Bartosz ; Ditkowski

Paulina Troc

Katarzyna Masiewicz

Magdalena Donczew

Keith F Chater

Jolanta Zakrzewska-Czerwinska

Dagmara Jakimowicz
email: dagmara.jakimowicz@uni.wroc.pl

Ludwik

Paulina Troć

Katarzyna Ginda-Masiewicz

Jolanta Zakrzewska-Czerwińska

The actinobacterial signature protein

Keywords: chromosome segregation, ParA, bacterial cytoskeleton, Streptomyces, sporulation

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

Introduction

ParA homologues (such as Soj in B. subtilis) are part of the machinery responsible for the rapid movement of bacterial plasmids or chromosomal origin regions (oriC) toward cell poles soon after the start of replication [START_REF] Gerdes | Plasmid and chromosome partitioning: surprises from phylogeny[END_REF][START_REF] Gerdes | Bacterial mitotic machineries[END_REF]Hayes and Barilla, 2006;[START_REF] Toro | Bacterial chromosome organization and segregation[END_REF]. Soj also plays a role in the regulation of chromosome replication and control of sporulation [START_REF] Marston | Dynamic movement of the ParA-like Soj protein of B. subtilis and its dual role in nucleoid organization and developmental regulation[END_REF]Quisel et al., 1999, Murray and[START_REF] Murray | Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA[END_REF]. ParA homologues generally act in collaboration with ParB, a protein encoded by the second gene of the same operon [START_REF] Gerdes | Plasmid and chromosome partitioning: surprises from phylogeny[END_REF]. ParB is also required for efficient plasmid and chromosome segregation, forming high-order nucleoprotein complexes at partitioning sites (parS) near ori [START_REF] Lin | Identification and characterization of a bacterial chromosome partitioning site[END_REF][START_REF] Jakimowicz | The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome[END_REF][START_REF] Leonard | Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus[END_REF].

ParA homologues are Walker A cytoskeletal ATPases. Thermus thermophilus ParA forms a sandwich dimer (Leonard et al., 2005a) and can oligomerise into filaments in the presence of DNA. The weak ATPase activity of ParA proteins is elevated in the presence of ParB homologues [START_REF] Easter | ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities[END_REF]Barilla et al., 2005;Leonard et al., 2005a;[START_REF] Bouet | Polymerization of SopA partition ATPase: regulation by DNA binding and SopB[END_REF]. Filaments formed by the ParA homologue of plasmid pTP228 (ParF) are remodelled in the presence of its ParB homologue due to enhancement of ParF ATPase activity (Barilla et al., 2005;2007). Polymerisation and depolymerisation of ParA underpin its dynamic localisation pattern in the cell [START_REF] Ebersbach | Bacterial mitosis: partitioning protein ParA oscillates in spiralshaped structures and positions plasmids at mid-cell[END_REF][START_REF] Lim | Bacterial DNA segregation by dynamic SopA polymers[END_REF][START_REF] Fogel | A dynamic, mitotic-like mechanism for bacterial chromosome segregation[END_REF]2006[START_REF] Ringgaard | Movement and equipositioning of plasmids by ParA filament disassembly[END_REF]. Several ParA homologues (SopA from F plasmid and ParA from plasmid pB171, chromosomal ParA from B. subtilis and V. cholerae) have been reported to oscillate within the cell as a helical spiral. This dynamic localisation can be disrupted by mutations in the nucleotide binding site or in parB [START_REF] Marston | Dynamic movement of the ParA-like Soj protein of B. subtilis and its dual role in nucleoid organization and developmental regulation[END_REF][START_REF] Quisel | Control of development by altered localization of a transcription factor in B. subtilis[END_REF][START_REF] Autret | Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein[END_REF][START_REF] Fogel | A dynamic, mitotic-like mechanism for bacterial chromosome segregation[END_REF]. ParA has been postulated to supply energy for the movement of ParB segregation complexes (Leonard et al., 2005b). Disassembly of the ParA polymer of plasmid pB171, triggered by interaction with the segregation complex, moves the plasmids along the cell [START_REF] Ringgaard | Movement and equipositioning of plasmids by ParA filament disassembly[END_REF]. Similarly, in V. cholerae a dynamic band of ParAI shifts the segregating ParBI/parS complex to the cell pole to anchor the chromosome there [START_REF] Fogel | A dynamic, mitotic-like mechanism for bacterial chromosome segregation[END_REF].

The mycelial growth mode of Streptomyces coelicolor and its multiple synchronous septation during sporulation provide new perspectives on the mechanisms of chromosome segregation. During vegetative growth, hyphae elongate by tip extension and branching, while occasional septa separate adjacent multigenomic compartments (Flardh, 2003). Sporulation, triggered by nutrient depletion, starts with rapid growth of aerial hyphae, which involves intensive chromosome replication (Ruban-Osmialowska et al., 2006). In aerial hyphae 50 or more non-segregated chromosomes may be present in one long tip compartment. Conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronous segregation of multiple chromosomes and synchronous regular placement of Z-rings along the compartment [START_REF] Mccormick | Growth and viability of Streptomyces coelicolor mutant for the cell division gene ftsZ[END_REF]Flardh and Buttner, 2009). In contrast to the movement of chromosomes toward poles in rod-shaped bacteria, dozens of Streptomyces chromosomes are condensed and uniformly aligned along the hyphal tip compartment, ensuring that each prespore receives a single copy.

ParAB proteins are important for proper distribution of chromosomes in aerial hyphae before septation. The parAB operon is developmentally regulated, with one of its two promoters strongly upregulated shortly before sporulation septation [START_REF] Kim | Partitioning of the linear chromosome during sporulation of Streptomyces coelicolor A3(2) involves an oriC-linked parAB locus[END_REF][START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF]. Deletion of parA or parB results in frequent anucleate spores (about 24% or 15%, respectively), but does not visibly affect colony growth or sporulation. Elimination of ParA also strongly affects sporulation septation. Before DNA segregation and septation, ParB binds to numerous parS sites near oriC to form large complexes, which are arrayed regularly along the aerial hyphal tip compartment, disassembling soon after septation is completed [START_REF] Jakimowicz | The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome[END_REF][START_REF] Jakimowicz | Developmental-stagespecific assembly of ParB complexes in Streptomyces coelicolor hyphae[END_REF].

ParA has a complex localisation pattern, dependent on the growth stage. In vegetative hyphae and young aerial hyphae it is found at the tips, but during further aerial growth it forms extended helical ParB was the only known interaction partner of ParA until recent evidence that B. subtilis Soj interacts with replication initiator DnaA, controlling its activity [START_REF] Murray | Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA[END_REF]. Here, we show that a novel interaction partner of Streptomyces ParA, ParJ, influences ParA filament stability.

Remarkably, ParJ is one of a very small number of signature proteins confined to, but ubiquitous among, Actinobacteria, whose members include many organisms of medical, environmental and industrial importance [START_REF] Gao | Structural and phylogenetic analysis of a conserved actinobacteria-specific protein (ASP1; SCO1997) from Streptomyces coelicolor[END_REF].

Results

ParA interacts with a conserved, actinobacteria-specific protein

Interesting properties of S. coelicolor ParA, which include formation of elongated filaments in aerial hyphae, encouraged us to search for its interaction partners, using a bacterial two hybrid system (BTH) to screen an expression library of S. coelicolor DNA (a kind gift from Professor Justin Nodwell, McMaster University, Canada, unpublished). One of the positive clones contained sco1662, encoding a conserved hypothetical protein SCO1662 found exclusively within Actinobacteria. In S. coelicolor, sco1662 is located downstream of the glycerol utilisation operon and was thought to be the last gene of the operon (gylX), with an unknown function [START_REF] Smith | Cloning and transcription analysis of the entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) and identification of a closely associated transcription unit[END_REF]. However, comparison of gene arrangement in different Streptomyces species revealed that the position of the sco1662 homologues downstream of the gyl operon is not conserved (Fig S1), suggesting that SCO1662 is not associated with glycerol catabolism. Indeed, the sco1662 orthologues are highly unusual among conserved Streptomyces genes in their non-syntenous locations in the genomes of different species.

