Belen Ferrer

Francisco Javier Bermúdez-Silva

Ainhoa Bilbao

Lily Alvarez-Jaimes #

Irene Sanchez-Vera

Andrea Giuffrida

Antonia Serrano

Elena Baixeras

Miguel Navarro

Loren H Parsons #

Daniele Piomelli
email: piomelli@uci.edu

Fernando Rodríguez De Fonseca

Regulation of brain anandamide by acute administration of ethanol

Keywords: Cannabinoid, Alcohol, Anandamide, rat, hippocampus, fatty acid amidohydrolase 2

The endogenous cannabinoid anandamide participates in the neuroadaptations associated with chronic ethanol exposure. However, no studies describe the acute actions of ethanol on anandamide production and degradation. In the present study we investigated the time-course of the effects of the peripheral administration of ethanol (4 gr/kg, i.p.) on the endogenous levels of anandamide in central and peripheral tissues. Acute ethanol administration decreased anandamide in the cerebellum, the hippocampus and the ventral striatum, as well as in plasma and adipose tissue. Parallel decreases of a second acylethanolamide, palmithylethanolamide were observed in the brain. Effects were observed 45-90 min after ethanol administration. In vivo studies revealed that anandamide decreases were associated with a remarkable inhibition of the release of both anadamide and glutamate release in the ventral striatum. There were no changes in the expression and enzimatic activity of the main enzyme that degradates anandamide, the fatty acid amidohydrolase. Acute ethanol did not change neither the activity of n-acyltransferase, the enzyme that catalyzes the synthesis of the anandamide precursor, nor the expression of NAPE-PLD, the enzyme that releases anandamide from membrane phospholipid precursors. These results suggest that receptor-mediated release of acylethanolamide is inhibited by the acute administration of ethanol, and that this effect is not derived of increased fatty acid ethanolamide degradation.

Several research lines have established a role for both the endogenous cannabinoid anandamide (AEA) and the cannabinoid CB1 receptor in ethanol dependence [1 -3].

While administration of cannabinoid CB1 receptor agonists promotes ethanol intake [START_REF] Colombo | Stimulation of voluntary ethanol intake by cannabinoid receptor agonists in ethanol-preferring sP rats[END_REF], the administration of a cannabinoid CB1 receptor antagonist decreases ethanol self administration [START_REF] Arnone | Selective inhibition of sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors[END_REF], especially in animals with a history of ethanol dependence [START_REF] Rodriguez De Fonseca | Long-lasting increase in voluntary ethanol consumption and transcriptional regulation in the rat brain after intermittent exposure to alcohol[END_REF] or in alcohol preferring rat lines [7]. Following these findings, cannabinoid CB1 receptor knockout mice were found to display decreased ethanol preference, a phenotype that resembled that of wild-type littermates treated with a cannabinoid receptor antagonist [START_REF] Wang | Endocannabinoid signaling via cannabinoid receptor 1 is involved in ethanol preference and its agedependent decline in mice[END_REF].

Molecular studies have shown that chronic ethanol administration is associated with an increased formation of both AEA and its membrane precursor Narachidonylphosphatidylethanolamine [8]. Chronic alcohol exposure also resulted in stimulation of 2-arachidonylglycerol, a second endocannabinoid [9]. Animal studies also revealed that chronic exposure to ethanol downregulated cannabinoid CB1 receptors in the brain [10]. Finally, a recent gene screening study has identified the cannabinoid CB1 receptor as one of the genes whose expression is permanently affected by serial cycles of ethanol dependence and withdrawal [11]. These data indicate a role for the endogenous cannabinoid system as a relevant contributor to alcoholism. Human genetic studies support this experimental hypothesis since a linkage between clinical forms of alcoholism and polymorphisms and/or mutations of the genes encoding either the cannabinoid CB1 receptor [START_REF] Comings | Cannabinoid receptor gene (CNR1): association with i.v. drug use[END_REF][START_REF] Schmidt | Association of a CB1 cannabinoid receptor gene (CNR1) polymorphism with severe alcohol dependence[END_REF] or the fatty acid amidohydrolase (FAAH) [START_REF] Sipe | A missense mutation in human fatty acid amide hydrolase associated with problem drug use[END_REF], the enzyme responsible for AEA inactivation [START_REF] Cravatt | Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides[END_REF] have been described.

Despite the extensive information on the effects of chronic exposure to alcohol, no information is available on the acute effects of ethanol on AEA formation and degradation. We addressed this issue by analyzing the time-course of the effects of acute ethanol on AEA and the related mediator palmithylethanolamide (PEA) formation in the brain. We selected three different brain areas characterized by the sensitivity to ethanol effects: the ventral-striatum nucleus accumbens, where intrinsic neurons and glutamate release are inhibited by ethanol [START_REF] Criado | Sensitivity of nucleus accumbens neurons in vivo to intoxicating doses of ethanol[END_REF][START_REF] Yan | Effects of systemic ethanol on basal and stimulated glutamate releases in the nucleus accumbens of freely moving sprague-dawley rats: a microsialysis study[END_REF]; the dorsal hippocampus, where ethanol inhibits acetylcholine release [START_REF] Henn | Stimulatory and inhibitory effects of ethanol on hippocampal acetylcholine release[END_REF], as well as glutamate release and glutamate-dependent depolarization [19 -21]; and the cerebellum, where ethanol modifies the firing pattern and the glutamate response of Purkinje neurons [START_REF] Franklin | Acute ethanol alters the firing pattern and glutamate response of cerebellar Purkinje neurons in culture[END_REF]. These brain areas display relevant densities of cannabinoid CB1 receptors where they participate on presynaptic control of neurotransmitter release [23 -26]. We also monitored the in vivo and in vitro actions of ethanol on the enzymatic activities of Nacyltransferase (NAT) and FAAH, enzymes involved in AEA formation and degradation [START_REF] Marzo | Formation and inactivation of endogenous cannabinoid anandamide in central neurons[END_REF][START_REF] Cadas | Biosynthesis of an endogenous cannabinoid precursor in neurons and its control by calcium and cAMP[END_REF][START_REF] Cravatt | Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides[END_REF], as well as the mRNA expression of the nacylphosphatidylethanolamide-specific phospholipase D, the recently cloned enzyme that cleaves anandamide from its membrane precursor [START_REF] Okamoto | Molecular characterization of a phospholipase D generating anandamide and its congeners[END_REF]. The results suggest that acute administration of ethanol markedly inhibits in vivo formation of AEA and its related endocannabinoid palmithylethanolamide in the rat brain and that this effect is not dependent on the activity of FAAH or NAT enzymes.

MATERIAL AND METHODS

Animals

Male Wistar rats (Charles River, Wilmington, MA and Barcelona, Spain), weighing 350 ± 50 g were housed individually with food and water available ad libitum.

Animals were kept in an standard 12 h light/dark photoperiod (Lights on 8:00 to 20:00 a.m). They were handled daily for a week before starting the experiments, and they received i.p saline injections, once a day along the three days that preceeded the experimental administration of ethanol. All animal procedures met the National Institutes of Health guidelines for the care and use of laboratory animals, and the European Communities directive 86/609/EEC regulating animal research.

Chemicals

Unlabeled anandamide (AEA) and palmithylethanolamide standards and [ 2 H 4 ]-labeled anandamide palmithylethanolamide and were synthesized as described [START_REF] Giuffrida | Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry[END_REF].1,2-Di[ 14 C]palmityl-sn-glycerophosphocholine (108 mCi/mmol) was purchased from Amersham Bioscences (Barcelona, Spain), and [1-3 H] ethanolamine hydrochloride (60 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO).

Vehicle and ethanol administration: Blood and tissue sampling

Ethanol was dissolved in sterile saline (sodium chloride, 0.9% w/v) before i.p. injection. Rats received either ethanol solution (15% w/v, 4, 2 or 0.75 gr/kg) or a similar volume of a 0.9% saline solution (control group). In order to avoid potential circadian variations on endocannabinoid levels, as previously described [START_REF] Valenti | Differential diurnal variations of anandamide and 2-arachidonoyl-glycerol levels in rat brain[END_REF] all the experiments took place between 9:00 to 13:00 a.m. during the light phase of the cycle.

Since it has been described that restraint stress may affect brain anandamide levels in specific brain areas [START_REF] Patel | Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling[END_REF], we first explore the potential effects of injection procedure on the brain levels of this endocannabinoid. To this end, we analyzed whether a single saline injection affected anandamide concentrations in the ventral striatum, cerebellum and dorsal hippocampus. A group of injection-habituated animals was anesthetized with methoxyflurane (Schering-Plough, Union, NJ) 0, 30 and 60 min after the injection of 0.9% saline (1 ml/kg). The brains were collected after rapid decapitation and immediately frozen in dry ice. Dissection of frozen brains was performed at -10ºC. Brains were placed in acrylic rat brain matrices, and 2 mm thick slices were obtained using brain matrix razor blades. The target brain region were collected using a scalpel (cerebellum and dorsal hippocampus) or a sample corer (ventral striatum).

The ventral-striatum-accumbens, dorsal hippocampus and cerebellum were dissected in 2 mm-thick frozen coronal cuts, corresponding to plates 9-13 (ventral striatumaccumbens), 26-36 (dorsal hippocampus) and 56-65 (cerebellum) of the atlas of Paxinos and Watson [START_REF] Paxinos | The rat brain in stereotaxic coordinates[END_REF].

In order to evaluate the effects of ethanol on blood and brain endocannabinoid content, two more experiments were performed. For blood and visceral sampling, a group of animals was anesthetized with methoxyfluran 0, 45, 90 and 240 min after the injection of ethanol. Blood (2 ml) was collected from the heart of the animal with a syringe filled with 1 ml of Krebs-Tris buffer (in mM: NaCl 136, KCl 5, MgCl 2 1.2, CaCl 2 2.5, glucose 10, Trizma base 20; pH 7.4). Blood samples were centrifuged in accuspin tubes (Sigma, St. Louis, MO) and plasma layers used for monitoring plasma AEA as described [START_REF] Giuffrida | Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry[END_REF]. Samples were obtained from perirenal fat, liver and ileum and immediately frozen in dry ice. A second group of animals were sacrificed following the above described protocol 0, 45, 90 and 240 min after the injection of ethanol. The brains were collected and immediately frozen in dry ice. The ventral-striatumaccumbens, dorsal hippocampus and cerebellum were dissected as described above.

