinical practice guidelines of the American Society of Clinical Oncology[END_REF][START_REF] Harris | Consensus Statement on postmastectomy radiation therapy[END_REF][START_REF] Rutqvist | A systematic overview of radiation therapy effects in breast cancer[END_REF]. However, despite reducing loco-regional recurrences and increasing disease-free survival [START_REF] Truong | Optimizing locoregional control and survival for women with breast cancer: a review of current developments in postmastectomy radiation[END_REF], post mastectomy radiotherapy may negatively affect reconstruction outcome. As a consequence, several studies have demonstrated that immediate breast reconstructions in irradiated patients are associated with the potential for significant postoperative morbidity [START_REF] Wong | Incidence of major corrective surgery after post-mastectomy breast reconstruction and radiation therapy[END_REF][START_REF] Anderson | Low complication rates are achievable after postmastectomy breast reconstruction and radiation therapy[END_REF]. As a result, breast reconstruction may be delayed until the final pathological results are available from the mastectomy specimen and the indication for radiotherapy can be established. Therefore, the objective of this meta-analysis is to examine the published evidence on whether patients requiring post-mastectomy radiotherapy should have an immediate or delayed reconstruction and whether a prosthesis or autologous reconstruction is associated with the optimum outcome in terms of postoperative morbidity.

Immediate

METHODS:

Identification of studies

MEDLINE

Statistical Analysis

For post-operative complications in each study, the odds ratio (O.R.) of the simple proportions of events was estimated with its variance and 95% CI.

Heterogeneity between the O.R.s for the same outcome between studies was assessed by use of the X 2based Q statistic [START_REF] Lau | Quantitative synthesis in systematic reviews[END_REF]. Data were then combined across studies by the use of general variance methods with fixed and random effects models [START_REF] Kroll | Immediate reconstruction: Current status in cancer management[END_REF]. Analyses were conducted using StatsDirect version 2.5.6

(StatsDirect Ltd, Chesire, United Kingdom) and SPSS version 12.0 (SPSS, Inc, Chicago, IL). All statistical tests were two tailed.

RESULTS:

Eligible Studies 20 potentially eligible studies were identified that examined the effects of radiotherapy on immediate or delayed breast reconstructions. 9 studies were excluded from the meta-analysis due to low numbers (e.g. n < 15) or incomplete data set regarding postoperative morbidity. Of the 11 studies selected, 4 were studies that examined the effects of RT on immediate BR using implant or expanders. 4 were studies that examined the effects of RT on immediate BR using either a prosthesis or autologous flap. Finally 4 remaining studies evaluated the effects of RT on immediate versus delayed autologous BR.

(See Consort flow of study selection (Figure 1)).

A

DISCUSSION:

The findings of this meta-analysis, that an immediate BR using a prosthesis only in the presence of RT is associated with an increased risk of postoperative complications ((OR) = 4.2; 95% CI, 2.4-7.2(no PMRT vs. PMRT)), is supported by Aschermann et al, who conducted a retrospective review of 104 patients (123 breasts) who underwent mastectomy followed by implant breast reconstruction and demonstrated that complications requiring prosthetic device removal or replacement, as well as total complications, were more frequent in breasts that received radiation than breasts that did not (18.5 percent versus 4.2 percent for complications requiring prosthetic removal or replacement, p < or = 0.025, and 40.7 percent versus 16.7 percent for total complications, p < or = 0.01) [START_REF] Ascherman | Implant reconstruction in breast cancer patients treated with radiation therapy[END_REF]. On analysis of table 1 and 2 below, it is apparent that patients with immediate implant based reconstructions, receiving radiotherapy have significantly greater incidences of complications, compared to those who did not receive radiotherapy.

Immediate breast reconstruction following a skin-sparing mastectomy has a number of advantages over the delayed reconstruction. Firstly, it provides a more enhanced aesthetic result due to preservation of the infra-mammary fold, allowing a more natural appearance and there is also the option to adjust the position of the scar [START_REF] Kroll | Immediate reconstruction: Current status in cancer management[END_REF]. More importantly for the patient, it provides enormous psychosocial benefits by restoring femininity and improving vitality, sexuality and quality of life [START_REF] Kroll | Immediate reconstruction: Current status in cancer management[END_REF]. In the absence of implants, immediate breast reconstruction has a very favourable morbidity profile even when exposed to adjuvant radiotherapy [START_REF] Huang | Comparison of local recurrence and distant metastases between breast cancer patients after postmastectomy radiotherapy with and without immediate TRAM flap reconstruction[END_REF]. Moreover, it is oncologically safe with an acceptable local recurrence rate [START_REF] Huang | Comparison of local recurrence and distant metastases between breast cancer patients after postmastectomy radiotherapy with and without immediate TRAM flap reconstruction[END_REF]. Despite this, advocates of delayed breast reconstruction suggest that there are two main problems with immediate breast reconstruction in the presence of radiotherapy. Firstly, radiation therapy can adversely affect the cosmetic outcome and cause increased postoperative complications [START_REF] Motwani | The impact of immediate breast reconstruction on the technical delivery of postmastectomy radiotherapy[END_REF].

Secondly, immediate breast reconstruction can impair the efficacy and delivery of radiotherapy [START_REF] Motwani | The impact of immediate breast reconstruction on the technical delivery of postmastectomy radiotherapy[END_REF]. Autologous reconstructions have a more predictable response than implants to radiotherapy however the exact sequencing of this therapy is contentious. Kronowitz et al, suggested the concept of the delayed-immediate breast reconstruction as a potential solution [START_REF] Kronowitz | Delayed-immediate breast reconstruction[END_REF]. This involves placing a tissue expander at the time of a skin-sparing mastectomy and waiting for the final pathological results of the specimen. If radiotherapy is not required, an immediate reconstruction is proposed and if radiotherapy is required, a delayed reconstruction is advised. Unfortunately, this requires two operations, which are associated with significant psychological and cost implications.