The protein was named ParJ for the reasons that are given below.

The ParA-ParJ interaction was verified in vitro using purified ParA [START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF] (or 6His-ParA) and recombinant ParJ purified in two expression systems: 6His-ParJ and ParJ (36 kDa) cleaved from a glutathione S-transferase (GST) fusion (Fig. S2). In one approach, a strong, concentration-dependent interaction of 6His-ParA with ParJ (cleaved from GST) immobilised on a microtitre plate was detected with an anti-ParA antibody (Fig. 1). In a second approach, using surface plasmon resonance (SPR), ParA was immobilised on a CM5 chip, and tested for interaction with increasing concentrations of 6His-ParJ (empty flow cell served as negative control for unspecific binding to the chip surface). The SPR studies showed binding with a K D of about 30 nM (calculated from steady state affinity and from kinetic parameters) (Fig. 2A). Additionally the kinetic analysis

Deletion of parJ affects the formation of aerial hyphae, sporulation septation and chromosome partitioning into spores

Since ParA extended filament is important for efficient chromosome segregation and regular septation in sporulating aerial hyphae, we expected that the newly identified ParA partner might also have a specific function at this stage. The question regarding its role in development and its connection with ParA could be answered by the construction of DJ548, a parJ deletion mutant. The mutant showed normal colony morphology on MS solid medium, but its aerial growth and sporulation started somewhat earlier than in the wild type, particularly on the rich medium R2YE, which generally slows development. Aerial hyphae, as observed by the white colony phenotype, developed after 44-48 h of growth, compared with 70-90 h for the wild type strain (Fig. 3A). Introduction of a wild type copy of parJ with its putative native promoter (297 bp of upstream DNA) into the chromosome of parJ deletion mutant, DJ548 (yielding strain DJ556) restored the normal rate of development (Fig. 3B).

Developmental acceleration was independent of ParA, since the same phenotype was shown by a parJparA double deletion strain, DJ555 (Fig. 3A).

Further studies of mutant strains DJ548 (parJ deletion) and DJ555 (parJparA double deletion) used fluorescent microscopy to analyse chromosome segregation and septation. DNA and cell wall staining (with DAPI and wheat germ agglutinin, WGA-Texas Red conjugate, respectively) revealed that 8% of prespore compartments were anucleate in the parJ deletion mutant (Fig. 4, Table 1). In addition, prespore compartments were more uneven in size, compared to wild type, with about 18% being minicompartments (shorter than 0.7 µm, half of the normal length) (Table 1). Chains of "early" spores of strain DJ548 (parJ deletion) were also more susceptible to cell wall staining with WGA than the wild type, indicating that their cell wall was less fully mature (Fig. S3). However, the mature spores were as resistant to SDS, temperature and lysosyme treatment as the wild type. The segregation and septation defects were both complemented in DJ556 (Table 1). Altogether, these results showed that ParJ is involved in sporulation-associated chromosome segregation and cell division.

Irregular septation and a large fraction of minicompartments, and a somewhat more severe segregation defect, were previously observed in a parA deletion strain [START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF], Table 1). The parJparA double mutant, DJ555, had a phenotype intermediate between the two single mutants (anucleate prespore compartments 13%, minicompartments about 13%, see Table 1). Thus, the phenotypes were not additive -instead, parJ deletion partially suppressed the segregation and septation defects of the parA deletion. The effects of parA overexpression in the parJparA deletion background were also investigated by integrating pIJ6902parA, containing parA controlled by the inducible tipA promoter, into DJ555 (strain DJ557). Remarkably, parA overexpression partially complemented the irregular septation and segregation phenotype of parJ deletion, giving 1-2% of anucleate prespore compartments and 8-10% minicompartments, similar to the phenotype of parA overexpression in a parA single deletion background (DJ553). In summary, these genetic interactions reinforce the notion that the two proteins interact in vivo but also suggest that some parJ function(s) are independent of its partner protein ParA.

ParJ foci accompany ParA filaments in aerial hyphae

We investigated the cytological distribution of ParJ, using a strain DJ558 in which parJ was replaced by a parJ-egfp gene fusion in its chromosomal locus. ParJ-EGFP was almost fully functional as demonstrated by DNA and cell wall staining, which showed only a very slight segregation/septation defect in DJ558 (Table 1). Fluorescence microscopy revealed very weak EGFP fluorescence in DJ558 vegetative mycelium, but in some aerial hyphae ParJ-EGFP formed numerous small foci (Fig. 5A).

We analysed 180 unseptated, possibly nascent aerial hyphae and 122 septated hyphae. About 40% of the unseptated hyphae showed no fluorescence, 34% contained only sparsely spaced and rather faint foci, and 25% contained intense and densely spaced foci. Intense foci were present in most (67%) of the septated hyphae foci. Thus, ParJ foci form in aerial hyphae at or close to the end of growth, and before sporulation septation, and persist somewhat beyond the establishment of the septa.

Foci in prespore compartments were slightly more densely packed than in nonseptated hyphae, with the respective average inter-foci distances of 0.95 µm and 1.05 µm both being less than the length of a prespore compartment (about 1.3 -1.4 µm) (Fig. 5B). Overall, 67% of prespore compartments had a single focus, about 26% contained two, and almost 5% three foci. A few compartments had more than three foci. Thus, the distribution of the foci is not closely related to the positions of septa.

To correlate the temporal patterns of subcellular localisation of ParJ and ParA, we performed immunolocalisation of ParA in strain DJ558 expressing parJ-egfp (Fig. 6A). In young aerial hyphae ParA localises close to the tips, and later in aerial hyphal growth its signal extends toward the hyphal base, resembling a helical filament. Both faint and intense ParJ foci could be accompanied by partially or fully extended ParA filaments. This implies that ParJ foci form during the extension of ParA filaments, though the technical limitations of immunofluorescence impeded analysis of the dynamics of the two processes together in real time. Since ParA filaments were absent from septated hyphae, ParJ foci are longer-lived than ParA filaments.

The formation of ParJ-EGFP foci was not much affected by the elimination of ParA. The intensity and average distances between foci formed in J3306 (parA deletion) were the same as in the wild type, except that inter-foci distances varied slightly more (Fig. 6B). Thus, if there is any influence of ParA on ParJ distribution, it is small. In a reciprocal experiment, we did not observe any differences from the wild type in ParA localisation in parJ deletion strain DJ548 (data not shown).

Taken together, our results showed that ParJ forms foci in aerial hyphae at the time of extension of ParA filaments but the formation of foci does not depend on ParA. Transcription analysis (Fig. S4) showed that the developmental assembly of ParJ foci is underpinned by developmental induction of

ParJ induces ParA depolymerisation in vitro

ParA polymerisation and its ATPase activity are crucial for its function in chromosome segregation. Previously it was found that ATP binding is required for ParA polymerisation, while during disassembly of the filament ATP is presumably hydrolysed (Leonard et al., 2005b). To The influence of ParJ on ParA filament assembly/disassembly in real time was studied using dynamic light scattering (DLS), which assesses both abundance and size of particles. For a ParA solution (3 µM) in the presence of ATP, the recorded average intensity of light scattering increased up to about 2000 kilocounts/second (kct/s) and the particle size up to 500 nm. In the presence of 6His-ParJ (1.5 µM) and ATP both parameters were reduced, to about 500 kilocounts/second (kct/s) and to 100 nm, respectively. Thus ATP-dependent polymer formation by ParA was inhibited by ParJ (Fig. 7B). Moreover, addition of 6His-ParJ (0.75 -7 µM) to earlier polymerised ParA filaments led to an immediate, ParJ concentration-dependent, decrease of light scattering and particle size, indicating rapid polymer disassembly (Fig. 7C) To find out if the observed ParA depolymerisation effect could result from stabilisation of the monomer form, we tested the interaction of ParJ with ParAK44E, which contains a mutation in the Walker A motif and can neither bind ATP nor dimerise [START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF]. BTH and SPR studies showed that ParJ interacts with ParAK44E, albeit less strongly than with the wild type (Fig. 2B). To test if ParJ affects ATPase activity of ParA we employed an assay using radioactive ATP. Surprisingly, we found that ParJ itself (cleaved from GST) exhibited ATPase activity (Fig 8,inset).