Plasma ethanol levels

Plasma ethanol levels were measured enzymatically using a commercial kit (Sigma, St. Lous, MO, USA). Assays were run following the manufacturer's instructions.

High-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses of tissular contents of acylethanolamides

AEA and PEA were solvent-extracted from plasma, brain areas (accumbens, dorsal hippocampus and cerebellum) and peripheral tissues (perirenal fat, liver and ileum), fractionated by column chromatography and quantified by HPLC/MS with an isotope dilution method [START_REF] Giuffrida | Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry[END_REF][START_REF] De Fonseca | An anorexic lipid mediator regulated by feeding[END_REF].

Real-time PCR for relative quantification of CB1, FAAH, NAPE-PLD and GAPDH mRNA expression

Total RNA from ventral striatum-accumbens, hippocampus and cerebellum was obtained using Trizol reagent (Gibco BRL Life Technologies, Baltimore, Maryland) according to the manufacturer's instructions. All RNA samples showed A260/280 ratios between 1.8 and 2.0. First strand synthesis from each sample was done using random hexamer primer and M-MuLV reverse transcriptase (Roche Applied Science, Indianapolis, USA) according to manufacturer's instructions. Negative control reactions omitted reverse transcriptase. Resulting cDNAs were used as the template for real-time quantitative PCR, which was performed on the LightCycler instrument (Roche) with the SYBR Green I detection format.

The following primers were used (Accession numbers from NCBI database in parenthesis): CB1 (NM_012784) forward: 5'-agacctcctctacgtgggctcg-3', reverse: 5'gtacagcgatggccagctgctg-3' (314 bp product); FAAH (U72497) forward: 5'gttacagagtggagagctgtc-3', reverse: 5'-gagggttactgcagtcaaagc-3' (344 bp product); NAPE-PLD (AB112351) forward: 5'-ggagcttatgagccaaggtg-3', reverse: 5'actctccgtgcttcaggatg-3' (223 bp product); GAPDH forward, 5'-ggtgatgctggtgctgagta-3',GAPDH reverse, 5'-actgtggtcatgagcccttc-3' (272 bp product). Endogenous control: beta-glucoronidase (GUS, NM_017015) forward: 5'-tcctgtacaccacccctacc-3', reverse 5'-gccatcctcatccagaagac-3' (146 bp product). Primers were obtained from Proligo (Proligo France SAS, Paris, France).

Quantification was carried out on the basis of standard curves run simultaneously than samples [START_REF] Hansson | Genetic impairment of frontocortical endocannabinoid degradation and high alcohol preference[END_REF]. CB1, FAAH, NAPE-PLD, GADPH and GUS standards were generated by PCR-amplification from control samples. The PCR product was run in a 1% agarose gel electrophoresis for checking the fragment size and the absence of other contaminant fragments, quantified by 260 nm absorbance, and serially diluted to 10 -5 pg/ l. Several 10-fold dilutions (10 -1 -10 -5 ) were checked for optimal cycling on the LightCycler and three of them were selected for standard curves taking in consideration mRNA abundance. Each reaction was run in duplicate and contained 3 l of cDNA template, 3 mM Cl 2 Mg and 0.5 M of primers in a final reaction volume of 20 l. Cycling parameters were 95ºC for 10 min to activate DNA polymerase, then 30-40 cycles of 95ºC for 10 s, annealing temperature for 10 s (CB1: 62ºC, FAAH: 41ºC, NAPE-PLD: 57ºC, GADPH: 66ºC; GUS: 61ºC ) and a final extension step of 72ºC for 16 s in which fluorescence was acquired. Melting curves analysis were performed to ensure only a single product was amplified. Data for CB1, FAAH, NAPE-PLD and GAPDH were normalized for expression of GUS.

Preparation of rat tissue subcellular fractions.

Tissues were homogenized in 50 mM Tris buffer, pH 8, containing 0.32 M sucrose.

Homogenates were centrifuged first at 1000 × g (5 min), the pellet was then discarded and the supernatant centrifuged at 45,000 × g (30 min). The pellets obtained were solubilized at 0-4°C in Tris buffer. The protein content in the membrane fraction was measured with the Bradford method. All tissue samples and membrane fractions were stored at -70ºC until use.

Enzymatic determinations

We assayed membrane-bound FAAH activity using arachidonoyl-[1-3 H]-ethanolamide as a substrate, and measuring metabolized [ 3 H]anandamide (as [ 3 H]ethanolamine) in the aqueous phase after chloroform extraction, as described [START_REF] Hansson | Genetic impairment of frontocortical endocannabinoid degradation and high alcohol preference[END_REF][START_REF] De Fonseca | An anorexic lipid mediator regulated by feeding[END_REF]. Microsomal NAT activity assays were performed using 1,2-Di[ 14 C]palmityl-sn-glycerophosphocholine as substrate [START_REF] Cadas | Occurrence and biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine, in rat brain[END_REF]. Both enzymatic assays were run under conditions that were linear in time and protein concentration.

In vivo microdialysis of anandamide and glutamate

Drug-naïve male Wistar rats (n = 11; 300-350g; Charles River, Wilmington, MA USA) were anesthetized (Isofluorane 1-2% vapor) and implanted with microdialysis guide cannula (20-gauge, SciPro Inc., Sanborn NY, USA) aimed at the nucleus accumbens shell (from Bregma: AP +1.6 mm, ML +/-0.8 mm, and DV -5.7 mm from dura; [START_REF] Paxinos | The rat brain in stereotaxic coordinates[END_REF][START_REF] Giuffrida | Dopamine activation of endogenous cannabinoid signaling in dorsal striatum[END_REF]). Seven days of post-operative recovery period was allowed before testing. On the day before microdialysis, animals were anesthetized (Isofluorane 1-2% vapor) and a microdialysis probe (2 mm PES membrane, 15 kDA MW cutoff, SciPro Inc., Sanborn NY, USA) was inserted and secured to the previously implanted guide cannula. Probes were perfused overnight with artificial cerebrospinal fluid (aCSF; 145 mM NaCl, 2.8 mM KCl, 1.2 mM MgCl 2 , 1.2 mM CaCl 2 , 5.4 mM D-glucose, 0.25 mM ascorbic acid, pH 7.2-7.4) at a rate of 0.2 l/min. The following morning, the perfusate was switched to an aCSF containing 30% (w/v) hydroxypropyl-cyclodextrin (HB-CD) and the flowrate was increased to 0.6 µl/min. Inclusion of HB-CD in the perfusate provides a substantial increase in the dialysis recovery of eCBs (similar to the findings by Walker and colleagues [START_REF] Walker | Pain modulation by release of the endogenous cannabinoid anandamide[END_REF]). Following a 90 minute equilibration period dialysate samples were collected at 10-minute intervals during a 40 minute baseline period, for 30 minutes following vehicle injection (ethanol:emulphor:saline, 1:1:18; 1ml/kg) and finally for 120 minutes after ethanol administration (0.75 or 2 g/kg, i.p.; delivered as 20% (w/v)). Dialysate samples were frozen immediately following collection and stored at -80°C until analysis for eCB content using liquid chromatography coupled with mass spectrometry (LC-MS) as described below.

A second group of male Wistar rats (n = 5) were surgically implanted with microdialysis guide cannulae aimed that the NAC for the analysis of ethanol-induced alterations in interstitial glutamate (GLU) levels. The same protocol as described above was followed with the exception that the dialysis perfusate did not contain HB-CD (this additive is not necessary for the recovery of monoamines and amino acids) and a faster perfusate flowrate was employed (1 µl/min) to allow sampling splitting for the monoamine and amino acid analyses (see below).

LC-MS Analysis of dialysate endocannabinoid content

Dialysate levels of AEA were determined using liquid chromatography coupled with ESI mass spectrometry (LC-MS). 5 µl microdialysate aliquots were spiked with 5 µl of 100 nM of (S)-(+)-arachidonyl-2'-hydroxy-1'-propylamide (S-2 methanandamide) and loaded onto a pre-column (0.5 x 2.5 mm, Haisil HL C18 5µm; Higgins Analytical Inc., Mountain View, CA) using a 30% MeOH (v/v) mobile phase delivered at 70 µl/min. Following a 2 min wash period, mobile phase flow through the precolumn was reversed via a switching valve and the eCBs were delivered to a 0.3 x 50 mm microbore analytical column (Haisil HL C18 3µm; Higgins Analytical Inc., Mountain View, CA) using an isocratic mobile phase consisting of 70% MeOH (v/v) delivered at 5 µl/min. The analytical column eluent was delivered via a nanoelectrospray interface into the mass spectrometer (Agilent 1100MSD) that was run in positive Selected Ion Monitoring mode to maximize sensitivity. Similar to findings by others [START_REF] Giuffrida | Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry[END_REF] we find that sodium adducts of these molecules provide greater sensitivity than do their protonated forms. The following mass/charge (m/z) ratios were used: AEA, 370.3 (M+1Na); S-2 methanandamide, 384.3 (M+1Na). External calibration curves were constructed from a minimum of three standard concentrations (each run in duplicate) and were generated daily. Under these conditions the limits of quantitation were approximately 0.1 nM.

Analysis of dialysate glutamate

Microdialysis samples were split into two 5 µl aliquots for quantification of glutamate content by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Specific details on these analyses have been previously published [START_REF] Hansen | Characterization of glutamate-induced formation of N-acylphosphatidylethanolamine and N-acylethanolamine in cultured neocortical neurons[END_REF].

Statistics

Statistical significance was assessed by one-way ANOVA. Following a significant F value, post hoc analysis (Student-Newman-Keuls) was performed. Calculations were made using the BMDP statistical package.

RESULTS

Acute injection of saline does not affect brain anandamide levels

The acute injection of saline did not affect brain anandamide levels in the ventral striatum, cerebellum or dorsal hippocampus, (See Table 1). Since saline injection in habituated animals is a mild stressor, the lack of effects of this procedure on brain endocannabinoid content is in agreement with previous reports [START_REF] Patel | Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling[END_REF] which described a decrease in anandamide only after severe stress induced by restraint, and only in the amygdala (and not in the cerebellum or the forebrain, including the nucleus accumbens).