McKeown et al, demonstrated that patient satisfaction with cosmetic outcome was similar between patients undergoing immediate and delayed reconstruction (autologous LD) plus radiotherapy [START_REF] Mckeown | Weiler-Mithoff The timing of autologous latissimus dorsi breast reconstruction and effect of radiotherapy on outcome[END_REF]. Interestingly, they also noted that most patients in retrospect would have preferred an immediate reconstruction [START_REF] Mckeown | Weiler-Mithoff The timing of autologous latissimus dorsi breast reconstruction and effect of radiotherapy on outcome[END_REF]. In a prospective study by Thomson that assessed 73 women post immediate implant-assisted LD or autologous LD reconstruction with or without radiotherapy, there was no difference between the groups in terms of overall cosmetic outcome as determined by the patients. When a panel of independent cosmetic assessors reviewed the photographic evidence at different intervals over a defined time period, they concluded that while radiotherapy had an adverse effect on aesthetic outcome (p=0.0002) this was more obvious in the implant-assisted LD group [START_REF] Thomson | A prospective longitudinal study of cosmetic outcome in immediate latissimus dorsi breast reconstruction and the influence of radiotherapy[END_REF].

Kroll and colleagues, reviewed 1,384 free-flap procedures performed for reconstruction of the breast or of head and neck defects. They assessed the effects of prior irradiation of the recipient site on the incidence of total flap loss which were more common in flaps transferred to previously irradiated sites [START_REF] Kroll | Does prior irradiation increase the risk of total or partial freeflap loss?[END_REF].

It is evident from this study (table 5 and6) that exposure of an autologous flap to radiotherapy increases the postoperative complication rates irrespective of whether an immediate or delayed reconstruction is performed (30.1% versus

32.1% respectively).

The potential limitations of this study are that patient selection criteria for either prosthetic-based or autologous BR were poorly defined and may have differed between centers and time periods. There was also variation in the RT treatments used in terms of both dose and use of a boost. It is possible that the incidence of postoperative BR complication rates varies with the dose of RT used and this could not be assessed in our analysis.

Finally, in addition to selecting the appropriate timing of radiotherapy and reconstruction, appropriate patient selection is also paramount to consistently obtain successful outcomes. A BMI of less than 30 is associated with a better outcome in all reconstructive surgical procedures [START_REF] Chang | Effect of obesity on flap and donor site complications in free transverse rectus abdominis myocutaneous flap breast reconstruction[END_REF]. A history of smoking and diabetes are also poor prognostic indicators for myocutaneous flap viability [START_REF] Chang | Effect of smoking on complications in patients undergoing free TRAM flap breast reconstruction[END_REF].

Patient selection for reconstruction was not stated in many of the studies involved in this meta-analysis and therefore could not be included.

Conclusion:

Post-mastectomy radiation, irrespective of the method of reconstruction, increases the incidence of postoperative complications however, this study has demonstrated that in this setting, an autologous flap offers a more favourable outcome in terms of morbidity than expander/implant reconstruction. There is a paucity of high quality conclusive data regarding the correct sequencing of breast reconstruction and radiotherapy. The majority of studies involve small numbers of patients in single centres with retrospective anaylsis. Multicenter randomised controlled trials with longer follow-up times and better specified parameters are necessary to validate any future strategies regarding the optimum timing of radiotherapy and breast reconstruction.

Unfortunately, these studies are difficult to perform, as it is difficult to ethically justify demanding that patients undergo an immediate versus a delayed reconstructive procedure due to a randomisation process. Therefore, in the absence of level I evidence, the current data suggests that immediate breast reconstruction with PMRT may be undertaken though morbidity is higher with either immediate or delayed technique. The timing and effect of radiotherapy on breast reconstruction must be discussed to ensure informed opinion and consent of the patient. The patient's expectations, preferences, motivations as well as their level of understanding should be explored to enhance postoperative satisfaction and quality of life [START_REF] Saulis | A retrospective analysis of patient satisfaction with immediate postmastectomy breast reconstruction: comparison of three common preocedures[END_REF]. Odds ratio meta-analysis plot of Immediate BR + RT v Delayed BR + RT.

  breast reconstruction without radiotherapy, offers enhanced aesthetic and safe oncological outcomes, is more cost-effective and provides a positive psychological effect [12-16]. Despite this, less than 20% of patients having a mastectomy have immediate breast reconstruction in the United States [2]. This may be due to patient choice or possibly apprehension on the part of the surgeon that radiotherapy maybe required postoperatively which potentially could compromise the reconstruction. Radiotherapy can cause unpredictable changes in all tissues and prosthetic materials [17]. It has a biphasic nature with the acute effects occurring over days to weeks and a delayed response, which can occur from months to years after completion of the therapy [18]. The acute phase usually involves acute inflammatory changes which may lead to desquamation or even necrosis of tissue [19]. The delayed phase involves atrophy, fibrosis and inhibition of normal wound healing mechanisms [19]. Furthermore, opponents of immediate breast reconstruction, suggest it may alter chest wall anatomy and therefore distort the geometrics of the radiation field design leading to under/overdosing the targeted and underlying tissues [20, 21]. However, Stralman et al demonstrated a locoregional recurrence rate of 6% in 100 patients who had a mastectomy with immediate (implant/autologous) reconstruction followed by radiotherapy with a mean follow up of 108+/-26 months. This suggests, there is no significant decrease in efficacy or delivery of radiation post-immediate implant or autologous reconstruction [22]. Furthermore, Huang et al, compared the incidence of local recurrence and distant metastasis of postmastectomy radiotherapy for breast cancer patients with and without immediate TRAM flap reconstruction. They reported no statistical differences in the incidences of locoregional recurrence or distant metastasis between the TRAM flap and the non-TRAM flap patients [23].

Figure 1 .