Additionally, the rate of ATP hydrolysis for ParA, 6His-ParJ and mixture of the proteins was monitored in the quantitative assay by measurement of released phosphate (PiPer Phosphate Assay Kit, Molecular Probes). These experiments showed that 6His-ParJ also had ATPase activity (thus, activity was independent of the purification method) and was more efficient in hydrolysing ATP than ParA (Fig. 8). When both proteins were mixed, the ATPase activity was similar to the summed activities of the two separate proteins (Fig. 8), thus ParJ presumably does not enhance ParA activity. However, further studies will be required to elucidate in detail any reciprocal effects on ATPase activity. Since

Discussion

An actinobacterial signature protein is a partner of the cytoskeletal protein ParA

We have identified a completely novel ParA interaction partner, ParJ, that is necessary for efficient chromosome partitioning during sporulation septation. A recent extensive phylogenomic analysis of 19 Actinobacteria revealed that sco1662 is one of only five signature genes for the phylum, while a second of the five is also a parJ paralogue [START_REF] Gao | Structural and phylogenetic analysis of a conserved actinobacteria-specific protein (ASP1; SCO1997) from Streptomyces coelicolor[END_REF]. In S. coelicolor this protein, SCO1997 (which shows 25% identity and 43% similarity to ParJ), has been crystallised [START_REF] Gao | Structural and phylogenetic analysis of a conserved actinobacteria-specific protein (ASP1; SCO1997) from Streptomyces coelicolor[END_REF], revealing that it forms a trimer with a large internal cavity surrounded by three pronged, clawlike domains. The electrostatic surface potential of each claw is negative, generating an overall acidic surface in this area that might interact with other, positively charged, protein(s). The sequence similarity of ParJ to SCO1997 and modelling studies suggest that it may have the same trimer structure. Conserved lysines and arginines of the C-terminal region of ParA could provide an interaction interface with ParJ. Additionally, divalent cation binding was predicted for SCO1997, which is consistent with our finding of ParJ ATPase activity. Since the SCO1997 structure does not contain any known nucleotide binding site, further studies will be required to characterise the ATP binding motif. The structures of SCO1997 and ParJ do not match any known proteins, so elucidation of their function is likely to provide novel insights into physiological characteristics that are unique to Actinobacteria, and which might be investigated in the context of disease therapy (the loss of ParA from Corynebacterium and from Mycobacterium severely reduces growth rate: [START_REF] Donovan | Subcellular localization and characterization of the ParAB system from Corynebacterium glutamicum[END_REF], Nisa et al. 2010, Masiewicz et al. manuscript in preparation).

ParJ is responsible for ParA disassembly

Previously, several lines of evidence, mainly from plasmid systems, have shown that the dynamics of ParA polymerisation is affected by ParB proteins (Moller-Jensen et al., 2003;[START_REF] Garner | Dynamic instability in a DNA-segregating prokaryotic actin homolog[END_REF]Barilla et al. 2005;[START_REF] Bouet | Polymerization of SopA partition ATPase: regulation by DNA binding and SopB[END_REF]) but our finding that ParJ brings about ParA filament disassembly seems to be the first report of a different regulator of ParA polymerisation. All studied ParB homologues appear to enhance nucleotide hydrolysis by cognate ParAs [START_REF] Easter | ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities[END_REF]Leonard et al., 2005a[START_REF] Bouet | Polymerization of SopA partition ATPase: regulation by DNA binding and SopB[END_REF][START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF]. Leonard et al. (2005a) (Barilla et al., 2005). Enhanced ParA filamentation was also reported for plasmid proteins SopA/SopB [START_REF] Bouet | Polymerization of SopA partition ATPase: regulation by DNA binding and SopB[END_REF] and suggested for the ParM-ParR system, where interaction with ParR stabilizes the ParM filament, facilitating its continued polymerisation (Moller-Jensen et al., 2003;[START_REF] Garner | Dynamic instability in a DNA-segregating prokaryotic actin homolog[END_REF].

Our data also showed ParJ interacted with non-dimerising ParAK44E, which is not able to bind ATP, albeit less strongly than with the wild type protein. Thus, ParJ-mediated inhibition of ParA polymer assembly could result from ParA monomer stabilisation. However, we could not exclude ParJdependent induction of ATPase activity of ParA as an explanation for ParA polymer destabilisation.

ParJ is required for efficient chromosome segregation and septation.

Elongated ParA filaments formed during the formation of ladders of Z-rings in aerial hyphae, depolymerising at the stage of synchronous septation [START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF]. The timing of formation of ParJ foci, particularly their presence in septated hyphae, is consistent with their role during final ParA filament disassembly. Our failure to detect any change in ParA filament dynamics in the parJ deletion mutant may be due to limitations of the immunofluorescence technique. In a parB deletion background (data not shown) ParJ foci still formed but, surprisingly, seemed less distinct than in the wild type, presumably either because of some alteration of ParA filaments in the absence of ParB, or because of disturbed regulation of ParJ levels. Analysis of a set of mutant strains provided evidence that the functions of both proteins during septation and segregation are interlinked. The segregation defect of the double parJparA, deletion strain was less severe than that resulting from ParA elimination. This partial phenotype could be a reflection of an increase in the amount of ParJ available in the parA deletion mutant to interact with, and sequester, some other element of the segregation/cell division machinery. This would be overcome by deleting parJ. Moreover, the level of parJ transcript -and therefore probably of ParJ -is elevated in a parA deletion mutant (Fig. S4). We tentatively suggest that ParJ interacts with some other subsequent element of segregation/cell division machinery. Our preliminary BTH tests for interaction of ParJ with some known division proteins (SsgB, FtsZ, MreB) did not give any positive result, so further studies are required to find protein(s) or subcellular structures that interact with ParJ.

The hypothesis of a more complex ParJ function than solely regulation of ParA filament dynamics is supported by the accelerated development of parJ deletion colonies. Since the same effect was observed in the parA deletion background it suggests some additional regulatory function for ParJ.

At present we have no strong candidates for ParJ targets in the potential regulatory circuit, although one possibility would be the parAB operon itself. For pelleting assays, 6.66 µM ParA and 7 µM 6His-ParJ were incubated in reaction buffer in a total of 40µl (50mM Tris-HCl, 150 mM NaCl, 5 mM MgCl 2 ) in the presence of ATP (2 mM). After incubation at 30°C for 30 min. the samples were centrifuged at 20°C at 100 000 rpm. for 30 min in a TLA100 rotor (Beckman, ultra TLX).

The supernatants were removed and mixed with gel-loading buffer. The pellets were resuspended in 15-20 µl of reaction buffer and mixed with gel-loading buffer. The whole pellet fraction and half of the supernatant fraction were subjected to SDS-PAGE and stained with Coomassie blue.