Acute ethanol decreases plasma anandamide levels

The acute i.p. administration of 4 gr/kg of ethanol led to a rapid rise in plasma ethanol levels, which then remained stable for 4 hr after the administration (Fig 1A).

Clearance of plasma ethanol was evident 8 hr after the injection and was complete within the first 24 hr. Plasma AEA levels were affected after acute ethanol exposure (Fig 1B ): AEA was not detectable in plasma 45 min after acute ethanol exposure, returned to normal levels within the following 45 min, peaked at 4 hr and returned to control levels at 8 hr (1.3 ± 0.6 pmol/ml, n = 8) and 24 hr (0.7 ± 0.5 pmol/ml, n = 8).

In peripheral tissues, ethanol decreased AEA levels in perirenal fat, but resulted in a marked increase in the small intestine and liver 90 min after the injection (Table 2).

Based on both the presence of stable ethanol levels and on the changes in plasma AEA, we selected the time points of 0, 45, 90 and 240 min for the biochemical analysis of both AEA and PEA and the enzymatic activities of FAAH and NAT in the brain.

Acute ethanol decreases anandamide and palmithylethanolamide in

Acute ethanol does not increase FAAH activity

In order to investigate whether the decrease on the levels of AEA and PEA observed after acute ethanol might be derived of changes in the activity of the enzyme involved in the degradation of these acylethanolamides -FAAH -, we tested its activity in membrane fractions obtained from animals acutely exposed to ethanol. FAAH activity was not affected by ethanol neither in membranes from ventral-striatum nor from cerebellum (Figure 4A and4B). However, FAAH activity was decreased 45 min after alcohol administration in the hippocampus, normalizing thereafter. Additional studies performed in vitro studies using cerebellar membranes indicated that FAAH activity was not affected by the presence of ethanol in the buffer at concentrations in the range of those found in plasma after the acute administration of ethanol (10 -9 to 10 -2 M, data not shown).

Acute ethanol does not afect the expression of CB,1 FAAH and NAPE-PLD mRNA in the hippocampus and the cerebellum

Since we observed that ethanol decreased FAAH activity in the hippocampus, we monitored the expression of FAAH mRNA as well as that of the n-acylphosphatidylethanolamide-specific phospholipase D, the recently cloned enzyme that cleaves anandamide from its membrane precursor (Fig 5A to 5D). Any of the endocannabinoid-related genes were affected by acute ethanol administrtion either at 2 or 12 hr after the injection. However, GADPH expression was clearly increased after acute alcohol exposure. This pattern was observed also both in the hippocampus and in the cerebellum. These results point against an activation of degradation as the main origin of the ethanol-induced decrease of anandamide in the brain.

Acute ethanol does not affect NAT activity

The activity of the enzyme involved in the synthesis of the AEA precursor Narachydonoyl phosphatidyl ethanolamine -NAT -was not affected in cerebellar membranes after acute i.p. administration of ethanol (Table 3). We could not measure NAT activity on individual hippocampus because the technique may require pooling of various samples in order to reach sufficient amount of protein.

Acute ethanol decreases anandamide in the ventral striatum

Baseline dialysate AEA concentrations were 2.03 ± 0.5 and 2.12 ± 1.1 nM for the 0.75 g/kg (n = 6) and 2.0 g/kg (n = 5) ethanol groups, respectively. Although vehicle injection produced no significant alteration in dialysate AEA levels, intraperitoneal ethanol administration led to a significant and dose-dependent decrease in dialysate AEA concentrations (F(1,141) = 25.094, p < 0.001; see Figure 6A). Administration of 0.75 g/kg ethanol produced a subtle and non-significant decrease in dialysate AEA levels. However, injection of 2 g/kg ethanol produced a significant reduction in dialysate AEA (F 12, 52 = 2.621; p<0.001) which was evident within 40 minutes after administration and persisted at approximately 60% of pre-ethanol baseline levels for the remainder of the 120 minute post-ethanol sample collection period.

Acute ethanol decreases glutamate in the ventral striatum

Baseline dialysate glutamate levels in the same samples were 602 ± 21 nM.

Glutamate levels in the dialysate samples were significantly reduced following 2 g/kg ethanol (F 12,82 = 4.679; p<0.0001) which was evident within 30 minutes after administration and persisted at approximately 70% of pre-ethanol baseline levels for the remainder of the 120 minute post-ethanol sample collection period (Figure 6B).

DISCUSSION

These results suggest, first, that acute exposure to ethanol affects AEA formation in the brain and peripheral tissues and, second, that this effect is not dependent on ethanol-induced activation of AEA degradation by FAAH. The effects on AEA were replicated for a second acylethanolamide, PEA, that shares with AEA biochemical pathways for synthesis or degradation, suggesting a common mechanism for the decrease of both acylethanolamides. In vivo microdialysis experiments suggest that the effect on AEA release might be dependent on the inhibitory actions of ethanol on glutamate release, one of the main triggers for AEA formation Ethanol induced a marked decrease in the formation of AEA in the brain, plasma and adipose tissue, which was time-limited to the first 90 min of study (Fig 1 and2). Opposite effects were observed in the liver and, especially, in the small intestine, where a marked increase in AEA release was also observed 90 min after ethanol administration. These effects suggest the existence of tissue-selective endogenous cannabinoid targets for ethanol. Increases in AEA levels in tissues may be dependent either on changes in the synthesis of AEA precursor n-arachydonylphosphatidylethanolamide (NAPE), or on the cleavage of NAPEs species by receptor-activated phospholipase D (PLD), or by blockade of FAAH-dependent enzymatic degradation of AEA [START_REF] Cadas | Biosynthesis of an endogenous cannabinoid precursor in neurons and its control by calcium and cAMP[END_REF][START_REF] Cravatt | Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides[END_REF][START_REF] Marzo | Formation and inactivation of endogenous cannabinoid anandamide in central neurons[END_REF]. As there was no activation of FAAH by in vivo or in vitro ethanol, and since initial observations indicate no changes in NAT activity (Table 2), we propose that the decrease in AEA and PEA release observed may be dependent on reduced receptor-activated NAPE-PLD activity, a major source for the release of acylethanolamides [START_REF] Giuffrida | Dopamine activation of endogenous cannabinoid signaling in dorsal striatum[END_REF][START_REF] Hansen | Characterization of glutamate-induced formation of N-acylphosphatidylethanolamine and N-acylethanolamine in cultured neocortical neurons[END_REF][START_REF] Piomelli | Receptor-dependent formation of endogenous cannabinoids in cortical neurons[END_REF]. Acute ethanol might operate then through the modulation of neurotransmitters capable of activate specific receptors coupled to NAPE-PLD. This mechanism may not request a substantial modification of NAPE-PLD production because the inhibition of the release of neurotransmitters will solely result in reduced endocannabinoid production. Following this rationale, we observed that the expression of this synthetic enzyme is not affected 2 hr after the administration of ethanol, although further research is needed to clarify whether chronic administration of alcohol might affect its expression and activity, leading to the increased production of endocannabinoid described in animal models of alcoholism.

Several neurotransmitters may contribute to a decrease in PLD-mediated NAPE breakdown and the subsequent lowering of AEA and PEA levels. First, acute ethanol may decrease glutamate and acetylcholine-dependent PLD activation through its welldescribed inhibitory effects on presynaptic release of these neurotransmitters [START_REF] Maejima | Presynaptic inhibition caused by retrograde signal from metabotropic glutamate to cannabinoid receptors[END_REF][START_REF] Wilson | Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses[END_REF].

In this regard, the microdialysis eperiments confirms this hypothesis, since we found parallel decreases in extracellular levels of both, AEA and glutamate, in the ventral striatum (Figure 6) after acute ethanol exposure. Chronic ethanol exposure will result in the development of tolerance and ultimately to increased release of both transmitters [START_REF] Dahchour | Taurine blocks the glutamate increase in the nucleus accumbens microdialysate of ethanol-dependent rats[END_REF]42], which will contribute to the enhanced AEA formation associated with chronic ethanol exposure [START_REF] Hungund | Ethanol, endocannabinoids, and the cannabinoidergic signaling system[END_REF]. Second, ethanol may directly inhibit glutamate NMDA and metabotropic receptors involved in PLD-dependent AEA release, leading to a decrease in NAPE breakdown [START_REF] Lovinger | Ethanol inhibits NMDA-activated ion current in hippocampal neurons[END_REF][START_REF] Maejima | Presynaptic inhibition caused by retrograde signal from metabotropic glutamate to cannabinoid receptors[END_REF][START_REF] Hansen | Characterization of glutamate-induced formation of N-acylphosphatidylethanolamine and N-acylethanolamine in cultured neocortical neurons[END_REF]. Although there is direct evidence that ethanol might disrupt PLD dependent signaling, acting as a metabolic substrate that shifts PLD activity to the production of the phospholipid phosphatidylethanol [43], this hypothesis can not be used to explain the effects observed in the present study since recent reports indicate that NAPE-PLD does not use ethanol as a substrate [START_REF] Okamoto | Molecular characterization of a phospholipase D generating anandamide and its congeners[END_REF].

An additional factor contributing to a decrease in anandamide formation might be the stress response activated by acute ethanol exposure [44]. Since ethanol can markedly increase the activity of the hypothalamus-pituitary-adrenal axis, the rise in corticosterone might affect endocannabinoid production. However, and based on the the fact that restraint , a severe form of experimental stress in rats, only reduced anandamide formation in the amygdala, but not in other areas such as the nucleus accumbens and cerebellum, the potential contribution of ethanol-induced stress to the inhibition of acylethanolamides release may be of less importance than that originated by ethanol-induced inhibition of both glutamate and acetylcholine release. Indeed, this experimental hypothesis needs to be confirmed. Finally, the effects observed were tissue-selective, indicating the existence of a complex network of signals regulating endocannabinoid production. The fact that the dorsal hippocampal area is more sensitive to the inhibitory effects of ethanol on AEA and PEA formation may be dependent on the coexistence of a very important glutamatergic and cholinergic innervation, two neurotransmitters that cooperatively stimulate AEA release [START_REF] Piomelli | Receptor-dependent formation of endogenous cannabinoids in cortical neurons[END_REF]. Indeed, all these mechanisms may interact together in a tissue-selective fashion, in agreement with the proposed role for acylethanolamides as local mediators. The local specificity of mechanisms regulating AEA formation is supported by the activator role of ethanol in the small intestine. We have recently reported that the small intestine produces large amounts of AEA in starved animals [45]. The effects of acute ethanol on AEA production resemble those found after 18 h of food deprivation, probably because ethanol may act as a functional hypoglycemiant drug through its interference with glucose metabolism [46].

