Arg-129 plays a specific role in the confirmation of antithrombin and in the enhancement of factor Xa inhibition by the pentasaccharode sequence of heparin

Biochimica et Biophysica Acta, 1225 (1994) 135-143

135

© 1994 Elsevier Science B.V. All rights reserved 0925-4439/94/$07.00

BBADIS 61323

Arg-129 plays a specific role in the conformation of antithrombin and in the enhancement of factor Xa inhibition by the pentasaccharide sequence of heparin Saloua Najjam, Gilliane Chadeuf, Sophie Gandrille and Martine Aiach

*

Groupe de Recherche sur la Thrombose, INSERM CJF 91-01, UFR des Sciences Pharmaceutiques et Biologiques, 4Avenue de l'Observatoire, F-75270 Paris Cedex 06 (France)

(Received 8 February 1993)

Key words: Antithrombin; Protein conformation; Factor Xa; Protein inhibition; Arginine; Heparin activation; Affinity chromatography

Small amounts of a variant antithrombin (AT) bearing an Arg-129 to Gin mutation were purified from plasma by means of affinity chromatography on insolubilized heparin at very low ionic strength. As a control, two variant antithrombins, one bearing a Pro-41 to Leu mutation and the other an Arg-47 to His mutation, were purified in the same way. The biochemical characterization of the variants and the kinetic study of thrombin and activated factor X (F Xa) inhibition in the presence of heparin and heparin derivatives suggest that Arg-129 plays a specific role in AT conformation and F Xa inhibition enhancement. Indeed, the purified variant adopted the locked conformation described for AT submitted to mild denaturing conditions (Carrell, R.W., Evans, D.Li. and Stein, P.E. (1991) Nature 353, 576-578) and resembling the latent form of plasminogen activator inhibitor (PAI) (Mottonen, J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geoghegan, K.F., Gerard, R.D. and Goldsmith, E.J. (1992) Nature 355, 270-273). Moreover, the mutant AT was partially reactivated by heparin for thrombin inhibition, but did not respond to the specific pentasaccharide domain of heparin for F Xa inhibition.

Introduction

The main physiologic thrombin inhibitor, antithrombin (AT), is a 432-amino-acid single-chain polypeptide with three internal disulfide bonds. Like other proteins belonging to the family of serine-proteinase inhibitors (serpin), A T forms stable equimolecular complexes with its cognate proteinases, i.e., thrombin and other activated (a) coagulation factors (F), mainly F Xa, F IXa and F XIa (for reviews, see Refs. 3 and 4). The conformation of cleaved A T resembles that of al-antitrypsin, a typical member of the serpin family [5,6]. Recent observations have suggested that serpins act as suicide substrates for their target enzymes [7]. The proteinase first cleaves a surface-reactive center loop after a P1 residue which, in AT, is an Arg located at position 393 of the amino-acidsequence. Cleavage of the P1-PI' bond is followed by the insertion of the amino-terminal part of the reactive center into a large A-sheet that forms the main feature of the molecule

* Corresponding author. SSDI 0925-4439(93)E0087-R

[8]. The enzyme thereby induces a modification of the inhibitor conformation which is responsible for irreversible trapping of the target proteinase [9]. Heparin enhances the inactivation of coagulation proteinases by A T and must bind the inhibitor in order to exert its catalytic effect. Only about one-third of the heparin chains possess a specific A T binding sequence [10], a pentasaccharide segment that contains a single trisulfated D-glucosamine residue and that has been fully synthesized [11]. Among several serpins interacting with heparin, A T is the only one to require the presence of this pentasaccharide sequence, which induces a conformational change of the inhibitor. The molecular mechanism underlying the transformation of A T into a more effective inhibitor by heparin is still a matter of debate [12]. One of the more puzzling issues is the molecular mass dependence of A T activation by heparin in the presence of two groups of coagulation proteinases, the paradigms of which are F Xa and thrombin. The pentasaccharide increases F Xa inhibition but has minimal effects on thrombin inhibition. Only heparin chains containing the pentasaccharide sequence plus at least 13 additional saccharides accelerate thrombin inhibition. Kinetic data suggest that heparin might act as a template interacting with both