Dynamic light scattering (DLS)

Polymerisation of ParA in the presence of ParJ was measured by DLS (DynaPro Wyatt Technology). To observe the effect of ParJ on ParA polymerisation 14 µl of protein mixture (3.26 µΜ ParA and 1.5µΜ 6His-ParJ in 50 mM Tris-HCl pH 7.0, 150 mM NaCl, 5 mM MgCl 2 ) was preincubated at room temperature for 5min. Then ATP (2mM) was added to the protein solution, which was placed in the quartz cuvette and incubated at 30°C in the DynaPro chamber. In a complementary approach 6His-ParJ (0.75, 1.5, 3, 7 µM) was added after 5 min of ParA incubation at 30 °C in the chamber. Measurements were taken every 2 s.

ATPase activity assays

The ATPase activity of ParA and 6His-ParJ was assayed by the spectrophotometric detection of inorganic phosphate using a P i Per Phosphate Assay Kit (Molecular Probes) according to the manufacturer's instructions.

ATP binding assay

For the assay 6HisParJ and ParJ cleaved form GST were used. Prior to binding of ATP ParJ was dialysed against 50 mM Tris (pH 7.4), 150 mM NaCl, 10 mM EDTA, 5% glycerol to remove bound Mg 2+ and ATP and later against 50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 5% glycerol. The reaction mixtures contained 6 µM protein, 2.5 µM [α-32P]ATP (specific activity, 0.2 Ci/mmol) in the 50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 5% glycerol. Binding was performed for 30 min at room temperature followed by UV cross-linking (8 J) done on ice for 30 min using a UV Crosslinker (Hoefer Scientific Instruments). Reactions were terminated by addition of an equal volume of SDS sample buffer, and the samples were electrophoresed on a 10% SDS-polyacrylamide gel. The gel was fixed, stained with Coomassie blue, dried, and exposed to a phosphorscreen.

F

  compartments, as ParB complexes form[START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF]. ParA mediates efficient assembly of ParB complexes in vivo and in vitro, and ParA ATPase activity is crucial for its dimerisation and interactions with ParB, though not for ParA localisation in vivo. We have postulated that ParA provides scaffolding for proper distribution of ParB complexes and consequently controls synchronised segregation of several dozens of chromosomes, possibly mediating a segregation and septation checkpoint.

  slow dissociation rate (k d =8.3x10 -4 1/s). Thus the interaction of ParJ and ParA was confirmed.

  promoters, with parJ transcription apparently being negatively regulated by ParA at later time points.

  determine whether ParJ affects ParA polymerisation in vitro, we monitored filament formation by ParA using pelleting assays, in which polymerised and unpolymerised proteins were separated into pellet and supernatant fractions, respectively, and further analysed by SDS/PAGE. Using this assay we found that in the presence of ATP Streptomyces ParA, like the other ParA homologues (but not requiring presence of DNA), formed higher order complexes that would be consistent with filament formation: the protein was present only in the pellet fraction (Fig 7. A, Ditkowski unpublished). However, in the presence of ATP and an equimolar amount (7 µM) of 6His-ParJ, ParA was retained in the supernatant fraction, suggesting that ParJ either inhibited ParA polymerisation or induced depolymerisation (Fig 7. A).

  ParJ does not contain any recognisable ATP binding motif, and in order to exclude potential contamination with ATPase, we confirmed its binding of ATP to ParJ in an independent UVcrosslinking experiment(Fig 8B). The observed binding was Mg 2+ dependent and was observed for both ParJ cleaved from GST and 6HisParJ.

  suggested two possible mechanisms of ATPase activation of ParA by ParB. In the first model, favoured by Leonard et al., activation of the ATPase activity of ParA polymer subunits leads to destabilisation of the polymer; alternatively, according to the second model, enhanced ATPase activity might result from promotion of nucleotide exchange in the ParA-ADP monomer, shifting the equilibrium to the , polymerisation-proficient dimer. There are also several lines of evidence showing that dynamics of polymer formed by ParA homologues is affected by ParB proteins. For example, in the case of plasmid pTP288 ParF, addition of ParG (ParB homologue) resulted in extensive remodelling of the polymer in vitro

Fig. 8 .Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .Fig. 7 .

 81234567 Fig. 8. ATPase activity of ParJ A. ATPase activity of 6His-ParJ, ParA and mixed 6His-ParJ and ParA proteins, all at 1µM concentration. The control reactions (ATP hydrolysis, and internal protein control) were subtracted. Inset, hydrolysis of 32 P-ATP by ParJ. B. UV cross-linking of ParJ binding [α-32P]ATP. Following cross-linking, samples were analysed by SDS-polyacrylamide gel electrophoresis and the gel was exposed to a phosphorscreen. Top panel shows Coomassie blue-stained SDS-polyacrylamide gel of UV cross-linked samples, bottom panel -autoradiography image from the UV cross-linking experiment 1, ParJ (cleaved from GST) in the presence of MgCl 2 , 2, ParJ (cleaved from GST) in the absence of MgCl 2 , 3, 6His-ParJ in the presence of MgCl 2 , 4, negative control -ParB in the presence of MgCl 2 .

Table 1 . Statistical analysis of segregation/septation defects in parJ mutant strains

 1

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 . Strains used in this study

 2 Top panel, change in polymer average size (nm). Bottom panel, changes in light-scattering intensity, expressed as kct/s.

	Strain	Relevant genotype

Acknowledgements:

We thank Justin Nodwell for providing S. coelicolor expression library in bacterial two-hybrid system, Jerzy Majka for help with BIAcore data analysis and Gabriella Kelemen for helpful discussion. This work was supported by the Ministry of Science and Higher Education (Grant N N301 285437). B.D. was supported by a scholarship of the President of Polish Academy of Sciences and British Council Young Investigator Programme.

Concluding remarks

In summary, we have described the first regulator of ParA polymerisation other than ParB. Taking into consideration the conservation of both ParJ and polar growth among Actinobacteria and their absence from other bacterial phyla, we suggest a polar growth-related regulatory function for ParJ. ParJ may contribute to special ParA filament dynamics required for polar growth. Immediate questions are whether the conserved ParJ paralogue SCO1997 is also involved in ParA dynamics, and whether there is some interplay between ParJ and SCO1997. Presumably, any such interplay would be found throughout the Actinobacteria. More broadly, it is likely that in other bacterial phyla similarly complex molecular mechanisms govern the formation of supramolecular structures by ParA.

Experimental procedures

DNA manipulations, bacterial strains, media and growth conditions, and sample preparation for microscopy, were as described before [START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF][START_REF] Kieser | Practical Streptomyces Genetics[END_REF]. Application of the bacterial two hybrid (BTH) system [START_REF] Karimova | A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli[END_REF] and library screening are described in Supplementary material (SI Text).

PCR targeting was used for the construction of mutant strains (Table 2). The PCR targeting procedure was applied as described before [START_REF] Jakimowicz | Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae[END_REF][START_REF] Gust | PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin[END_REF], details of the construction are given in Supplementary material (SI Text). Protein purification followed published procedures, details are provided in Supplementary material (SI Text).

Surface plasmon resonance (SPR) analysis

For SPR analysis, untagged ParA protein (500 response units [RU]) was immobilized on the CM5 Sensor Chip in presence of ATP according to the manufacturer instructions. As a control, a flow channel without immobilised protein was used. SPR analysis was performed on a BiaCore 3000, by applying increasing concentrations of 6His-ParJ (as indicated) solutions in binding buffer (10 mM HEPES/KOH, pH 7.4, 150 mM NaCl, 50 µg/ml BSA, 5 mM MgCl 2 , 0,05% Tween 20), when indicated supplemented with 2 mM ATP. Binding was measured for 3 min at flow rate 20 µl/min at room temperature in SPR running buffer (10 mM HEPES/KOH pH 7.4, 150 mM NaCl, 0.05% Tween 20). The ParJ protein injections were followed by two to four injections of 1-5 M NaCl to remove all bound analyte from the sensor chip. The results were plotted as sensograms after subtraction of the background response signal obtained in a control experiment. The BIAevaluation ver.4.1 program (Pharmacia Biosensor AB) was used for fitting the curves to calculate dissociation rate k d and dissociation constant K D . K D was additionally calculated from steady state equilibrium, and was consistent with the results delivered by BIAevaluation software.