All the effects on AEA and PEA formation in the brain described in the present study were observed while substantial amounts of ethanol remained unmetabolized, as reflected in its plasma levels (Fig 1A). Its rapid disappearance suggests a tight regulation of acylethanolamide production, again in agreement with its role as a local mediator of intercellular signaling. Inhibition of anandamide formation is regulated by rapid homeostatic counterregulatory responses that, after repeated exposure to ethanol, lead to an activation of NAT, an enhanced production of NAPEs (AEA and PEA precursors) and an increased tissular release of AEA [8, 9, 2].

In summary, acute exposure to ethanol inhibits acylethanolamide production in the brain through mechanisms that do not involve fatty acid amidohydrolase activity but may be linked to neurotransmitter release. The relevance of this finding for the acute effects of ethanol, and for the neuroadaptions associated with its chronic administration will be further investigated.

Cannabinoid receptor antagonist SR141716A decreases operant ethanol self administration in rats exposed to ethanol-vapor chambers. Zhongguo Yao Li Xue Bao. 120, 1109-14.

  the ventral striatum, cerebellum and hippocampus Acute injection of ethanol decreased AEA levels in the ventral striatum (Fig 2A), cerebellum (Fig 2B) and hippocampus (Fig 2C). This effect was observed in all brain areas 90 min after ethanol administration. In the hippocampus, the reduction of AEA levels was observed at all time points. Similarly, ethanol decreased PEA in the ventral striatum (Fig 3A) 45 min after its injection, the cerebellum at 45 and 90 min (Fig 3B) and the hippocampus in all time points studied (Fig 3C).

  7. Cippitelli A, Bilbao A, Hansson AC, del Arco I, Sommer W, Heilig M, Massi M, Bermudez-Silva FJ, Navarro M, Ciccocioppo R, and Rodriguez de Fonseca F (2005) The European TARGALC Consortium. Cannabinoid CB1 receptor antagonism reduces conditioned reinstatement of ethanol-seeking behavior in rats. Eur J Neurosci. 21, 2243-51. 8. Basavarajappa BS and Hungund BL (1999) Chronic ethanol increases the cannabinoid receptor agonist anandamide and its precursor Narachidonoylphosphatidyl-ethanolamine in SK-N-SH cells. J Neurochem. 72, 522-8. 9. Basavarajappa BS, Saito M, Cooper TB and Hungund BL (2000) Stimulation of cannabinoid receptor agonist 2-arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons. Biochim Biophys Acta. 1535, 78-86. 10. Basavarajappa BS, Cooper TB and Hungund BL (1998) Chronic ethanol administration down-regulates cannabinoid receptors in mouse brain synaptic plasma membrane. Brain Res. 793, 212-8. 42. Imperato A, Dazzi L, Carta G, Colombo G and Biggio G (1998) Rapid increase in basal acetylcholine release in the hippocampus of freely moving rats induce by withdrawal from long-term ethanol intoxication. Brain Res 784, 347-350. 43. Kötter K and Klein J (1999) Ethanol inhibits astroglial cell proliferation by disruption of phospholipase D-mediated signaling. J Neurochem 73, 2517-2523. 44. Rivier C. (1993) Acute interactions between cytokines and alcohol on ACTH and corticosterone secretion in the rat. Alcohol Clin Exp Res. 17, 946-950. 45. Gómez R, Navarro M, Ferrer B, Trigo JM, Bilbao A, del Arco I, Cippitelli A, Nava F, Piomelli Dand Rodríguez de Fonseca F. (2002) A peripheral mechanism for CB1 cannabinoid receptor-dependent modulation of feeding. J Neurosci 22, 9612-9617. 46. Williams HE (1984) Alcoholic hypoglycemia and ketoacidosis. Med Clin North Am 68, 33-8.

FIGURE 1 TimeFIGURE 2 FIGURE 3 Time

 123 FIGURE 1

FIGURE 4 TimeFIGURE 5 FIGURE 6 33 Biochemical

 45633 FIGURE 4

  FIGURE 1

  FIGURE 2

  FIGURE 5

FIGURE 6 -

 6 FIGURE 6

TABLE 1

 1 Effects of acute administration of vehicle (0.9% saline solution, injected 1ml//kg i.p.) on anandamide levels (pmol/g tissue) in the ventral striatum (nucleus accumbens) , cerebellum and dorsal hippocampus, measured in injection-habituated animals 0, 30, and 60 min after the injection. Data are the means ± SEM of 5-8 determinations per group. (*) P < 0.01 versus time 0 group, Newman-Keuls.

		Anandamide (pmol/g)
	Time after the	0'	30'	60'
	injection (min)
	Ventral Striatum	119 ± 20	98 ± 40	82 ± 29
	Cerebellum	16.5 ± 1.4 14.7 ± 1.8 26.7 ± 1.9
	Dorsal	137 ± 51	134 ± 63	119 ± 41
	Hippocampus

TABLE 2

 2 Effects of acute administration of ethanol (4 gr/kg i.p.) on anandamide (AEA) levels

	(pmol/g tissue) in the perirenal fat, liver and small intestine, 0, 45, 90 and 240 min
	after the injection. Data are the means ± SEM of 5-8 determinations per group. (*) P
	< 0.01 versus time 0 group, Newman-Keuls.
		0'	45'	90'	240'
	Fat	16.2 ± 1.7	11.9 ± 0.9	19.1 ± 1.2	6.4 ± 0.6 *
	Liver	3.7 ± 1.8	3.7 ± 1.8	21.9 ± 3.7 *	4.8 ± 2.9
	Small	33.9 ± 13.2	63.4 ± 23.5	285.1 ± 13.2 *	62.8 ± 12.8
	intestine

Biochemical Journal Immediate Publication. Published on 15 Feb 2007 as manuscript BJ20061898 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 Feb 2007 as manuscript BJ20061898 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 Feb 2007 as manuscript BJ20061898 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work has been supported by Plan Nacional Sobre Drogas (F.R.F.) and National Institute on Drug Abuse (D.P., L.H.P.) and FIS 02/001, REDES G03/05, CO3/08, MEC SAF 2004/07762 and and 5 th Framework Programme, grants QLRT-2000-01691 and QLRT-2001-01048 (M.N. and F.R.F.)


Katrine S Larsen

Henrik Østergaard

Jais R Bjelke

Ole H Olsen

Hanne B Rasmussen

Leif Christensen

Birthe B Kragelund

Henning R Stennicke

Engineering the Substrate and Inhibitor Specificities of Human Coagulation Factor VIIa

Keywords: Serpin, ATIII, specificity, profiling, protease, FVIIa activated coagulation factor IXa, FX, coagulation factor X, FXa, activated coagulation factor X, LMW, low molecular weight, MUGB, 4-methylumbelliferyl p-guanidinobenzoate, rFVIIa, recombinant activated coagulation factor VII, PS-SCL, positional scanning-substrate combinatorial library, serpin, serine protease inhibitor, sTF, soluble tissue factor

The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For factor VIIa (FVIIa), the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. Here, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning-substrate combinatorial libraries and evaluated the role of selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa showed P1 specificity exclusively towards Arg and Lys. In the S2 pocket Thr, Leu, Phe and Val were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed, and as a consequence, the optimal substrate for FVIIa could not be derived directly from a sub-site directed specificity screen. To evaluate the role of the active site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (c99), which is considered one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and reduced rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate factor X demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.

INTRODUCTION

Coagulation factor VIIa (FVIIa) is a trypsin-like serine protease responsible for triggering blood coagulation when it associates with its cofactor tissue factor (TF) which is exposed upon vascular injury [START_REF] Davie | The coagulation cascade: initiation, maintenance, and regulation[END_REF][START_REF] Perona | Structural basis of substrate specificity in the serine proteases[END_REF]. TF-bound FVIIa is an efficient activator of both factor IX (FIX) and X (FX) which on the platelet surface ultimately results in a burst of thrombin, fibrin deposition, and the formation of a haemostatic plug [START_REF] Davie | The coagulation cascade: initiation, maintenance, and regulation[END_REF]. Thus, initiation of blood coagulation represents a powerful and highly amplified system and for obvious reasons it must be tightly controlled in order to restrict thrombosis to the site of vessel injury and prevent complete occlusion of the damaged vessel. The enzymatic activity of FVIIa is regulated by two physiological inhibitors.

The FVIIa-TF complex is efficiently inhibited by the Kunitz inhibitor tissue factor pathway inhibitor (TFPI) in the presence of FXa by forming an inhibitory TFPI-FXa-FVIIa-TF quaternary complex [START_REF] Broze | The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action[END_REF]. FVIIa can also be inhibited by the serine protease inhibitor (serpin) antithrombin III (ATIII) and the inhibition is accelerated many folds by sulphated glycosaminoglycans lining the vascular wall [START_REF] Olson | Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombinproteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement[END_REF]. Interactions taking place at regions remote from the active site cleft, at so-called exosites, also influences the rate of TFPI inhibition of the FVIIa-TF complex and FXa [START_REF] Petersen | Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor[END_REF], whereas the direct interaction between ATIII and its target proteases is mediated primarily through active site interactions and few exosite interactions [START_REF] Chuang | The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation[END_REF].

From a molecular perspective, the interaction between FVIIa and TF is characterized by a large interface between their extracellular domains [START_REF] Banner | The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor[END_REF]. The activation of FIX and FX by this complex is mediated by extensive interactions involving both the Gla-EGF1 region and the protease domains of FIX and FX. Thus, the activation is largely considered to be controlled by exosite interactions remote from the active site [START_REF] Shobe | Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking[END_REF][START_REF] Baugh | Exosite interactions determine the affinity of factor X for the extrinsic Xase complex[END_REF] and thus, ground state stabilization (K m ) for the activation reaction may be largely independent of the active site. Consequently, we hypothesized that the inhibitory profile and the ability to act as an initiator of coagulation may constitute two independent paradigms in terms of substrate recognition. Thus, when altering substrate specificity there are a number of different strategies available among which the most obvious are: 1) blocking sites where large residues are preferred and 2) generating new cavities [START_REF] Bone | Structural plasticity broadens the specificity of an engineered protease[END_REF][START_REF] Bone | Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters[END_REF].