 1 Figure 1. Quorom diagram: Consort flow of study selection.

TOR=

  Type of reconstruction, RT = Radiotherapy, Fib = Fibrosis, FN = Fat necrosis, Inf = Infection, Re-op = Re-operation, Immed = Immediate reconstruction, Delay = Delayed reconstruction, N/A = Not available.

Figure 4 .

 4 Figure 4. Meta-analysis of immediate versus delayed autologous breast

morbidity was defined in terms of capsular contracture, infection, fat necrosis, fibrosis and the necessity to re-operate on the patient. The rates of these complications are displayed in tables 2, 4 and

	Postoperative complications in BR in the presence of RT
	Patients undergoing PMRT and immediate BR (n = 196) are more likely to suffer
	morbidity when compared to patients not receiving PMRT (n = 229) ((OR) = 4.2;

total of 1,105 patients were identified from 11 selected studies [10, 11, 15, 22, 25-31]. These were subsequently divided into 3 cohorts for subgroup analyses. The first group (n = 424) were patients' with immediate BR using a prosthesis alone with/without RT. The second subgroup (n = 380) compared patients with immediate autologous BR versus prosthesis alone in the presence of RT. The last subgroup analysis (n = 301) compared the effects of RT on immediate versus delayed autologous BR. 95% CI, 2.4-7.2(no PMRT vs. PMRT) see figure 2.). Finally reconstruction technique was examined when PMRT was delivered after BR and this demonstrated that autologous reconstruction is the superior reconstruction technique in terms of postoperative morbidity ((OR) = 0.20; (95% CI, 0.1-0.4

(autologous vs. implant based) see figure

3

). Postoperative 6. Interestingly, the effect of delaying BR until after PMRT had no significant effect on outcome ((OR) =0.87; 95% CI, 0.47-1.62 (delayed vs. immediate) see figure

4

.).

Table 1 .

 1 Effect of radiotherapy on immediate implant/expander based breast reconstruction.

	20 studies of BR +	9 studies excluded
	RT	due to insufficient
		data
	11 studies of BR +	4 studies of BR +
	RT	RT with
		implants/expanders
	7 studies of BR with autologous flaps + RT	4 studies of implant versus autologous flaps + RT.
	3 studies comparing
	immediate versus
	delayed autologous
	flap recon + RT.

Table 2 : Postoperative complications in patients undergoing immediate breast reconstruction with an expander/implant with or without post- mastectomy radiotherapy.

 2

	Author	MOR Patients	RT	CC %	Inf. %	Nec. % Reop%
	Tallet	E/I	55	Yes	29%	9%	12.7%	3.6%
	[25]
			22	No	0%	9%	0%	4.5%
	Krueger	E/I	19	Yes	26%	37%	N/A	37%
	[26]
			62	No	10%	19%	N/A	8%
	Cordeiro	E/I	81	Yes	1.2%	3.7%	N/A	5%
	[27]
			75	No	3.7%	0.9%	N/A	0.7%
	Whitfield	E/I	41	Yes	19.5%	N/A	N/A	N/A
	[28]
			69	No	0%	N/A	N/A	N/A
	MOR = Method of reconstruction, RT = Radiotherapy, CC = Capsular
	contracture, Inf = Infection, Nec = Necrosis, Re-op = Reoperation.

Table 3 :

 3 Effect of radiotherapy on implant versus autologous breast reconstruction.

	Author	TOR	Patie	MOR	Radiot	Complic
			nt No		x	s.
	Anderson [11] Immediate 35	Autolog.	Yes	0%
		Immediate 50	Implant/Ex	Yes	5%
	Jhaveri [29]	Immediate 23	Autolog.	Yes	8.7%
		Immediate 69	Implant	Yes	55%
	Wong [10]	Immediate 47	Autolog	Yes	9%
		Immediate 15	Implant based Yes	40%
	Stralman [22] Immediate 59	Autolog	Yes	13.5%
		Immediate 82	Implant	Yes	34%
	TOTAL	Immediate 164	Autolog	Yes	7.8%
	TOTAL	Immediate 216	Implant	Yes	33.5%
	TOR = timing of reconstruction, MOR = Method of reconstruction, Ex =
	Expander, Autolog. = Autologous reconstruction.

Table 4 : Postoperative complications in patients undergoing post- mastectomy radiotherapy with either immediate implant or immediate autologous breast reconstruction.

 4

	Author	N= MOR RT	CC.%	Inf.%	Fib % FN%	Reop%
	Anderson	35	Autol Yes 0%	0%	5.7%	17.2% 0%
	[11]
		50	E/I	Yes 4%	4%	0%	0%	33%
	Jhaveri	23	Autol Yes 0%	0%	N/A	N/A	0%
	[29]
		69	E/I	Yes 42.9% 28.6%	N/A	N/A	20.3%
	Wong	47	Autol Yes 0%	8.5%	N/A	N/A	21.3%
	[10]
		15	E/I	Yes 40%	13.3%	N/A	N/A	66.6%
	Stralman	59	Autol Yes 0%	N/A	N/A	N/A	13.5%
	[22]
		82	E/I	Yes 17%	N/A	N/A	N/A	34.1%
	N = Number

of patients, MOR = Method of reconstruction, RT = Radiotherapy, CC = Capsular contracture, Inf = Infection, Fib = Fibrosis, FN = Fat necrosis, Reop = Re-operation. Figure 3:

  Meta-analysis of data comparing immediate autologous versus prosthetic BR in the presence of postoperative RT.