ELISA

ParJ (or BSA) was bound to Greiner Microtiter and Maxisorp NUNC ELISA plates at 10 µg/ml in 100 µl of coating buffer (0.1 M NaHCO 3 pH 9.2) and incubated at 4°C overnight. After rinsing with wash buffer (phosphate-buffered saline, 0.5% Tween 20) and blocking (phosphate-buffered saline, 0.5% Tween 20, 5% BSA), 10-5000 ng of 6His-ParA diluted in 100 µl of blocking buffer was added to each well and incubated at 30°C for 1 h. After washing, a rabbit ParA antiserum and alkaline phosphatase-conjugated goat anti-rabbit IgG antiserum were used for detection of bound ParA. The absorbance of the chromogenic conversion of substrate disodium p-nitrophenyl phosphate hexahydrate (1mg/ml) was read at 405 nm in an ELISA plate reader.

Figure Legends

Fig. 1. Interaction of ParA with SCO1662 (ParJ)

Binding of 6His-ParA (10-5000 ng) to ParJ (cleaved from GST), or BSA, bound to a microtiter ELISA plate was detected with anti ParA antibody conjugated with alkaline phosphatase, whose activity was detected by measuring absorbance of reaction product at 405 nm. Inset: Bacterial two hybrid analysis of interaction of T25-ParA with T18-ParJ. Intense colony colour indicates interaction of the analysed proteins.


Dr Laura Green
email: l.green@ucl.ac.uk

Andrew S Lawrie

Shelain Patel

Fahad Hossain

Andrew Chitolie

Fares Ian J Mackie

S Haddad

Sam J Machin

Fares; Haddad

F S Haddad

The impact of elective knee/hip replacement surgery and thromboprophylaxis with Rivaroxaban or Dalteparin on Thrombin Generation

Keywords: dalteparin, knee/hip replacement surgery, rivaroxaban, in vivo/ex vivo thrombin generation dalteparin, hip/knee replacement surgery, rivaroxaban, in vivo/ex vivo thrombin generation

come

Introduction

Without thromboprophylaxis, the incidence of venous thromboembolism (VTE) developing after major orthopaedic surgery is estimated to be 40-60%. Factors such as reduced mobility, presence of proximal lower extremity injury, previous VTE, BMI of >30 kg/m 2 , advanced age and active cancer, are all contributing factors for the high VTE risk [START_REF] Geerts | Prevention of venous thromboembolism: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition)[END_REF]. In addition, haemostatic changes occurring during total hip and knee replacement (THR and TKR respectively) surgery further contribute to the patho-physiological mechanism of VTE development. There is evidence that elevation of the in vivo markers of thrombin generation (prothrombin fragments 1+2 [F1+2]), thrombin-antithrombin complex (TAT), and fibrinolysis (D-dimer) begin during surgery with the greatest risk of clotting activation occurring at the time of joint insertion (Sharrock et al, 1995a;Sharrock et al, 1995b).

Although activation of coagulation is known to occur during orthopaedic surgery, there generally remains a discrepancy in the timing of thromboprophylaxis initiation between Europe and the USA due to different approaches to: minimising peri-/post-operative bleeding; avoiding presurgical hospital admissions; and simplifying conventions adopted for the administering of anaesthesia. In Europe, thromboprophylaxis is generally started 12 hours before major orthopaedic surgery whereas in North America it is initiated 12-24 hours after surgery. It is hoped that the availability of more sensitive clotting tests and more predictable oral anticoagulant agents would reduce some of the above discrepancies and bring about a greater consensus in the timing of thromboprophylaxis therapy.

Low molecular weight heparins (LMWH), unfractionated heparin (UFH) or the synthetic pentasaccharide fondaparinux are the recommended pharmacological prophylaxis for VTE prevention after major orthopaedic surgery [START_REF] Geerts | Prevention of venous thromboembolism: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition)[END_REF]NICE 2010) Rivaroxaban is an oral, highly selective and potent direct inhibitor of factor Xa (FXa). It inhibits not only free FXa but also the prothrombinase and clot bound FXa [START_REF] Perzborn | In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939--an oral, direct Factor Xa inhibitor[END_REF][START_REF] Gulseth | Rivaroxaban: an oral direct inhibitor of factor Xa[END_REF]. Its terminal half-life is approximately 5-9 hours in healthy young subjects [START_REF] Kubitza | Safety, pharmacodynamics, and pharmacokinetics of BAY 59-7939--an oral, direct Factor Xa inhibitor--after multiple dosing in healthy male subjects[END_REF] and approximately 13 hours in healthy elderly subjects [START_REF] Laux | Direct inhibitors of coagulation proteins -the end of the heparin and low-molecular-weight heparin era for anticoagulant therapy?[END_REF]. Around 60% of the drug is excreted via the renal route (~ 1/3 unchanged) with the rest being eliminated via the fecal-biliary tract (Weinz et al, 2009). Rivaroxaban inhibits thrombin generation (TG) [START_REF] Graff | Effects of the oral, direct factor xa inhibitor rivaroxaban on platelet-induced thrombin generation and prothrombinase activity[END_REF] and prolongs prothrombin time (PT) and activated partial thromboplastin time (APTT) in a dose-dependent manner. Due to the predictable dose-response relationship, it is suggested that laboratory monitoring is not required [START_REF] Kubitza | Safety, pharmacodynamics, and pharmacokinetics of BAY 59-7939--an oral, direct Factor Xa inhibitor--after multiple dosing in healthy male subjects[END_REF].

LMWH exhibit their overall anticoagulant activity via the antithrombin-mediated anti Xa and anti IIa effects and through the release of tissue factor pathway inhibitor (TFPI) from the endothelium [START_REF] Hoppensteadt | The role of tissue factor pathway inhibitor in the mediation of the antithrombotic actions of heparin and lowmolecular-weight heparin[END_REF]. Although the relative anti Xa:IIa ratio varies between different LMWH preparations, depending on their molecular size distribution [START_REF] Baglin | Guidelines on the use and monitoring of heparin[END_REF], their clinical efficacies in the prevention of VTE after surgery are similar (White & Ginsberg, 2003). Being composed of smaller molecules than UFH, LMWH have better bioavailability, longer plasma half life (approximately 4 hours) and better anticoagulant response predictability; thus rendering laboratory monitoring unnecessary in most instances [START_REF] Hirsh | Parenteral anticoagulants: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition)[END_REF]. However in selected groups, e.g. obese or underweight patients, during pregnancy, children, those at increased risk of bleeding, and patients with severe renal impairment, measurement of LMWH antithrombotic activity is of utmost importance. The currently available assays of anti-Xa activity are limited in their ability to predict bleeding and/or thrombotic risks [START_REF] Greaves | Limitations of the laboratory monitoring of heparin therapy. Scientific and Standardization Committee Communications: on behalf of the Control of Anticoagulation Subcommittee of the Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis[END_REF][START_REF] Leizorovicz | Factor Xa inhibition: correlation between the plasma levels of anti-Xa activity and occurrence of thrombosis and haemorrhage[END_REF], thus highlighting the need for more physiologically relevant tests.

All anticoagulant agents directly or indirectly inhibit TG. Recent studies have shown that ex vivo measurement of an individual's potential to generate thrombin over time (via the TG test), may be more sensitive than the current tests at measuring the effect of anticoagulation [START_REF] Al Dieri | Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time[END_REF]2006). Furthermore, to our knowledge no studies investigating ex vivo TG during orthopaedic surgery have yet been published.