Numerous attempts at engineering specific enzyme-substrate

Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901

© 2007 Biochemical Society interactions have been described in the literature; the general conclusion reached from these studies have been that major changes to substrate specificity often require global changes to the protein structure [START_REF] Hedstrom | Serine protease mechanism and specificity[END_REF]14]. In contrast to the challenge associated with generating new substrate specificities via the introduction of specific interactions, the generation of FVIIa variants that do not interact with ATIII, but maintain the ability to activate FX, appears to be of an entirely simpler nature.

In order to identify the most optimal sub-site for altering the FVIIa specificity we first evaluated the substrate specificity of FVIIa using positional scanning-substrate combinatorial libraries (PS-SCLs). One aspect that such libraries do not always fully address is the potential interdependence between the different sub-sites. Thus, in order to get a more detailed insight into the primary substrate selectivity of FVIIa we generated and analyzed a number of purified peptide substrates as well as generated a three dimensional structure of FVIIa-sTF in complex with a chloromethyl ketone (cmk) derivative mimicking an optimal substrate based on screening of the peptide substrates. Based on the findings from these studies a number of variants were generated by substituting residues within the active site cleft that form direct interactions with the side chains of the substrate [START_REF] Chang | Engineered recombinant factor VII Q217 variants with altered inhibitor specificities[END_REF][START_REF] Rezaie | Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity[END_REF]. However, for reasons of simplicity only one position will be evaluated at present. From other trypsin family members such as tPA, FXa, APC and thrombin [START_REF] Rezaie | Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity[END_REF][START_REF] Vindigni | Role of residue Y99 in tissue plasminogen activator[END_REF][START_REF] Rezaie | Role of Leu99 of thrombin in determining the P2 specificity of serpins[END_REF] position 99 (chymotrypsin numbering) has proven important for the S2 specificity of this family. Consequently, in order to reduce the rate of inhibition of FVIIa by circulating inhibitors while retaining proteolytic activity towards FX, we initially focused on this position in our attempts to alter the primary specificity of FVIIa.

Biochemical

Mutagenesis and protein expression and purification-

The wild-type FVII expression plasmid pLN174 [START_REF] Persson | Site-directed mutagenesis but not gammacarboxylation of Glu-35 in factor VIIa affects the association with tissue factor[END_REF] was used as template for site directed mutagenesis using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). Plasmids were prepared using the QIAprep spin miniprep kit (QIAGEN) and the mutations were verified by DNA sequencing. Baby hamster kidney (BHK) cells were transfected with the FVII expression plasmids using the FuGENE6

Transfection Reagent (Roche) and the FVII variants purified as previously described [START_REF] Persson | Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor[END_REF].

After auto-activation the concentration of the FVIIa variants was determined by ELISA. All FVIIa variants were active site titrated with the ester substrate 4-methylumbelliferyl pguanidinobenzoate (MUGB) essentially as described [START_REF] Payne | Effect of soluble tissue factor on the kinetic mechanism of factor VIIa: enhancement of pguanidinobenzoate substrate hydrolysis[END_REF].

Positional scanning-synthetic combinatorial libraries-ACC-based PS-SCLs were synthesized at Novo Nordisk A/S by solid phase on Fmoc-7-amino-4-carbamoylmethylcoumarin resin essentially as previously described [START_REF] Persson | Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor[END_REF]. The four synthesized PS-SCLs consisted of 18 sublibraries with each sub-library containing a mixture of tetrapeptide substrates incorporating one of the 18 proteinogenic amino acids (Met and Cys excluded) at P1, P2, P3 or P4. In the randomized. In the P1 library the P1 residue was varied and the P2, P3 and P4 residues were all randomized. The concentration of the libraries was determined by amino acid analysis and absorbance measurements at 325 nm. The optimal emission and excitation parameters for assaying the PS-SCLs were determined by cleaving one of the P4 sub-libraries with trypsin.

Each sub-library was assayed at final concentrations of 3, 6, 9 and 12 µM in black 96-well fluorescence plates in a total reaction volume of 200 µl. The assays were performed at room temperature in a buffer containing 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 0.01% Tween80, pH 7.4. The hydrolysis reactions were initiated by addition of a final concentration of 100 nM of FVIIa variant and 150 nM of sTF. Kinetics was monitored with a SpectraMax Gemini EM fluorescence microplate reader with excitation at 380 nm, emission at 455 nm and cutoff at 420 nm. The tetrapeptide substrates in each sub-library were assayed at a concentration far below the expected K m and thus, linear fitting of the initial rates yielded a set of apparent second order rate constants for hydrolysis of each sub-library which were normalized to the rate of the sub-library with the highest activity.

Assay of ACC-tetrapeptides-23 ACC-tetrapeptides were synthesized on Fmoc-7-amino-4carbamoylmethylcoumarin resin essentially as previously described [START_REF] Maly | Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carbamoylmethylcoumarin (ACC) fluorophore[END_REF]. The concentration of the peptides was determined by amino acid analysis and RP-HPLC.

[ ] [ ] [ ]0 m 0 D 0 Lim obs I ) K S (1 K I k k + + = (Equation 2)
where K D is the dissociation constant for binding of FVIIa to the ATIII-heparin complex to form the non-covalent Michaelis-complex and k Lim is the observed rate constant at saturation for formation of the covalent protease-inhibitor complex.

The apparent second-order rate constant of inhibition, k inh , is given by k inh = k Lim /K D . Crystallization conditions-The complex between WYTRcmk-FVIIa and sTF 1-209 was purified using a Superdex75 16/60 prep-grade column (Amersham Biosciences, Uppsala, Sweden) and concentrated to approximately 10 mg/ml using a Centriprep centrifugation device with a M r cutoff of 50 kDa. A buffer composition of 10 mM Tris, 100 mM NaCl, 2 mM CaCl 2 , pH 7.5 was used. Crystal plates were obtained using the hanging drop vapor diffusion method using 0.1 M sodium citrate, 16.5-15.5 % (w/v) PEG4000, 12 % (v/v) 1-propanol, pH 5.6 as precipitating agent. A final protein concentration in the drop of approximately 3 mg/ml was used. Seeding was performed using crystal needles obtained as previously described [START_REF] Banner | The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor[END_REF].

FVIIa-ATIII complex breakdown-Covalent

Crystal data collecting and processing-Crystallographic data collection was performed at the Swiss Light Source synchrotron beam line PXI. Data reductions were performed with the XDS software package [24]. The structure was solved by the molecular replacement method using the MR facility of the CCP4 software package and the PDB entry 1DAN as a search model [25].

Model building was performed using Coot software [26] and iterative refinement (initially performed as a rigid body refinement) using RefMac5 of the CCP4 software package [25]. the expected primary specificities [START_REF] Snipas | Characteristics of the caspase-like catalytic domain of human paracaspase[END_REF]. Specificity profiling of rFVIIa in the presence of sTF showed as expected that rFVIIa only accepts Arg and Lys in the S1 pocket with Arg being favoured three times over Lys. In the S2 pocket β-branched or hydrophobic amino acids are allowed with Thr, Leu and Phe being the preferred residues (see Figure 1). In the S3 position the large aromatic residues Phe, Tyr and Trp are preferred, while the S4 sub-site appears highly promiscuous although a slight preference for Trp can be detected. An identical specificity profile of rFVIIa was obtained in the absence of sTF indicating that sTF do not alter the substrate specificity of rFVIIa (data not shown).

RESULTS

Specificity profiling of rFVIIa-The

Assay of proteases with the PS-SCLs provides a complete specificity profile in the non-prime sites of the active site cleft. However, only one substrate-binding pocket is profiled at a time and therefore no steric considerations or interdependency between the sites are investigated.

To elucidate any interdependency between the S1-S4 binding pockets, rFVIIa was assayed with a range of single ACC-tetrapeptides having Arg in P1; Thr or Leu in P2; Trp, Tyr, Leu or Ala in P3; and Trp, Phe or Asn in P4. The substrates were selected partly to address different levels of activity based on the specificity profiles and partly to explore the space around the natural FX cleavage site NLTR-IVGG. For technical reasons the NYTR-ACC peptide was omitted and thus rFVIIa was assayed with a total of 23 different ACC-tetrapeptides in the presence of sTF.

The obtained k cat /K m values for cleavage of the ACC-tetrapeptides by rFVIIa confirmed the preference for aromatic residues in both P3 and P4 (Table 1). The highest k cat /K m values were obtained for the WATR-ACC and WYTR-ACC peptides. The best peptide, WATR-ACC, was P3-Trp residue. For all peptides, rFVIIa was more active towards P2-Thr peptides than P2-Leu peptides by a factor of approximately 2.5. Thus, the clear P2-Thr specificity was independent of the nature of the P3 and P4 residue of the peptides.

The discrimination between two substrates is determined by the relative binding of their transition states to the enzyme. The difference in transition state stabilization energy, ∆∆G T ‡ , of the peptides was calculated using the relative difference in k cat /K m values [START_REF] Gron | Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching[END_REF] where ∆∆G T ‡ is negative if the XXXR peptide is a better substrate than the YYYR peptide:

(YYYR) m cat (XXXR) m cat T /K k /K k -RTln = ∆∆G ‡
Using this equation it was possible to evaluate the impact of a single substitution on the overall transition state stabilization energy as well as to assess whether there was interdependence between the different subsites. Interestingly, even with the relatively small number of substrates tested there appear to be quite a significant degree of interdependence as is evident from Figure 2. Thus, the effect on ∆∆G T ‡ of a single amino acid substitution is highly context dependent, e.g., in the extreme case the ∆∆G T ‡ for the P4 N->W substitution in the NATR-ACC peptide was -7.3 kJ ⋅ mol -1 while the same substitution in the NWTR-ACC peptide was only -0.4 kJ ⋅ mol -1 . For reference, in a system with no sub-site interdependence a ∆∆G T ‡ of -3.2 kJ ⋅ mol -1 would have been expected based on the sub-site profiling.