	Author	TOR	Patient	MOR			Radiotx Comp.
			No
	Odds ratio meta-analysis plot Immediate Autologous BR +RT v Prosthetic BR + RT Williams Immediate 19 pTRAM Yes 31%
	[30]
	Anderson	Delayed	108	pTRAM		Yes		25%	0.27 (0.00, 7.61)
	Tran [31]	Immediate	32	fTRAM		Yes		9.4%
	Jhaveri								0.10 (0.01,
		Delayed	70	fTRAM		Yes		0.46) 14.3%
	Spear [15]	Immediate	34	pTRAM		Yes		50%
	Wong								0.14 (0.02,
		Delayed	38	pTRAM		Yes		0.75) 57.1%
	TOTAL	Immediate 85	TRAM		Yes		30.1%
	Stralman								0.30 (0.11,
	TOTAL	Delayed	216	TRAM		Yes		0.77) 32.1%
	Combined								0.20 (0.11,
	[fixed]								0.39)
	0.01	0.1	0.2	0.5	1	2	5	10
	odds ratio (95% confidence TOR = timing of reconstruction, MOR = Method of reconstruction, Radiotx =
			interval)
	Radiotherapy, Comp = complications, pTRAM = pedicled TRAM, fTRAM = free
	TRAM.

Table 6 : Postoperative complications in patients undergoing either immediate or delayed TRAM breast reconstruction with radiotherapy.

 6

	Author	TOR	Patients RT	Fib	FN	Inf	Re-op
	Williams	Immed 19	Yes	10.5% 15.8% 5.3%	N/A
	[30]
		Delay	108	Yes	0%	17.6% 6.6%	N/A
	Tran	Immed 32	Yes	75%	43.8% N/A	9.4%
	[31]
		Delay	70	Yes	0%	8.6%	N/A	0%
	Spear	Immed 34	Yes	N/A	23.7% 5.3%	0%
	[15]
		Delay	38	Yes	N/A	23.8% 4.8%	2.4%

Data Extraction and Outcomes

The following information regarding each eligible trial was recorded: authors' names, journal, patient numbers, timing and method of reconstruction, addition of radiotherapy and the post-operative complication rate. The primary end point of


Sarah Jones

Richard J Evans

Martyn P Mahaut-Smith

ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation

Keywords: Platelets, aggregation, calcium, ectonucleotidases, ADP

come     Extracellular Ca2+ modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation

Introduction

ADP is an important platelet agonist during haemostasis and thrombosis, exerting its effects through two G-protein-coupled receptors P2Y 1 and P2Y 12 . P2Y 1 is coupled to G αq , leading to an increase in cytosolic calcium through stimulation of phospholipase C β (PLC β ) (Jin, et al 1998[START_REF] Savi | Role of P2Y1 purinoceptor in ADP-induced platelet activation[END_REF], whereas P2Y 12 is coupled to G αi , leading to activation of phosphatidylinositol 3-Kinase (PI3-K) [START_REF] Jackson | PI 3-kinase p110beta: a new target for antithrombotic therapy[END_REF][START_REF] Trumel | A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase[END_REF] and inhibition of adenylate cyclase. For platelet-platelet adhesion, and thus thrombus formation, activation of the fibrinogen receptor α IIb β 3 is required, a process that depends upon concomitant stimulation of G αq and G αi signalling pathways (Jin and Kunapuli 1998). Several platelet agonists bind to receptors coupled to G αq , including thrombin, thromboxaneA 2 and ADP. However, ADP is the only platelet agonist that stimulates G αi signalling at physiological concentrations and is therefore an essential co-stimulus to achieve full functional responses for all known platelet agonists [START_REF] Gachet | P2 receptors, platelet function and pharmacological implications[END_REF][START_REF] Paul | Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways. Role of p160 Rhoassociated coiled-coil-containing protein kinase in platelet shape change[END_REF]. Despite the central role of ADP in platelet aggregation and thrombogenesis, it is normally considered to be a weak platelet agonist due to the reversible nature of the aggregation response observed in vitro at physiological levels of external Ca 2+ [START_REF] Gachet | P2 receptors, platelet function and pharmacological implications[END_REF]. Extremely low levels of extracellular Ca 2+ abolish fibrinogen binding to α IIb β 3 integrin, however at micromolar extracellular calcium concentrations, ADP-evoked aggregation is enhanced compared to physiological Ca 2+ levels and not readily reversible [START_REF] Mustard | Factors responsible for ADP-induced release reaction of human platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF]. Although this paradoxical effect was reported more than two decades ago, the underlying basis whereby extracellular calcium modulates ADP-evoked aggregation remains unclear. It has been proposed that millimolar Ca 2+ levels inhibit TXA 2 generation via altered ERK phosphorylation [START_REF] Garcia | Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets[END_REF], leading to loss of secondary aggregation [START_REF] Mustard | Factors responsible for ADP-induced release reaction of human platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF], however exactly how Ca 2+ achieves this effect is not known. Following stimulation of platelet P2Y receptors with ADP, the duration and amplitude of the response can be regulated by two principal mechanisms, firstly desensitization of the P2Y receptors preventing further signalling, and secondly removal of ADP by ectonucleotidases.

Ectonucleotidases comprise a large family of extracellular nucleotide degrading enzymes including ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (E-NPPs), alkaline phosphatases and 5' nucleotidase [START_REF] Zimmermann | Extracellular metabolism of ATP and other nucleotides[END_REF]. ADP derived from platelets and other blood cells is thought to predominantly be metabolised by E-NTPDase1 (CD39), a membrane-bound enzyme expressed by endothelial cells, lymphocytes and macrophages [START_REF] Kansas | Expression, distribution, and biochemistry of human CD39. Role in activation-associated homotypic adhesion of lymphocytes[END_REF][START_REF] Marcus | The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39[END_REF], as well as microparticles that originate from these cell types [START_REF] Atkinson | Ecto-nucleotidases of the CD39/NTPDase family modulate platelet activation and thrombus formation: Potential as therapeutic targets[END_REF][START_REF] Banz | CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation[END_REF]. CD39 converts ADP to AMP, which is subsequently converted to adenosine, an inhibitor of platelet function, by 5'nucleotidase (CD73) expressed on endothelial cells and in plasma [START_REF] Coade | Metabolism of adenine nucleotides in human blood[END_REF][START_REF] Heptinstall | Adenine nucleotide metabolism in human blood--important roles for leukocytes and erythrocytes[END_REF][START_REF] Zimmermann | Extracellular metabolism of ATP and other nucleotides[END_REF].There is also evidence that soluble E-NPPases in plasma can degrade ADP directly to adenosine [START_REF] Birk | Role of a novel soluble nucleotide phospho-hydrolase from sheep plasma in inhibition of platelet reactivity: hemostasis, thrombosis, and vascular biology[END_REF][START_REF] Cauwenberghs | Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation[END_REF], thus ectonucleotidases can convert prothrombotic mediators into inhibitors of platelet activation.