The objectives of this study were to: (a) prospectively assess the peri-operative effects of elective total knee/hip replacement surgery on TG, F1+2, TAT and D-dimer; (b) compare the impact of prophylactic doses of dalteparin and rivoraxaban on TG 24 hours after surgery. PT, APTT, antithrombin and anti-Xa levels were also measured.

Methods and Materials

Patients

The study was approved by the local ethics and Research and Development Office at University College London Hospitals (UCLH) NHS Trust. After informed consent, a total of 51 patients, age ≥ 50 years with no upper limit, were recruited from July to September 2009 for the dalteparin group, and from October 2009 to January 2010 for the rivaroxaban group.

Inclusion criteria were eligibility to receive thromboprophylaxis therapy after elective THR or TKR surgery. Exclusion criteria were: acquired/inherited bleeding disorders, history of VTE recurrence, platelets of < 100 x 10 9 /L and active malignancies. Aspirin and/or warfarin were stopped 7 and 5 days respectively prior to surgery. Mechanical VTE prophylaxis with thigh length graduated compression stocking was given to all patients from the time of admission. In UCLH chemical prophylaxis is administered post-operatively in order to avoid: peri-/postoperative bleeding; pre-surgical hospital admissions; and the risk of spinal haematoma.

Therefore thromboprophylaxis with dalteparin (subcutaneously) or rivaroxaban (orally), once a day, was administered 6-8 hours after surgery, in 25 and 26 patients respectively. A description of patient demographics and their renal function prior to surgery is presented in Table 1. In 3/25 patients weighing <50kg, the dose of dalteparin given was 2500 units and 21/25 received 5000 units. The remaining patient, who received dalteparin 5000 units post-operatively, declined to provide a blood sample 24 hours after the surgery; however the pre-and the peri-operative samples have been included in this analysis. All patients in the rivaroxaban group received the dose of 10 mg. Surgeries were performed at UCLH by a team of 3 senior surgeons using similar surgical techniques.

Blood sample collection

Venous blood samples for measurement of haemostatic markers were obtained: (1) immediately pre-operatively, in the anaesthetic room; (2) peri-operatively at the time when the joint was inserted; and (3) a mean of 16.4 hours (range 15 -18) after thromboprophylaxis had been administered (i.e. approximately 24 hours after surgery). Blood was taken using a 21 gauge It is well recognised that contact activation can interfere with TG assay and therefore immediately after the venepuncture corn trypsin inhibitor (a factor XIIa inhibitor [Cambridge Bioscience, Cambridge, UK]) was added to the whole blood for TG measurement, at a final concentration of 18.3 µg/mL [START_REF] Luddington | Clinical measurement of thrombin generation by calibrated automated thrombography requires contact factor inhibition[END_REF]. Within 1 hour of collection platelet poor plasma (PPP) was prepared by double centrifugation at ambient temperature (2000g for 12 minutes) and aliquots of PPP were frozen to -80ºC. On the day of assay samples were thawed to 37ºC.

Laboratory analysis

Full blood counts (FBC) were performed using a KX-21 analyser (Sysmex UK Ltd, Milton Keynes, UK). Unless otherwise stated coagulation assays were performed on a CS-2000i automated coagulation analyser (Sysmex UK Ltd), and all reagents were from Siemens Healthcare Diagnostics (Marburg, Germany). Innovin and Actin FS were used in PT and APTT tests respectively. D-Dimer (Innovance D-Dimer) was measured by an immunoturbidometric technique. F1+2 and TAT complexes were measured by ELISA (Enzygnost F1+2 and Enzygnost TAT respectively). Antithrombin assay and dalteparin/rivaroxaban-anti-Xa activities were performed by chromogenic assay using Berichrom® Antithrombin kit and Berichrom® Heparin kit respectively. Standard curves for anti-Xa activities were constructed by adding known concentration of dalteparin (Pfizer Pharmaceutical) or rivaroxaban (Bayer Schering Pharma) to the standard human plasma (Siemens Healthcare Diagnostics) obtaining a final concentration of 1.0 IU/ml anti-Xa activity for dalteparin and 0.6 µg/ml anti Xa activity for rivaroxaban (the limit of detection for LMWH was 0.05 IU/mL and for rivaroxaban 0.01 µg/ml). The relative potencies of the anticoagulants in patient's plasma were derived from the appropriate calibration curve.

Thrombin generation

The TG was performed using the Calibrated Automated Thrombogram (CAT) system (Thrombinoscope BV, Maastricht, The Netherlands) as described by [START_REF] Hemker | Calibrated automated thrombin generation measurement in clotting plasma[END_REF] in conjunction with the manufacturer's PPP-Low reagents, which gave reaction concentrations of 1 pmol/L tissue factor (TF) and 4 µmol/L Phospholipid (Thrombinoscope BV). We decided to use in order to reduce TG intra-assay variations, contact pathway activation was inhibited with CTI [START_REF] Chantarangkul | Thrombin generation assessed as endogenous thrombin potential in patients with hyper-or hypo-coagulability[END_REF]van Veen et al, 2008) and the pre-, peri-and post-operative samples from each patient were tested at the same time. All sample TG reactions and sample specific calibrators were tested in triplicate. The following parameters of the TG curve were measured: lag time; time to the peak (ttP); peak thrombin; area under the curve, known also as the endogenous thrombin potential (ETP); and the velocity-index-rate of TG calculated by the formula Peak/(ttP -lag-time) and expressed as nM/minutes. The mean intra-assay coefficient of variation (CV) for TG lag time, ETP, peak thrombin and ttP were: 4.9%, 6.8%, 6.5%, and 3.1%; whereas mean inter-assay CV were: 8.8%, 10.7%, 11.6% and 5.5% respectively.

Statistical analysis

Data analysis was performed using Analyse-It ® software version 2.03 (Analyse-It Software Ltd, Leeds, UK). The results are presented as a median with the 25 th and 75 th percentiles in parenthesis. Differences in haemostatic markers between the pre-, peri-and post-operative periods were analysed using the non parametric 'Wilcoxon signed rank' test. Comparisons between dalteparin and rivaroxaban groups were made using the un-paired 'Mann-Whitney' test whereas association between different variables were performed using the 'Spearman rank correlation coefficient'. A 2-tailed p value of < 0.05 was considered significant.

Results

Thrombin Generation test and anti Xa levels

The median TG lag time and ttP decreased from the pre-operative to the peri-operative period while ETP, peak thrombin and velocity-index-rate increased, indicating enhanced coagulability during the peri-operative period (Figure 1). The increase in TG was similar between THR and TKR surgery and did not correlate with the body mass index (BMI) (data not shown).

Although there was no statistical difference between the TG pattern seen in 3 patients who received 2500 units of dalteparin and those who received 5000 units, we have excluded the former from the peri-vs. post-operative analysis. Post-operatively not all patients on dalteparin exhibited the same pattern of TG. Allowing for the TG intra-assay CV of 10%, we define a "favourable" TG response as exhibiting both: (a) reduction of the ETP and peak Analysis of the TG parameters for all dalteparin patients (n=21), from the peri-to post-operative period, showed a significant increase in the lag time and decrease in ETP (Figure 1); from the pre-to post-operative period, no significant changes were seen in TG parameters. No correlation was seen between the TG changes and: (a) the BMI or (b) renal function tests (data not shown).

The pre-operative antithrombin levels of the dalteparin patients were within the normal reference range (80-130 iu/dL) and at 24 hours post-operatively the median dalteparin anti Xa activity was undetectable in all patients.