Three dimensional structure of WYTRcmk-FVIIa in complex with sTF 1-209 -To gain an increased insight into the manner in which an optimal substrate, as determined by the PS-SCL technique, binds to rFVIIa, the three dimensional structure of rFVIIa-sTF in complex with Trp-Tyr-Thr-Arg-chloromethyl ketone (cmk) was determined (Figure 3). The structure of the complex resolved to 2.05 Å based on 52,511 unique reflections observed giving 100 % completeness for the complete bin range 2.05 -39.16 Å. Overall, the structure is highly similar to the structure of FVIIa-TF inhibited with Phe-Phe-Arg-cmk published by Banner et al [START_REF] Banner | The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor[END_REF]. clearly visible in the electron density map, i.e. Arg, Thr and Tyr (see Figure 3). Thus, to secure that the inhibitor was intact, the complex was analysed by MALDI-TOF MS, which demonstrated that the inhibitor had the expected sequence (data not shown).

The arginine in the P1 position was strongly defined and showed a similar binding motif as observed in the structure by Banner et al [START_REF] Petersen | Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor[END_REF]. The P2 Thr residue is positioned adjunct to the catalytic triad with a distance of 3.4 Å to the catalytic His193 (c57), which also is the closest contact to any residue of the S2 pocket. The residue is within hydrogen bonding distance with the two water molecules W156 and W178 and could be part of a hydrogen-bonding network of the S2 pocket involving the para-positioned hydroxyl group of Tyr234 (c94), the carboxylate group of Asp196 (c60), the carbonyl of Thr238 (c98) and amides of both Gly237 (c97) and Thr238 (c98). No other contacts can be identified for the P2 residue.

The P3 Tyr residue interacts with the Gln313-Pro321 (c170-c170I) loop and the parapositioned hydroxyl group of the side chain of the tyrosine hydrogen bonds with the carbonyl of Asp319 (c170G), while the benzyl moiety interacts via hydrophobic contacts with Pro321 (c170I) and Trp364 (c215). The carbonyl of the Tyr hydrogen bonds with the amide of Gly367 (c216) as well as with water molecule W56 as earlier described. Thus, the observation that the preferred residue is Tyr, even compared to Phe and Trp, is reflected by the extra hydrogen bond donor of the side chain, which then leads to the more stabilized pocket as observed for the Gln313-Pro321 loop (c170-c170I). Enzymatic activity of FVIIa variants-The enzymatic activity of the FVIIa variants towards small peptidyl substrates was analysed with the chromogenic peptide S-2288 (D-Ile-Pro-Arg-pNA) and the fluorogenic peptide WATR-ACC (Table 2). The amidolytic activity towards S-2288 was highly reduced for variants T239A, T239G and T239Y (4.8-, 10.9-and 10.8-fold, respectively, relative to wild-type rFVIIa). For variant T239I the k cat /K m values for cleavage of S-2288 was only slightly decreased compared to wild-type FVIIa. The amidolytic activity of all the FVIIa variants was improved when measured with the WATR-ACC peptide but the k cat /K m values were still lower compared to wild-type rFVIIa.

Expression and characterization of FVIIa variants-

The FVIIa variants were analysed for their ability to activate FX in the presence of sTF (Table 2). The proteolytic activity was shown to be reduced 8-, 24-and 7-fold for variants T239A, T239G and T239Y, respectively, relative to wild-type rFVIIa whereas the k cat /K m value for activation of FX by variant T239I was comparable to that of wild-type rFVIIa.

The influence of residue 239 (c99) on substrate specificity-The effect of the introduced mutations on the substrate specificity was monitored with the PS-SCLs. Because of the limited amounts of purified FVIIa variants, T239A and T239G were only profiled with the P2 library whereas T239I and T239Y were profiled with both the P2 and P4 library. Phe, Thr and Leu were favourable P2 residues when Thr239 was substituted with either Ala or Gly but relative to wild-type rFVIIa the majority of the P2 sub-libraries were disfavoured. When Ile or Tyr was introduced in position 239 (c99) large changes in specificity were observed. T239I showed a T239Y showed a 2.5-fold increased preference towards P2-Gly relative to wild-type rFVIIa.

The P4 specificity was also affected by this mutation with all amino acids being nearly equally well tolerated as P4 residue and no clear preference for Trp was observed as seen in the P4 profile for wild-type rFVIIa. However, all the P4 sub-libraries were disfavoured for T239Y relative to wild-type rFVIIa.

Kinetic parameters for ATIII inhibition of FVIIa variants-The stoichiometry of inhibition (SI)

for inhibition of wild-type rFVIIa and the FVIIa variants by ATIII in the presence of LMW heparin could not be determined accurately since the titration curves moved to higher [I] 0 /[E] 0 ratios as a function of time indicating breakdown of the ATIII-rFVIIa complexes (data not shown). The apparent second-order rate constant of inhibition (k inh ) of wild-type rFVIIa and the FVIIa variants by ATIII was determined by the progress-curve method (Figure 5) with the results shown in Table 3. The wild-type rFVIIa-sTF complex was inhibited by ATIII in the presence of LMW heparin with an apparent second-order rate constant of inhibition of 26917 M -1 • s -1 . Compared to wild-type rFVIIa, variants T239A and T239G were inhibited very slowly by ATIII with k inh reduced 4.4-and 5.7-fold, respectively, reflecting their low enzymatic activities. In contrast, T239Y was inhibited slightly faster than wild-type rFVIIa. Interestingly, T239I showed a 40% reduction of k inh relative to wild-type rFVIIa but maintained almost full activity against both S-2288 and FX.

FVIIa-ATIII complex breakdown-

The first-order rate constant of covalent complex breakdown, k brkdn , was determined for wild-type rFVIIa and the FVIIa variants by measuring the restoration of amidolytic activity over 12 hours of pre-formed serpin-protease complexes.

Similar values of k brkdn were obtained when the covalent complexes were made with 2.5-and 5-fold excess of ATIII relative to FVIIa confirming complete inhibition prior to measuring the complex breakdown. The obtained t ½ for the complex of ATIII and wild-type rFVIIa was 5.6 hours (Table 3). The covalent complexes between ATIII and variants T239A, T239G and T239Y were more stable than the wild-type complex with t ½ of 6.8, 12 and 9. respectively. In contrast, T239I formed an unstable complex with ATIII having t ½ of 1.9 hours.

DISCUSSION

The serine proteases of the haemostatic system are hallmarked by a marked and distinctive specificity towards endogenous substrates. This is required to tightly regulate the specific proteolytic events that convert inactive precursors in blood to active enzymes in the reactions leading to blood coagulation and fibrinolysis. FVIIa plays a crucial role in the haemostatic system by initiating blood coagulation upon vascular injury. In the current study the substrate and inhibitor specificity of FVIIa has been evaluated using small peptidyl substrates, physiological macromolecular substrates as well as site-directed mutagenesis of key residues in the active site cleft of FVIIa. As expected, rFVIIa only accepted positively charged amino acids in the S1 pocket with a 3-fold higher preference towards Arg relative to Lys. In the P2 position hydrophobic or β-branched amino acids were favoured (Thr>Leu>Phe>Val) and aromatic amino acids were preferred in the S3 pocket. Thus, the specificity profile clearly demonstrates that rFVIIa is not a very discriminative protease with low degree of active site specificity. The selectivity index defined by the ratio between the apparent second order rate constant of the best and worst sub-library is 26 in the P2 specificity profile of rFVIIa. Thus, the P2 selectivity for rFVIIa appears to be more like trypsin, which has a selectivity index of 12, than FXa or thrombin which are characterized by selectivity indexes of 290 and 19000, respectively [START_REF] Bianchini | Mapping of the catalytic groove preferences of factor Xa reveals an inadequate selectivity for its macromolecule substrates[END_REF]. Recently, the P2 and P3 specificity of FVIIa has been profiled using fluorogenic peptide micro-arrays [START_REF] Gosalia | High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays[END_REF]. In that study Thr and Val were also found to be favourable P2 residues, however, in P3 the investigators found Gln, Arg, Asn and Pro to be preferred, in contrast to the preference towards aromatic amino acids found in the current study. The basis for this difference remains unclear, however, it may be due to a possible interdependence between S3 and S4 as the study by Gosalia et al utilizes Ala in P4 [START_REF] Gosalia | High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays[END_REF] and the present study uses a degenerated pool of amino acids.

Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society

In the P4 position there was a pronounced preference towards Trp which is in agreement with a study searching for extensions to the A-183 peptide [31], where it was found by Maun et al that the best sequence extending into the active site of rFVIIa also had a Trp in P4. From modelling studies, the P4 Trp was expected to make favourable π-π interactions with Trp364 (c215) in the bottom of the putative S4 pocket. However, to our surprise the structure of the WYTRcmk-FVIIa-sTF complex showed no electron density corresponding to the P4 Trp (see Figure 3) indicating that there is not a well-defined S4 pocket in FVIIa despite the clear preference observed within the libraries. This could be explained by suboptimal interactions between the P4 residue and the S4 pocket, which would result in a poorly defined electron density because of high temperature factors and suggest that FVIIa may not have a welldefined S4 sub-site. This notion is to some extend supported by the structure as well as the fact that the natural substrate for FVIIa is the N*LTR-IVGG site of FX which harbours a glycosylated Asn in the P4 position.

A review of a large number of proteases in the PDB database has revealed that proteases generally bind their substrates in an extended binding conformation which is to a large degree defined by β-strand type backbone interactions [START_REF] Tyndall | Conformational homogeneity in molecular recognition by proteolytic enzymes[END_REF]33]. The PS-SCLs profile the primary selectivity in the S1-S4 binding pockets of a protease independently without accounting for cross-talk between sub-sites and without the requirements for optimal alignment of the peptide backbone. To address this issue, interdependency between the S1-S4 binding sites of rFVIIa was investigated by determining the change in transition state stabilization energy, ∆∆G T ‡ , upon a single substitution in the substrate, e.g., WATR-ACC to FATR-ACC etc. As demonstrated by the derived data accumulated in Figure 2, there is a significant degree of interdependence between the different sub-sites as indicated by the relatively large differences associated with identical amino acid substitutions in different scaffolds, i.e., the ∆∆G T ‡ for a P4 substitution of Asn -> Trp or Phe is very different for substrates with different P3 residues, while only minor differences are observed between similar substrates with Leu or Thr in P2.

Thus, the S3 selectivity is likely to be driven or influenced by the P4 occupancy and this could Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901

© 2007 Biochemical Society potentially explain the difference between the two rather similar approaches used to describe FVIIa specificity.