In this study we have investigated further the mechanism(s) underlying the differential responses to ADP at physiological compared to low (micromolar) extracellular calcium concentrations. We demonstrate that degradation of ADP by Ca 2+ -dependent ectonucleotidases is an important factor in determining the amplitude and duration of platelet aggregation. The results have consequences for understanding the effectiveness of ADP as a platelet agonist, and in the selection of experimental conditions to explore P2Y receptor activation.

F

Materials and Methods

Materials

CHRONO-LUME was purchased from Labmedics (Manchester, UK), Pyruvate kinase was obtained from Roche Diagnostics Limited (East Sussex, UK), GF109203X and MRS2179 were purchased from Tocris (Bristol, UK). TXB 2 assay kits were purchased from Cambridge Bioscience LTD (Cambridge, UK). Cangrelor (ARC-69931MX) was a kind gift from AstraZeneca (Moindal, Sweden). ADP, apyrase (gradeVII), aspirin, phosphoenol pyruvate and all other chemicals were purchased from Sigma (Poole, UK).

Platelet preparation

Blood was obtained from healthy, aspirin-free, volunteers according to a protocol approved by the local ethical committee of the University of Leicester. Blood was drawn from the forearm by venepuncture into a syringe containing acid citrate dextrose anticoagulant (ACD: in mM 85 trisodium citrate, 78 citric acid, 111 glucose) 9:1 v/v. Platelet-rich plasma (PRP) was obtained by centrifugation at 700g for 5 minutes. When re-calcified, 20mM CaCl 2 (calculated using a Nomogram [START_REF] Hastings | The ionization of calcium, magnesium, and strong citrates[END_REF]) was added to citrated PRP to achieve [Ca 2+ ] o of approximately 2mM immediately prior to each experiment. The extracellular Ca 2+ in nominally Ca 2+ -free saline and in similar citrated plasma: saline mixtures has been estimated to be approximately 20µM and 17µM, respectively [START_REF] Packham | Thromboxane A2 causes feedback amplification involving extensive thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium[END_REF][START_REF] Rolf | Platelet shape change evoked by selective activation of P2X1 purinoceptors with alpha,beta-methylene ATP[END_REF].

To prepare washed platelet suspensions, apyrase (0.32 U/ml), and where stated aspirin (100µM or 1mM), were added to the PRP and platelets pelleted by centrifugation at 350g for 20 min. Platelets were then resuspended in a volume of nominally Ca 2+ free saline (in mM: 145 NaCl, 5 KCl, 1 MgCl 2 10 HEPES, 10 glucose, fibrinogen 1mg/ml pH 7.35) equal to that

Platelet aggregation

PRP or washed platelet suspensions with or without apyrase (0.32U/ml) (prior to substantial P2Y receptor desensitisation) were diluted (1:1) in saline and stimulated with ADP at 37°C under stirring conditions. Aggregation was measured using optical aggregometry (Model 400 lumi-aggregometer, Chronolog, Havertown, USA).

Platelet disaggregation

Washed, apyrase-free platelets were stimulated with ADP (10µM) at 37°C under stirring conditions in the presence of 2mM Ca 2+ . After 2 minutes, apyrase (0.32U/ml), the P2Y 1 receptor antagonist MRS2179 (10µM), the P2Y 12 receptor antagonist AR-C69931MX (1µM) or a saline control was added to the suspension. Disaggregation was assessed 3 minutes after the addition of the P2Y receptor antagonists or apyrase and calculated as a percentage of the peak ADP-evoked aggregation.

ADP measurement

The concentration of extracellular ADP was assessed by luciferin: luciferase luminescence measurements after conversion to ATP via a method adapted from [START_REF] Heath | Secretion from dense granules: luminescence method for adenine nucleotides Methods[END_REF]. Briefly, 2 minutes after addition of 10 µM ADP to plasma or apyrase-containing saline, with or without Ca 2+ , 50µl samples were removed and added to a mixture of 420µl TXB 2 measurements TXB 2 synthesis was measured as an indication of TXA 2 production due to the highly labile nature of TXA 2 . Washed platelets were stimulated with ADP (10µM) at 37°C under stirring conditions for 3 minutes in the presence and absence of apyrase (0.32U/ml), in both physiological and nominally Ca 2 -free conditions, and reactions terminated by snap freezing.

For analysis of TXB 2 , samples were thawed and centrifuged at 3000g for 10 minutes at 4°C.

The supernatant was diluted 1: 5 using the buffer supplied with the assay kit and TXB 2 determined according to the manufacturer's instructions (Cambridge Bioscience, UK).