In contrast 23/26 of rivaroxaban patients showed a favourable TG response from the peri-to the post-operative period with no adverse instances. The effect of rivaroxaban on TG was noted to be greatest on the lag time, ttP, the peak thrombin and velocity-index-rate for both pre-to postand peri-to post-operative periods (Table 2). The median rivaroxaban level was 0.03µg/ml (inter quartile range: 0.01-0.06). There was a good correlation between rivaroxaban levels and: (a) lag time (rs= 0.46, p= 0.01); (b) ttP (rs= 0.53, p= 0.005); (c) peak thrombin (rs = -0.59, p= 0.001) and velocity-index-rate (rs = -0.61, p=0.0009) at 24 hours after surgery. Comparison of the TG parameter changes between dalteparin and rivaroxaban groups show that the latter significantly reduced TG more than the former at 24 hours after surgery (Table 2).

Other haemostatic markers

FBC, PT and APTT were normal in all patients prior to surgery. PT and APTT in both groups were within the normal reference range (10.0 -12.0 seconds and 21.0 -31.5 seconds respectively) during the peri-and post-operative periods. D-dimer increased during and after surgery in all patients with no significant difference between dalteparin and rivaroxaban groups (Figure 2). F1+2 and TAT increased during surgery (n= 51); from the peri-to post-operative period these markers decreased significantly in the rivaroxaban group, but not in the dalteparin (Figure 2).

Clinical outcome

There was no peri-operative blood loss necessitating transfusion of red blood cells or platelets during this period. Two patients in each group required > 2 units of red blood cells from the time of administering thromboprophylaxis to the time when the final blood sample was taken.

Discussion

During THR surgery, a combination of venous stasis/hypoxia and local trauma to the vessels and bone causes the release of tissue factor leading to activation of the coagulation system (Sharrock et al, 1995a) which, in turn, generates large amounts of thrombin. In the case of TKR surgery, although there is evidence that activation of the clotting system begins during surgery (Sharrock et al, 1995b), the mechanism of its initiation is less clear. The amount of thrombin that is produced cannot be quantified directly and therefore in vivo surrogate markers, namely F1+2, and TAT complexes, are used in measuring TG. F1+2 is released during the activation of thrombin from its precursor prothrombin; TAT complexes are produced in proportion to the amount of thrombin generated (resulting in regulation of the latter). However these markers have a very short half-life (in minutes) and provide only a partial picture of the entire TG process. The TG test, on the other hand, measures the ex vivo potential of plasma to generate thrombin over time and provides more information on the initiation, propagation and decay phases of TG, leading to a more complete view of the coagulation process [START_REF] Hemker | Thrombin generation, a function test of the haemostatic-thrombotic system[END_REF].

The current study measured TG of 51 patients who underwent elective hip/knee replacement surgery. The peri-operative sample was taken at the time when the new hip/knee joint had just been inserted -believed to coincide with the moment of maximum surgical trauma. We, like others (Sharrock et al, 1995a;Sharrock et al, 1995b;[START_REF] Dahl | Sequential intrapulmonary and systemic activation of coagulation and fibrinolysis during and after total hip replacement surgery[END_REF], found that F1+2, TAT, and D-dimer increased during surgery. Moreover, for the first time, we demonstrated that ex vivo TG also increases during major orthopaedic surgery to a similar extent for THR and TKR. These results support the view that thrombogenecity in these orthopaedic patients begins during the surgery. Therefore it follows that initiation of thromboprophylaxis prior to orthopaedic surgery could be more effective at preventing or reducing these peri-operative changes.

Additionally, in the pre-operative period, 50%, 27% and 60% of all patients (n=51) had D-dimer, TAT and F1+2 levels above the normal range respectively. Advanced age and chronic [START_REF] Mari | Hemostasis factors and aging[END_REF].

As the risk of VTE is triggered during surgery and peaks about 3-7 days after, when clinical VTE first develops [START_REF] Planes | Risk of deepvenous thrombosis after hospital discharge in patients having undergone total hip replacement: double-blind randomised comparison of enoxaparin versus placebo[END_REF][START_REF] Scurr | Deep venous thrombosis: a continuing problem[END_REF], it is critical to deliver effective thromboprophylaxis in the immediate post-operative period. We compared the effect of prophylactic doses of dalteparin (n= 21) and rivaroxaban (n= 26) on TG approximately 24 hours after surgery. This timing corresponded to a mean of 16.4 hours after thromboprophylaxis had been administered. In the dalteparin group, from peri-to post-operative period, there was a large variation in individuals' responses in TG with ~ 30% of patients showing TG reduction (i.e. 'favourable' response as defined previously). The remaining patients, despite dalteparin, had no detectable anticoagulant effect in TG 24 hours after surgery. The effect of LMWH on TG has been investigated by several groups [START_REF] Petros | The effect of different anticoagulants on thrombin generation[END_REF][START_REF] Al Dieri | Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time[END_REF]Gerotziafas et al, 2007b). One in vitro study (Gerotziafas et al, 2007b), using healthy individuals' platelet rich plasma (PRP) and the CAT system, showed that at equivalent anti-FXa activity concentrations, the inhibitory effect of LMWH (enoxaparin, nadroparin and dalteparin) upon TG was different.

In the case of dalteparin a concentration of ≥0.4 anti-FXa IU/mL significantly reduced TG; however no significant changes were seen at lower concentrations (Gerotziafas et al, 2007b). In the dalteparin group the anti-FXa levels were undetectable: yet in ~30% of patients (through a mechanism unknown to us), a suppression of TG was seen. However, even though the anticoagulant mechanism of LMWH is not fully understood, its efficacy for VTE prophylaxis after orthopaedic surgery, being given as a once-daily dose, has been conclusively demonstrated in large clinical trials [START_REF] Geerts | Prevention of venous thromboembolism: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition)[END_REF]. This is largely due to its inhibitory effects at different stages of the coagulation pathway, making its resultant anticoagulant action quite complex and variable amongst different individuals, as reflected in our TG results.

In contrast, rivaroxaban reduced TAT, F1+2 and ex vivo TG more than dalteparin at ~24 hours after surgery. The higher inhibitory effect of rivaroxaban on TG could be explained by the highly specific and direct inhibition of FXa and its longer plasma half-life. Rivaroxaban inhibits not only free FXa but also the prothrombinase and clot bound FXa [START_REF] Perzborn | In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939--an oral, direct Factor Xa inhibitor[END_REF][START_REF] Gulseth | Rivaroxaban: an oral direct inhibitor of factor Xa[END_REF]. Therefore, as one should expect, it is highly effective in retarding the initiation and propagation phases of TG (in vivo and ex vivo) but has less effect on the ETP. This is confirmed by our in vivo findings, as well as those of Gerotziafas et al, who studied the in vitro TG effect (CAT system) of rivaroxaban on PRP (2007a); admittedly, in this study, the use of platelet poor plasma to perform TG is an unavoidable limitation. There was a good correlation between the rivaroxaban levels and the lag time, ttP, peak thrombin and velocity-index-rate. More interesting was the fact that small amounts of rivaroxaban which could not be detected by the traditional tests (PT) were manifested in TG. One in vivo study has even demonstrated that the TG test is more sensitive than FXa activity in detecting small amounts of rivaroxaban [START_REF] Graff | Effects of the oral, direct factor xa inhibitor rivaroxaban on platelet-induced thrombin generation and prothrombinase activity[END_REF].

This study and others [START_REF] Bendetowicz | The effect of subcutaneous injection of unfractionated and low molecular weight heparin on thrombin generation in platelet rich plasma--a study in human volunteers[END_REF][START_REF] Petros | The effect of different anticoagulants on thrombin generation[END_REF]Gerotziafas et al, 2007a;Gerotziafas et al, 2007b;[START_REF] Samama | Assessment of laboratory assays to measure rivaroxaban--an oral, direct factor Xa inhibitor[END_REF] have highlighted a few important findings:

(1) the different TG parameters are important for understanding the mechanism of action of different anticoagulants, (2) ETP alone should not be used to compare the efficacy of different anticoagulants, and (3) the TG test can be used as a tool to describe the pharmacodynamics of various anticoagulants. However further standardisation of the test and reagents are required before its use in clinical practice.