Interestingly, the effects of going from Asn in P4 of NATR-ACC to Phe or Trp far exceeds the predicted effects from the sub-site profiling as ∆∆G T ‡ values of -1.4 and -3.1 kJ ⋅ mol -1 , respectively, were expected rather than the observed -5 and -7 kJ ⋅ mol -1 , respectively. This is in contrast to the changes observed for the Phe to Trp substitution, which are very close to what should be anticipated from the profiling. One explanation for this observation is that optimal sub-site occupancy does not translate to one optimal substrate. As a result, if P3 contains Trp or Phe the influence of P4 is ablated in agreement with the observation that no electron density is visible for the P4 Trp in the X-ray structure of the WYTRcmk-FVIIa-sTF complex. Consequently, the effect of substituting Asn for a large aromatic amino acid in P4 will be underestimated in the PS-SCLs due to the requirement for a suboptimal P3 substituent for efficient catalysis.

Since the predominating specificity of FVIIa appears to be located to S1 and S2 we examined the S2 binding site for residues that may alter the substrate recognition. Thr239 (c99) is localized at the interface between the S2 and S4 binding pocket in the active site cleft and could potentially influence the substrate specificity in these binding pockets (see Figure 3).

The effect of substituting Thr239 with Ala, Gly, Ile and Tyr, respectively, was evaluated. All variants showed very low amidolytic and proteolytic activity relative to wild-type rFVIIa except variant T239I, which had an activity comparable to wild-type rFVIIa. Thus, just like FIXa [34],

FVIIa is highly sensitive to mutations in this position indicating that it is important in the and L281G (c99) where the apparent second-order rate constant was decreased 2-, 42-and 65-fold, respectively, indicating that the larger hydrophilic S2 pockets of FVIIa and APC are not optimal for interacting with ATIII [START_REF] Rezaie | Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity[END_REF]. T239I favoured large aromatic P2 residues revealed by a 2.5-and 4-fold increased preference towards P2 Trp and P2 Tyr, respectively, relative to wild-type rFVIIa. However, T239I also showed a slightly increased P2 Gly preference (1.5fold) but was inhibited slower by ATIII compared to T239Y and wild-type rFVIIa thus indicating that the inhibition of FVIIa by ATIII might be more complex. These findings nicely support the notion derived from the structure, which indicates that the preference for Thr is not associated with specific residue interactions.

Recent studies of FXa have demonstrated that the P2 Gly preference does not solely determine the specificity towards ATIII indicated by the findings that interactions at secondary sites outside the P6-P3' region also influence the inhibition of FXa by ATIII [START_REF] Chuang | Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparinactivated antithrombin[END_REF]. In addition, an exosite within strand 3 of β-sheet C of ATIII has been identified and shown to be important in the reaction of ATIII with FIXa and FXa [START_REF] Izaguirre | Localization of an antithrombin exosite that promotes rapid inhibition of factors Xa and IXa dependent on heparin activation of the serpin[END_REF]. The importance of residues within the putative S2 pocket of proteases to the inhibition by serpins has been confirmed by a mutagenesis study of APC [START_REF] Berg | Engineering the proteolytic specificity of activated protein C improves its pharmacological properties[END_REF]. Substitution of residues within the active site cleft of APC resulted in variants exhibiting resistance to inhibition by protein C inhibitor and α 1 -antitrypsin yet maintained anticoagulant activity. When Thr254 (c99) was substituted with Ser the half-life in human plasma was prolonged 2-fold due to reduced rates of inactivation by circulating serpins found in plasma. The effect was more pronounced when the T254S (c99) mutation was combined with mutations of residue Leu194 (c40) yielding up to 11-fold prolongation of the plasma half-life [START_REF] Berg | Engineering the proteolytic specificity of activated protein C improves its pharmacological properties[END_REF]. Thus, it appears that residue c99 in homologous enzymes as FVIIa and APC influences the inhibitor specificity towards different serpins.

This study of FVIIa and the APC variants described by Berg et al. [START_REF] Berg | Engineering the proteolytic specificity of activated protein C improves its pharmacological properties[END_REF] shows how it is possible to engineer the primary specificity of serine proteases without compromising their biological function in the propagation and regulation of the coagulation cascade. These interesting findings reinforce the notion that the in vivo specificity of proteases towards their macromolecular substrates is to a large degree dictated by exosite interactions. As a result, it is clearly possible to separately engineer the recognition of macromolecular substrates and inhibitors. Thus, by substituting key residues in the active site cleft of FVIIa the specificity towards peptidyl substrates as well as towards physiological inhibitors, e.g. ATIII, can be altered thereby influencing the rate of inactivation without significantly altering the ability to activate FX. the ∆∆G ‡ value anticipated based on the sub-site profiling. As it is evident there is significant impact of P3 on the substitution of P4 residues, but only minor influence of P2.

LEGENDS

  activated factor VIIa (rFVIIa) and soluble tissue factor (sTF, residues 1-219) was from Novo Nordisk A/S. Plasma-derived factor X (FX) and factor Xa (FXa) were from Enzyme Research Laboratories Ltd. Plasma-derived antithrombin III (ATIII) was from American Diagnostica inc. Low Molecular Weight (LMW) Heparin Sodium Salt from Porcine Intestinal Mucosa (molecular weight 5000 g/mol) was from Calbiochem. The chromogenic substrates S-2288 (D-Ile-Pro-Arg-p-nitroanilide) and S-2765 (benzyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide) were from Chromogenix. The inhibitor Trp-Tyr-Thr-Arg-cmk was synthesized by SynPep. All oligonucleotides were from MWG-Biotech AG. All restriction endonucleases were from New England Biolabs. SDS-PAGE was run on 4-12% NuPAGE Novex Bis-Tris gels from Invitrogen. All other reagents were of analytical grade from Sigma-Aldrich.

  complex breakdown was measured by making ATIII-FVIIa complexes at high concentrations of FVIIa and with ATIII in excess to ensure fast and complete inhibition. The reactions were then extensively diluted into a substratecontaining solution with [S] 0 >>K m and the restoration of protease activity was monitored continuously. The assay was performed in a buffer with 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 1 mg/ml BSA, 0.1% PEG 8000, pH 7.4. 0.5 µM of rFVIIa was incubated with 2 µM of sTF and 1.25 or 2.5 µM of ATIII. A 4-fold excess of LMW heparin over ATIII was used. The reactions were incubated at room temperature for one hour. Each reaction was diluted 400, 450 and 500 times into 200 µl reaction mixtures in a 96-well plate with a final concentration of 4 mM S-2288 and 100 µg/ml of Polybrene. The absorbance increase at 405 nm was measured for 12 hours in a SpectraMax 190 microplate reader. The first-order rate constant of complex breakdown, k brkdn , was determined by fitting the absorbance curves to a single exponential function. The observed first-order rate constants were converted to half lives, t 1/2 , according to the relationship t 1/2 =ln2/k brkdn . Data analysis-KaleidaGraph 3.6 from Synergy software or GraphPad Prism 4.0 was used to fit the data to the appropriate equations.

  positional scanning-substrate combinatorial libraries (PS-SCLs) were used to elucidate the substrate preference in the S1, S2, S3 and S4 binding pockets of rFVIIa. The libraries constitute a powerful tool for determination of protease specificity since a specificity profile for the individual sub-sites rapidly can be obtained by Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society monitoring the enzymatic liberation of the fluorogenic ACC. The PS-SCLs used in these studies were verified by amino acid analysis (data not shown) and the P1 library has previously been validated with three different proteases (trypsin, caspase-3 and subtilisin Carlsberg) showing

  cleaved 3.6-fold more efficiently than the NLTR-ACC peptide, which represented the P4-P1 cleavage site in the activation peptide of FX. Peptides with P4-Asn were cleaved slowly compared to peptides with P4-Trp or P4-Phe except the NWTR-ACC peptide, which was cleaved with a rate similar to the WWTR-ACC and FWTR-ACC peptides probably due to the favourable Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society

  The C α Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society r.m.s.d. between the protease domain of the two structures is 0.3 Å. The covalently bound inhibitor was identified in the active site in the initial electron density map and build unbiased by excluding it during initial refinements. Interestingly, only the three first residues were

  A number of FVIIa variants modified in position 239 (c99) were generated to explore the effect of different side chain sizes. The variants were expressed in BHK cells and purified to homogeneity by a combination of anion exchange chromatography and immunoaffinity chromatography. After complete autoactivation, the FVIIa variants appeared as two bands on SDS-PAGE under reducing conditions Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society identical to wild-type FVIIa without visible signs of degradation (not shown). All FVIIa variants were active-site titrated with MUGB. The active site concentrations determined by the MUGB titrations were >75% relative to the concentrations determined by ELISA for all FVIIa variants except T239G.

2. 5 -

 5 and 4-fold increased preference towards Trp and Tyr in P2, respectively, compared to wild-type rFVIIa. No changes in the P4 specificity were observed indicating that the introduced Ile residue was pointing into the S2 pocket and not influencing the specificity in the S4 pocket. Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society

  substrate and inhibitor recognition by FVIIa. Substitution of Thr239 with Tyr caused a 2.5-fold increased preference towards P2 Gly and a broader P4 specificity profile compared to wild-type rFVIIa. Interestingly, despite the very low catalytic activity of T239Y, this variant was inhibited by ATIII at a rate comparable to wild-type Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical SocietyrFVIIa. This is presumably due to the observed P2 Gly specificity of T239Y as ATIII has Gly in the P2 position of the reactive center loop. This is in accordance with FXa having Tyr in this position also showing a P2 preference towards Gly and being rapidly inhibited by ATIII, which indicated that the T239Y mutation in FVIIa transmitted a FXa-like P2 specificity. The increased propensity of P2 Gly specific proteases to inhibition by ATIII has also been demonstrated with gla-domainless APC T254Y (c99) showing an increased preference towards chromogenic substrates with P2-Gly residues and a 181-fold more efficient inhibition by ATIII compared to gla-domainless APC[START_REF] Rezaie | Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity[END_REF].Substitution of Thr239 in FVIIa with Ile resulted in a 40% decrease in the apparent secondorder rate constant of inhibition by ATIII relative to wild-type rFVIIa indicating an important role of residue 239 in the interaction with ATIII. Interestingly, the T239I mutation did not affect the proteolytic activity towards FX markedly. The general importance of residue c99 on the rate of inhibition by ATIII has been demonstrated with the thrombin variants L281Y, L281T

Figure 2 .