Statistics

Results

Extracellular Ca 2+ levels regulate ADP-evoked aggregation independently of thromboxaneA 2 synthesis ADP (10 µM) evoked a sustained aggregation of platelets in plasma anti-coagulated with citrate that reduces the extracellular Ca 2+ concentration ([Ca 2+ ] o ) to the micromolar range (Fig. 1a,e; average peak aggregation of 53.9 ± 3.4%). When the medium was recalcified to approximately 2mM free Ca 2+ , the aggregation was converted to a transient response that returned to baseline levels of transmission (-2.8 ± 2%) within approximately 2 minutes (Fig. 1a,e). Previous studies of this [Ca 2+ ] o -dependent aggregation response have shown that production of TXA 2 is enhanced at micromolar compared to millimolar [Ca 2+ ] o levels and concluded that secondary stimulation of TXA 2 receptors is responsible for the reversible nature of the ADP-evoked aggregation [START_REF] Garcia | Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets[END_REF][START_REF] Mustard | Factors responsible for ADP-induced release reaction of human platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF]. However, we observed a similar effect of [Ca 2+ ] o on aggregation when thromboxaneA 2 synthesis was blocked by aspirin (Fig. 1b,e; average values 50.7 ± 3% and 0.9 ± 0.3% in low and physiological Ca 2+ levels respectively). In platelets resuspended in a physiological saline, 10 µM ADP evoked a transient aggregation response in the presence of 2mM [Ca 2+ ] o , which was also converted to a sustained response by omission of CaCl 2 from the saline (Fig. 1c,e; transmission levels 2 min after ADP of 0.6 ± 2% and 53.2 ± 5.4%, respectively), in agreement with reports by other groups [START_REF] Mustard | Factors responsible for ADP-induced release reaction of human platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF]. As observed for platelets in the presence of plasma, aspirin did not block the sustained aggregation in salines with micromolar [Ca 2+ ] o (Fig. 1d,e; average values 55.9 ± 1.3% and 7.1 ± 4.2% in low and physiological Ca 2+ levels respectively). To ensure that TXA 2 generation was completely inhibited experiments were repeated in the presence of 1mM aspirin, which also had no significant effect on the ability of reduced [Ca 2+ ] o to enhance platelet aggregation (p>0.05 data not shown). Together, these data suggest that factor(s) other than altered TXA 2 production must contribute to the ability of reduced [Ca 2+ ] o to enhance ADP-evoked aggregation.

Prevention of ADP degradation abolishes the reversal of aggregation by calcium

Since our washed platelet preparation contained apyrase (E-NTPDase1 isolated from potato to prevent P2Y receptor desensitization) and PRP has been reported to contain endogenous ectonucleotidases, we considered whether degradation of ADP contributed to the transient nature of the ADP-evoked aggregation at millimolar Ca 2+ levels. Aggregation evoked by the hydrolysis-resistant analogue ADPβS was not significantly different in the presence or absence of extracellular Ca 2+ (Fig. 2a,d; aggregation at 2 minutes of 45.8±3.7% and 42.9±5.6% in normal and low [Ca 2+ ] o , respectively; p>0.05). Moreover, when platelets were resuspended in the absence of apyrase, and experiments performed rapidly to limit effects of desensitization, ADP-evoked aggregation was also sustained in the presence of 2mM extracellular Ca 2+ (Fig. 2b,d; 59.3±3.8 and 61.9±2.4% in normal and low Ca 2+ respectively).

ADP degradation is accelerated by millimolar calcium concentrations

To directly assess the extent of ADP degradation in millimolar versus micromolar [Ca 2+ ] o concentrations, ADP (10µM) was added to apyrase-treated saline or platelet-free plasma and the ADP concentration after 2 min measured by luminescence following conversion to ATP (see methods). The concentration of ADP remaining in nominally Ca 2+ -free saline was 1.61 ± 0.06µM, which was significantly reduced to 0.079 ± 0.03µM (p<0.001) in the presence of 2mM Ca 2+ , indicating accelerated nucleotidase activity by physiological [Ca 2+ ] o (Fig. 3a).

Similarly, ADP incubated with citrated plasma was degraded from 10µM to 2.1 ± 0.27µM by enzymes endogenous to plasma, whereas under recalcified conditions the ADP remaining was markedly lower, at 0.78 ± 0.14µM (p<0.001)(Fig. 3b). A significant effect of [Ca 2+ ] o on degradation of 10µM ADP was also detected at earlier time points, as shown by measurements after only 10s (see Supplementary Fig. 1). This suggests that platelets in the presence of millimolar Ca 2+ will be exposed to a reduced level of ADP throughout most of the experiment compared to at micromolar Ca 2+ levels. In contrast, addition of 2mM MgCl 2 to nominally Ca 2+ -free saline did not significantly affect ADP degradation or lead to a transient aggregation response (see supplementary Fig. 2). Together with the data in Fig. 2, these results support the conclusion that reduced ADP degradation substantially contributes to the paradoxical amplifying effect of reducing Ca 2+ on platelet aggregation. This is also consistent with the reported enhancement of ectonucleotidase activity at millimolar concentrations of calcium compared to its nominal absence or in the presence of a chelator such as EGTA [START_REF] Christoforidis | Purification and properties of human placental ATP diphosphohydrolase[END_REF][START_REF] Marcus | The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39[END_REF][START_REF] Strobel | Chicken oviductal ecto-ATP-diphosphohydrolase. Purification and characterization[END_REF].

Reversal of degradation is due to removal of ADP, not negative feedback by adenosine.

In plasma, 5'nucleotidases convert AMP generated by the degradation of ATP and ADP to adenosine [16][17], thus we also considered whether the transient responses to ADP in plasma involved inhibition via G αs -coupled adenosine A2a receptors. In citrated PRP, adenosine (10µM) inhibited ADP (10µM)-evoked responses, resulting in aggregation traces similar to those observed with ADP in physiological calcium concentrations (supplementary Fig. 3), an effect which is abolished by the addition of adenosine deaminase (1U/ml). In recalcified PRP however, the addition of adenosine deaminase had no effect on ADP-evoked aggregation (Fig. 4), indicating that negative feedback by the generation of adenosine does not contribute to the reversibility of ADP mediated responses.

Sustained aggregation to ADP requires constant P2Y 12 stimulation

We next sought to determine whether reversal of aggregation by Ca 2+ -dependent nucleotidases was due to loss of activation of P2Y 1 , P2Y 12 or both receptors. Disaggregation of ADP (10µM)-stimulated platelets was measured in response to apyrase (0.32 U/ml), the P2Y 12 antagonist cangrelor (1µM) or the P2Y 1 antagonist MRS2179 (10µM) (Fig. 5a).