The greater TG reduction of rivaroxaban would also support the results of the 4 phase III clinical trials (RECORD 1-4) which involved >12,500 patients [START_REF] Eriksson | Rivaroxaban versus enoxaparin for thromboprophylaxis after hip arthroplasty[END_REF][START_REF] Kakkar | Extended duration rivaroxaban versus short-term enoxaparin for the prevention of venous thromboembolism after total hip arthroplasty: a double-blind, randomised controlled trial[END_REF][START_REF] Lassen | Rivaroxaban versus enoxaparin for thromboprophylaxis after total knee arthroplasty[END_REF][START_REF] Turpie | Rivaroxaban versus enoxaparin for thromboprophylaxis after total knee arthroplasty (RECORD4): a randomised trial[END_REF]. These studies demonstrated that rivaroxaban at 10 mg od was significantly more effective for the prevention of VTE than enoxaparin 40 mg od (RECORD 1-3) [START_REF] Eriksson | Rivaroxaban versus enoxaparin for thromboprophylaxis after hip arthroplasty[END_REF][START_REF] Kakkar | Extended duration rivaroxaban versus short-term enoxaparin for the prevention of venous thromboembolism after total hip arthroplasty: a double-blind, randomised controlled trial[END_REF][START_REF] Lassen | Rivaroxaban versus enoxaparin for thromboprophylaxis after total knee arthroplasty[END_REF] and enoxaparin 30 mg twice daily dose (RECORD 4) [START_REF] Turpie | Rivaroxaban versus enoxaparin for thromboprophylaxis after total knee arthroplasty (RECORD4): a randomised trial[END_REF]. Clearly, one of the main concerns when prescribing chemical thromboprophylaxis in the first 24 hours post-operatively, and thereby reducing TG, is the bleeding risk. In the current study there were no clinical bleeding episodes but the size of the study is too small for us to draw any conclusions regarding its risk in general.

It is worth mentioning, nonetheless, that according to the four RECORD studies the major bleeding risks between rivaroxaban (starting 6-8 hours post-operatively) and LMWH were similar. Furthermore, in the RECORD 2 study, despite rivaroxaban being given for 3 weeks longer than enoxaparin, the incidence of major bleeding at 5 weeks was similar in both groups (0.1%) [START_REF] Kakkar | Extended duration rivaroxaban versus short-term enoxaparin for the prevention of venous thromboembolism after total hip arthroplasty: a double-blind, randomised controlled trial[END_REF].

Rivaroxaban prolongs PT in a dose dependent manner [START_REF] Kubitza | Safety, pharmacodynamics, and pharmacokinetics of BAY 59-7939--an oral, direct Factor Xa inhibitor--after multiple dosing in healthy male subjects[END_REF]. In our study the PT values, between 15-18 hours after rivaroxaban administration, were within the normal ranges. This is explained by the low plasma rivaroxaban levels (median 0.03µg/ml) and the thromboplastin reagent (Innovin) used. In the in vitro study performed by Samama and colleagues (2010) the concentration of rivaroxaban required to double the PT, when Innovin is used, was 0.7µg/ml; compared with other thromboplastin reagents (Neoplastin, Neoplastin plus, Recombiplastin, Thromborel S and TriniClot) this concentration was the highest, indicating that Innovin is the least sensitive PT-reagent.

In conclusion, TG (performed on platelet poor plasma using calibrated automated thrombogram and 1pM tissue factor), increases during orthopaedic surgery to a similar extent between total hip and knee replacement surgery. The effect of dalteparin on TG can be highly variable between different individuals at ~24 hours after major orthopaedic surgery. Rivaroxaban showed more TG consistency in its effect across individuals, and also inhibited in vivo (F1+2 and TAT) and ex vivo TG more than dalteparin post-operatively.

Disclosures

The work carried out in this study was supported by an unrestricted educational grant from Bayer Schering Pharma, UK. Data is presented as a median with the 25 th and 75 th percentiles in parenthesis THR, total hip replacement; TKR, total knee replacement; GFR, glomerular filtration rate *Normal BMI = 18.5-24.9; Overweight = 25-29.9; Obese ≥ 30; **All hip and knee replacement surgeries were unilateral; ¶ All previous THR or TKR surgeries were performed > 6 months ago and were not on the same joint. Box-Whisker plots with median values beneath their respective time periods. There were no significant differences between the data obtained from the pre-operative and perioperative samples for the dalteparin and rivaroxaban groups, therefore these were pooled (n= 51). Dalt, dalteparin; Riv, rivaroxaban Normal reference ranges: Thrombin-antithrombin complexes: 1.0 -4.1 µg/L; Prothrombin fragments 1+2: 69 -229 pmol/L; D-dimer: < 0.5 mg/L FEU

F

  . More recently in 2008 two new oral anticoagulant agents, namely dabigatran etexilate (Pradaxa® Boehringer-Ingelheim), a direct thrombin inhibitor, and rivaroxaban (Xarelto® Bayer Schering Pharma), a direct Xa inhibitor, were licensed in the European Union by the European Medicines Agency for use after THR and TKR and are currently under review by the Food and Drug Administration in the USA. In the UK they have been recommended by the National Institute for Clinical Excellence for similar indications (NICE 2010).

  minimal stasis, and drawn into 5ml citrate Vacutainers TM (BD Diagnostics, Oxford, UK) containing 0.5 ml of 0.109 M buffered tri-sodium citrate.

  of TF, as this concentration had been shown to increase the sensitivity of TG assays;

  b) increase of lag time and ttP (measured against peri-operative values); similarly the "adverse" TG response is defined as both: (c) increase of ETP and Peak thrombin and (d) shortening of lag time and ttP. Seven of 21 of dalteparin patients had a favourable TG response and only 1/21 had an adverse response. The remainder lack a clear TG pattern and were difficult to group.

  osteoarthritis are factors known to account for the hypercoagulability in these patients

Figure 1 .

 1 Figure 1. Thrombin generation during the pre-, peri-and post-operative period using 1pM Tissue Factor

Figure 2 .

 2 Figure 2. Haemostatic markers during the pre-, peri-and post-operative periods for dalteparin and rivaroxaban

Table 1 .

 1 van Veen,J.J.,Gatt,A., Cooper,P.C., Kitchen,S., Bowyer,A.E., & Makris,M. (2008) Corn trypsin inhibitor in fluorogenic thrombin-generation measurements is only necessary at low tissue factor concentrations and influences the relationship between factor VIII coagulant activity and thrombogram parameters. Blood Coagul.Fibrinolysis, 19, 183-189. Patient demographic

	o
	r
	P
	e e r
	R e v i e w

Table 2 .

 2 Comparison of thrombin generation parameters between dalteparin and rivaroxaban patients

			Pre-to Post-operative			Peri-to Post-operative
			Median % change					Median % change
		Lag time ttP	Peak	ETP	Velocity-	Lag time	ttP	Peak	ETP	Velocity-
				thrombin		index-rate			thrombin		index-rate
	Dalteparin	0.0	-2.0	+13.0	-4.1	+ 41	+12.7	+6.5	-3.7	-10.1	-17
	(n = 21)
	Rivaroxaban	+55.6	+38.5	-38.6	-14.9	-46	+80.8	+71.0	-58.2	-23.5	-64
	(n = 26)									-
	Dalteparin vs.	p <0.0001 p = 0.002 p =0.0001	NS	p = 0.001	p < 0.0001	p <0.001 p <0.0001 p = 0.04	p = 0.0003
	Rivaroxaban

ttP, time to peak; ETP, endogenous thrombin potential; NS, not significant

British Journal of Haematology