 2 Figure 2. Interdependence of FVIIa sub-sites as illustrated by the changes in transition state stabilization energies (∆∆G ‡ values) for cleavage of ACC-tetrapeptides (P4-P3-P2-Arg-ACC) for the substitution of P4 N->W (solid), N->F (empty) and F->W (hatched) as a function of the P3 residues with P2 = L (left panel) and P2 = T (right panel). The pred column reflects

Figure 3 .

 3 Figure 3.Crystal structure of WYTRcmk-FVIIa-sTF 1-209 obtained at 2.05 Å resolution.

Figure A is shown

  Figure A is shown in standard orientation. The orientation of figure B and C is slightly changed for optimal view. A) View of the protease domain in an electro-potential surface plot with the inhibitor shown with a 2F O F C electron density map with 2σ cut-off (Colour scales: negative charges red, positive charges blue, carbon grey, oxygen red and nitrogen blue). The S1, S2 and S3 pockets are indicated with arrows. Note the striking difference between S1 and S2 compared to S3 in which the formers are distinct cavities with defined boundaries and high degree of electro-negativity, while the S3 pocket is flat with an almost neutral electro-potential surface. No obvious S4 pocket can be identified surrounding the active site in accordance with the lack of electron density for the P4 residue. B) Close-up of selected residues within a 5 Å sphere of the covalently bound inhibitor shown with a 2F O F C electron density map with 2σ cutoff. As indicated, the electron density traced the active site residues well. Carbons of the inhibitor are coloured orange to distinguish it from protein carbons. C) Highlight of specific interactions of the selected active site residues and covalently bound inhibitor (view is slightly tilted compared to B). The S1, S2 and S3 composition is shown with brackets and residue identifications are given with chymotrypsin numbering. Hydrogen bonding networks are shown in orange dotted lines. The following secondary elements are coloured accordingly: Lys336 -Gly346 blue, His373 -Tyr377 green, Val362 -Ala369 yellow and Gln313 -Asn322 raspberry red. See main text for details.

Figure 4 .

 4 Figure 4. Specificity profiles of FVIIa variants modified in position 239 measured in the presence of sTF in 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 0.01% Tween80, pH 7.4. The columns in grey show the apparent second-order rate constants of substrate hydrolysis (M -1 • s -

Figure 5 .

 5 Figure 5. The rate of inhibition of rFVIIa and the FVIIa variants by ATIII was determined by the progress-curve method under pseudo-first order conditions measured in the presence of sTF and LMW heparin in 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 1 mg/ml BSA, 0.1% PEG8000, pH 7.4. k Lim and K D values were determined by plotting the k obs values against the concentration of ATIII and fitting the data to equation 2 (in Materials and Methods). The apparent second-order rate constant of inhibition, k inh , was determined as the ratio k inh = k Lim /K D . •, rFVIIa; ■, T239A; ♦, T239G; ▲, T239I; ▼, T239Y.

Figure 1

 1 Figure 1.

Figure 2

 2 Figure 2.

Figure 4 .

 4 Figure 4.

Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society

P2, P3 and P4 libraries Arg was fixed at P1 (preferred by FVIIa) and the last two positions were Biochemical Journal Immediate Publication.

Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society

	2288 (final concentrations 0.2-10 mM) in a total reaction volume of 200 µl in 96-well plates.
	The reactions were initiated by addition of 10 nM of each FVIIa variant in the presence of 100
	nM sTF. The absorbance increase was measured continuously at 405 nm in a SpectraMax 190
	microplate reader. k cat and K m values were determined by fitting the data to the Michaelis-
	Menten equation by non-linear regression. When K m >5 mM, only k cat /K m were determined by
	linear regression assuming [S] 0 << K m .
	The kinetic parameters of FX activation were determined by incubating 10 nM of each FVIIa
	variant with 100 nM of sTF and 0.1-6.4 µM of FX (all final concentrations) for 20 minutes at
	room temperature in a total reaction volume of 100 µl in 96-well plates. 50 µl of stop buffer
	(50 mM Hepes, 100 mM NaCl, 20 mM EDTA, pH 7.4) was added followed by addition of 50 µl
	of 2 mM substrate S-2765 and the absorbance increase was measured continuously at 405 nm
	in a SpectraMax 190 microplate reader. The k cat /K m values were determined and the amount
	of FXa generated was estimated from a standard curve generated with final concentrations of
	0.5-10 nM active-site titrated FXa.
	Kinetics of ATIII inhibition-The apparent second-order rate constant of inhibition, k inh , was
	determined by the progress curve method monitoring competing hydrolytic and inhibition
	Each peptide was assayed reactions [23]. All reagents were equilibrated at room temperature and the assay was
	in duplicate at final concentrations ranging from 0.78 µM to 50 µM in black 96-well performed in a buffer containing 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 1 mg/ml BSA,
	fluorescence plates in a total reaction volume of 200 µl. The assays were performed at room 0.1% PEG 8000, pH 7.4 in a total reaction volume of 200 µl in 96-well plates. To maintain
	temperature in a buffer containing 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 0.01% Tween80, pseudo-first order conditions during the assay a minimum 10-fold excess of ATIII over FVIIa
	pH 7.4. The hydrolysis reactions were initiated by adding a final concentration of 10 nM of was used. In a 96-well plate 3 µM LMW heparin was mixed with 50-750 nM ATIII. The
	rFVIIa and 100 nM of sTF and kinetics was monitored using a SpectraMax Gemini EM reactions were started by adding 20 µl of 2 mM S-2288 ensuring [S] 0 <K m followed by addition
	fluorescence microplate reader with excitation at 380 nm, emission at 455 nm and cutoff at of FVIIa and sTF to final concentrations of 5 and 50 nM, respectively. The absorbance increase
	420 nm. k cat /K m values were determined by linear fit of the data since [S] 0 << K m . was measured in a SpectraMax 190 microplate reader at 405 nm for 30 minutes to obtain
	progress curves at each inhibitor concentration. k obs values were determined by fitting the
	FVIIa activity assays-All proteins were diluted in 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 1 progress curves to equation 1:
	mg/ml BSA, pH 7.4. The amidolytic activity was assayed with the chromogenic substrate S-

Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society

  Lim and K D values were obtained by fitting the k obs values plotted against the inhibitor concentrations to a hyperbolic function which accounts for substrate binding (equation 2):

(Equation

1

) where [Pr] 0 and [Pr] are the accumulation of product at assay start (t=0) and at any time, respectively, visualized by a change in absorbance after addition of a chromogenic substrate. k

Table 1 .

 1 k cat /K m values (mM -1 • s -1 ) for cleavage of ACC-tetrapeptides of the format P4-P3-

	P2-Arg-ACC by rFVIIa in the presence of sTF measured in 50 mM Hepes, 100 mM NaCl, 5 mM
	CaCl 2 , 0.01% Tween80, pH 7.4. n.d., not determined.

Table 2 .

 2 k cat /K m values for cleavage of S-2288 and WATR-ACC as well as activation of FX by rFVIIa and the FVIIa variants in the presence of sTF measured in 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 1 mg/ml BSA, pH 7.4.

Table 3 .

 3 The apparent second-order rate constants for inhibition, k inh , of the FVIIa variants by ATIII determined as the ratio k inh =k Lim /K D measured in the presence of sTF and LMW heparin in 50 mM Hepes, 100 mM NaCl, 5 mM CaCl 2 , 1 mg/ml BSA, 0.1% PEG8000, pH 7.4. The first-order rate constants of complex breakdown, k brkdn , and t ½ determined as the average of six independent measurements. t ½ was calculated by the relation t ½ =ln2/k brkdn .

	Figure 1.	Specificity profile of rFVIIa in the presence of sTF measured in 50 mM Hepes,
	100 mM NaCl, 5 mM CaCl 2 , 0.01% Tween80, pH 7.4. In each library the apparent second-
	order rate constants of substrate hydrolysis were normalized to the sub-library with the
	highest activity. Full line: Mean value of all the columns. Dotted line: 1× standard deviation of
	all the columns.

Table 1 .

 1

	P2 = T

Table 2 .

 2

			k cat /K m
			(mM -1 ⋅ s -1 )
		S-2288	WATR-ACC	FX
	rFVIIa-sTF	26.12 ± 2.77	44.66 ± 0.11	550 ± 30
	T239A-sTF	5.45 ± 0.15	28.59 ± 0.08	69 ± 5
	T239G-sTF	2.40 ± 0.036	17.59 ± 0.10	23 ± 0.6
	T239I-sTF	19.71 ± 2.26	29.01 ± 0.07	461 ± 12
	T239Y-sTF	2.47 ± 0.18	6.08 ± 0.02	74 ± 5

Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical SocietyTable 3 .

 3

		k Lim	K D	k inh	k brkdn	t ½
		(10 -3 s -1 )	(10 -7 M)	(M -1 • s -1 )	(10 -5 s -1 )	(hours)
	rFVIIa	4.9 ± 0.32	1.81 ± 0.33	26917 ± 5237	3.43	5.6
	T239A	1.6 ± 0.10	2.60 ± 0.41	6160 ± 1058	2.85	6.8
	T239G	1.5 ± 0.14	1.14 ± 0.71	4688 ± 1155	1.61	12.0
	T239I	2.4 ± 0.12	1.45 ± 0.22	16338 ± 2564	10.20	1.9
	T239Y	5.3 ± 0.29	1.74 ± 0.26	30591 ± 4906	2.08	9.3

Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901

Biochemical Journal Immediate Publication. Published on 25 Apr 2007 as manuscript BJ20061901 © 2007 Biochemical Society

© 2007 Biochemical Society

Abbreviations

ACC, 7-amino-4-carbamoylmethylcoumarin; AMC, 7-amino-4methylcoumarin; APC, activated protein C; Arg152 (c15) etc, specific amino acid residues with chymotrypsin numbering in parentheses; ATIII, antithrombin III; BHK, baby hamster kidney cells; BSA, bovine serum albumin; cmk, chloromethyl ketone; ELISA, enzyme-linked immunosorbent assay; FVII, coagulation factor FVII; FVIIa, activated coagulation factor VII, FIX, coagulation factor IX;