Cangrelor reversed aggregation by 74.6 ± 5.9%, comparable to that observed with apyrase 74.9 ± 1.2% (Fig. 5b). In contrast, MRS2179 caused a more moderate reversal of aggregation (reduction of 24.0 ± 7.8%; Fig 5b). Thus, reversal of ADP induced aggregation by ectonucleotidases is largely due to loss of signalling through P2Y 12 receptors.

TXA 2 generation is enhanced at low extracellular calcium concentrations independently of altered ectonucleotidase activity.

To investigate whether reduced TXA 2 generation previously reported by others [START_REF] Harfenist | Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF], Packham, et al 1987) at millimolar [Ca 2+ ] o was a consequence of termination of ADP signalling by ectonucleotidases, the of presence of apyrase (0.32 U/ml) at micromolar and millimolar [Ca 2+ ] o was examined on TXB 2 production from washed platelets 3 minutes after stimulation with 10 µM ADP (Fig. 6a). As reported previously [START_REF] Harfenist | Effect of calcium ion concentration on the ability of fibrinogen and von Willebrand factor to support the ADP-induced aggregation of human platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF], Packham, et al 1987), TXB 2 production from apyrase-treated platelets was markedly reduced at physiological extracellular Ca 2+ concentrations compared to that observed in the nominal absence of Ca 2+ . However, similar results were observed in salines lacking apyrase. This indicates that although increased

Relative contribution of ADP degradation versus desensitisation in limiting platelet

responses to ADP P2Y 1 and P2Y 12 receptors are both susceptible to receptor desensitization after prolonged agonist stimulation [START_REF] Hardy | P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms[END_REF][START_REF] Mundell | Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function[END_REF]. To determine the relative importance of receptor desensitization versus ectonucleotidase activity in ADP-evoked aggregation, the pan-PKC inhibitor GF109203X was used to attenuate receptor desensitization and aggregation was measured in citrated PRP before and after recalcification (Fig 7a). An intermediate concentration of ADP (2 µM) was used for these experiments to unmask a clear potentiating effect of PKC inhibition, which increased the sustained aggregation at 3 min from 36.9 ± 6.5% to 60.5 ± 5.6% in micromolar [Ca 2+ ] o (Fig 7b). In contrast, after recalcification, aggregation in the presence of GF109203X or vehicle control, was not significantly different and returned to baseline levels of -3.5 ± 1.6% and 2.7 ± 1.2% respectively 3 min after stimulation (Fig 7b). Thus, ADP degradation overrides any contribution by P2Y receptor desensitisation to the reversal of aggregation in physiological external Ca 2+ concentrations.

Discussion

Reports of differential platelet responses to ADP in physiological versus nominally Ca 2+ -free conditions emerged over 20 years ago [START_REF] Mustard | Factors responsible for ADP-induced release reaction of human platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF]. These studies concluded that enhanced thromboxaneA 2 production accounts for the paradoxical amplifying effect of lowering Ca 2+ on ADP-evoked aggregation. The present study now shows that altered degradation of ADP can also contribute to this phenomenon. The known Ca 2+ -dependence of ecto-ADPases [START_REF] Marcus | The endothelial cell ecto-ADPase responsible for inhibition of platelet function is CD39[END_REF][START_REF] Zimmermann | Extracellular metabolism of ATP and other nucleotides[END_REF] provides the basis for the difference observed in millimolar versus micromolar [Ca 2+ ] o and this conclusion is supported by direct measurements of ADP. The sustained aggregation evoked by ADP is largely due to stimulation of P2Y 12 receptors, consistent with previous reports of the more crucial role of this G i -coupled pathway compared to P2Y 1 in amplifying responses to ADP, collagen and thrombin receptors [START_REF] Cosemans | Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization[END_REF][START_REF] Dorsam | Central role of the P2Y12 receptor in platelet activation[END_REF], Hechler, et al 1998[START_REF] Jackson | PI 3-kinase p110beta: a new target for antithrombotic therapy[END_REF][START_REF] Trumel | A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase[END_REF]. The present results also highlight the importance of controlling for nucleotide breakdown in studies of P2 receptor signalling when the external Ca 2+ concentration is modified. For example, it is common practice to include soluble apyrase to limit P2 receptor desensitisation within in vitro experiments and simply to vary the external [Ca 2+ ] to investigate the relative contribution of Ca 2+ entry versus release pathways in nucleotide-evoked signalling events.

The limited aggregation response at normal [Ca 2+ ] o has contributed to the view that ADP is a "weak platelet agonist". However, when metabolism of ADP is limited, the ability of this agonist to stimulate sustained aggregation, as shown in the present study, is more consistent with the substantial reduction in platelet activation observed in P2Y 1 and P2Y 12 receptordeficient mice [START_REF] Andre | P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries[END_REF][START_REF] Fabre | Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice[END_REF][START_REF] Leon | Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice[END_REF] and the major role of ADP in amplifying collagen and thrombin-evoked responses in vitro. It is possible that dense granule secretion evoked by collagen and thrombin provides a more sustained source of ADP compared to the single bolus application used in standard in vitro experiments. Furthermore, ATP (and thus also ADP) remains sustained for considerable time near sites of vascular injury [START_REF] Born | Contribution of blood platelets to the pathogenesis of myocardial infarction[END_REF], probably reflecting the continual recruitment and activation of platelets during the haemostatic process. Thus, the in vitro experimental condition that limits ADP degradation, such as micromolar [Ca 2+ ] o , may more closely represent the ability of this agonist to stimulate platelet function in vivo. Alternatively, use of a non-hydrolysable analogue such as ADPβS or repeated application of ADP to replace degraded agonist should be considered within in vitro studies designed to investigate mechanisms of ADP-dependent platelet activation.

Whilst ADP degradation significantly contributed to the transient nature of the responses to ADP in millimolar [Ca 2+ ] o , we agree with earlier studies [START_REF] Garcia | Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets[END_REF][START_REF] Packham | Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents[END_REF] that TXA 2 generation is lower at millimolar compared to micromolar [Ca 2+ ] o . In our experiments, the marked enhancement of TXB 2 generation by lowering [Ca 2+ ] o was also observed in apyrase-free saline, indicating that the Ca 2+ -dependent modulation of TXA 2 generation can occur independently of effects on ectonucleotidase activity. It has been reported that reduced TXA 2 synthesis in physiological calcium concentrations is the result of inhibited ERK phosphorylation [START_REF] Garcia | Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets[END_REF], however the process by which this is achieved is unclear, and whether these effects are downstream of an extracellular event or whether calcium influx is required, remains to be investigated. Despite there being no TXA 2 generation at millimolar [Ca 2+ ] o in the absence of apyrase, aggregation was indistinguishable from that in low [Ca 2+ ] o , suggesting that an initial application of 10 µM ADP can mediate sustained aggregation independently of secondary signalling. At lower concentrations of ADP, however, the release of secondary agonists is required to achieve full aggregation, therefore the effect of extracellular calcium on TXA 2 may be more significant, and Although we did not observe any difference in aggregation within the normal physiological range of extracellular calcium concentrations (0.5-2mM) (3 donors, data not shown), results from this study demonstrate the impact of variable ectonucleotidase activity on platelet function, which may have profound implications in certain clinical conditions. It has previously been reported that in blood from patients with elevated leukocyte counts, degradation of ADP is accelerated and aggregation in response to ADP is reduced due to increased NTDPase levels [START_REF] Glenn | Raised levels of CD39 in leucocytosis result in marked inhibition of ADP-induced platelet aggregation via rapid ADP hydrolysis[END_REF][START_REF] Pulte | CD39 activity correlates with stage and inhibits platelet reactivity in chronic lymphocytic leukemia[END_REF]. Moreover, in a rat model of cholestatic liver disease where plasma ectonucleotidase activity is enhanced, reduced aggregation is exhibited in response to ADP and low dose collagen (which is dependent on ADP secretion) [START_REF] Witters | ADP-degrading enzymes inhibit platelet activation in bile duct-ligated rats[END_REF]. Conversely, individuals demonstrating reduced ectonucleotidase expression may have more reactive platelets and be more susceptible to thrombotic events. Such patients may benefit from therapeutic intervention with soluble forms of NTPDase1.

In conclusion, the present study shows that reduced degradation of ADP by ectonucleotidases contributes to the paradoxical amplification of ADP-evoked aggregation at micromolar compared to millimolar extracellular Ca 2+ levels. The sustained inside-out activation of fibrinogen receptors that occurs in response to ADP at low [Ca 2+ ] o is likely to be more representative of the potential contribution of ADP to a developing thrombus in vivo, where a constant supply of this P2Y receptor agonist from activated platelets can override enzymatic clearance in the vicinity of a developing thrombus.

  plasma. In experiments performed at physiological calcium concentrations, 2mM CaCl 2 was added to the platelets immediately prior to use.

  Records of aggregation are from individual experiments, typical of ≥4 donors. Differences between means ± s.e.m were assessed using paired student t-tests and a p value of <0.05 considered to be significant. p values indicated at levels of <0.05 (*), <0.01 (**) and <0.001 (***).

  Finally, the [Ca 2+ ] o -dependence of ADP-evoked aggregation responses in plasma was abolished when platelets were resuspended in autologous heat-treated plasma (60 o C, 30 min) to destroy enzymatic activity (Fig 2c, d; 41.1±1.1 and 41.5±1.9% in millimolar versus micromolar [Ca 2+ ] o , respectively). Together, these observations are consistent with a role for Ca 2+ -dependent nucleotidase activity in the [Ca 2+ ] o -dependence to ADP-evoked sustained aggregation responses.

  ] o reduces TXB 2 synthesis, this is not dependent on the effect of Ca 2+ on ectonucleotidases.

  -evoked aggregation by[Ca 2+ ] o , likely results from a combination of both altered ectonucleotidase activity and TXA 2 production.

Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Prevention of ADP degradation leads to sustained aggregation at physiological

Figure 3 .

 3 Figure 3. ADP degradation by apyrase and endogenous ectonucleotidases present in

Figure 4 .

 4 Figure 4. Transient ADP-evoked aggregation responses resulting from Ca 2+ -dependent

  ADP (10µM) in nominally Ca 2+ free conditions or in the presence of 2mM Ca 2+ with or without adenosine deaminase (AD) 1U/ml.

Figure 5 .

 5 Figure 5. Reversal of aggregation by ADP degrading enzymes is largely due to the

Figure 6 .

 6 Figure 6. TXA 2 generation is enhanced at low extracellular calcium concentrations

Figure 7 .

 7 Figure 7. ADP degradation is the predominant mechanism regulating ADP-mediated platelet aggregation at physiological extracellular Ca 2+ concentrations. (a) Platelet

  after 10s. (a) Degradation of ADP (10µM) by apyrase (0.32U/ml) in nominally Ca 2+ free conditions or in the presence of 2mM Ca 2+ measured 10s after ADP addition. (b) Degradation of ADP (10µM) by ectonucleotidases present in plasma was measured before and after re-calcification, 10s after ADP addition. Online supplement 2. Millimolar concentrations of Mg 2+ do not accelerate ADP degradation. (a) Washed platelets stimulated with ADP (10µM) in nominally Ca 2+ free saline, or in the presence of 2mM Mg 2+ . (b) Degradation of ADP (10µM) by apyrase (0.32U/ml) present in the platelet saline buffer measured after 2 minutes in nominally calcium free saline or in the presence of 2mM Mg 2+ . Online supplement 3. Adenosine deaminase blocks the inhibition of ADP-mediated aggregation by adenosine. Aggregation of washed platelets stimulated with ADP (10µM) in the presence of adenosine (10µM) with or without prior addition of adenosine deaminase (AD) 1U/ml.

  Figure 1.

  Figure 3.

  Figure 4.

  Figure 5.

  Figure 6.

  Figure 7.

British Journal of Haematology