136

AT and thrombin, bridging the two reactants essential for enhanced thrombin inhibition [13-15]. Natural mutations of amino acids located in the N-terminal portion give rise to A T molecules whose antiproteinase activity enhancement by heparin is impaired [3,4]. This type of hereditary A T deficiency is characterized by the presence, in heterozygous patients, of two populations of A T molecules that can be separated on the basis of their affinity for insolubilized heparin. Substitution of residues at positions 7, 24, 41, 47, 99 and 129 of the amino-acid chain were found to be responsible for these phenotypes [16-23]. Arg-129 is located at the surface of the D helix rich in basic amino acids, which has been shown to play a major role in the heparin-AT interaction [21,24]. The substitution of Arg (R) 129 by Gin (Q) in A T Geneva led to the absence of binding to heparin-Sepharose at physiological ionic strength [23,25]. The variant, purified by means of immunoaffinity, presented decreased antithrombin activity, even in the absence of heparin, which suggested it was partially inactivated during the purification process [23]. We obtained a small amount of purified a R129Q A T from a second patient bearing the same mutation (AT Bayonne), using the property of the mutant protein to bind heparin-Sepharose at very low ionic strength. Two other natural variants were also purified by the same procedure from patients bearing a Pro (P) 41 to Leu (L) mutation (AT Colombes) and an Arg (R) 47 to His (H) mutation (AT Nice). During the purification process, R129Q A T was converted into a more stable and inactive inhibitor, while the two other variants remained fully active, suggesting that R129 plays a major role in keeping A T in a metastable active state. To clarify the respective roles of R129, P41 and R47 in the AT-heparin interaction, we compared the effect of various heparin derivatives on thrombin and F Xa inhibition by the mutant proteins. Heparin partially restored thrombin inhibition by the R129Q variant but had no effect on its ability to inactivate F Xa. The two other mutations moderately affected the stimulation of AT by heparin in the presence of both F Xa and thrombin. Materials and Methods

Materials. H u m a n thrombin, human and bovine F Xa were from Diagnostica Stago (Asni~res, France). The substrate for thrombin was H-D-Phe-Pip-Arg-pNA (S 2238, Kabi Pharmacia, St-Quentin-en-Yvelines, France). The substrate for F Xa was CH3SOz-D-LeuGly-Arg-pNA, A c O H (CBS 31-39) from Diagnostica Stago. Anti-AT and anti heparin cofactor II immune sera were from Diagnostica Stago. Heparin-Sepharose, concanavalin A (Con A) Sepharose, Mono Q column and Mono P column were

from Pharmacia. Fast protein-liquid chromatography (FPLC, Pharmacia) was used for all the purification procedures. All reagents were of the highest purity commercially available. Antithrombin purification and characterization. In two cases (AT Colombes and A T Nice), venous blood (150 ml) was obtained with the informed consent of the propositus, using 0.11 M sodium citrate (1:9) as anticoagulant. In the case of A T Bayonne, 500 ml of plasma was obtained by plasmapheresis. Normal A T was purified from plasma adsorbed to barium sulfate by heparin-Sepharose chromatography, with further purification by ion exchange on a Mono Q column [26]. The first step of A T purification from patient plasma was also performed using this method, except that plasma was adsorbed to dextran sulfate, as described by McKay [27]. The variant ATs were further purified by chromatography [28] on a Mono Q column in MES buffer (pH 6) and 0.02 M Tris buffer (pH 7.4), with a final step on a Mono P column for AT Bayonne. The homogeneity, molecular mass and thrombin complex formation of the three variants were assayed by means of SDS-PAGE (10% polyacrylamide) [28]. Immunoblotting for heparin cofactor II (HC II) [29] and A T were performed with samples containing 20300 ng of purified protein. Samples underwent SDSP A G E (10% polyacrylamide). The proteins were transferred onto a nitrocellulose membrane using a minisystern (Bio-Rad, Ivry-sur-Seine, France). The blot was treated as recommended by the manufacturer, made to react with an anti-HC II serum or with an anti-AT serum diluted 1:1000, thoroughly washed and incubated with a 1:3000 dilution of alkaline phosphateconjugated goat anti-rabbit-IgG IgG (Bio-Rad). A T antigen concentrations were measured by means of electroimmunodiffusion. Heparin cofactor activity was measured using ATPrest (Diagnostica-Stago) and expressed in U / m l , 1 U being the activity of 1 ml of normal plasma. Specific activity was taken as the ratio of heparin cofactor activity to the protein concentration, determined according to Peterson [30]. The heat stability of antithrombin was evaluated as described for proteinase-nexine by Evans [31]. Heparin preparations. UF-heparin (batch IBP:GW 87.0274, Rh6ne-Poulenc, kindly provided by Dr. Bouthier) had a specific activity of 173 I U / m g and a mean molecular mass of 12.5 kDa. HA-heparin was prepared by affinity chromatography on a column of human AT bound to Con A-Sepharose according to Denton [32]. Briefly, 3 mg of human purified AT, prepared as described above, was bound to 1 ml of Con A-Sepharose. 2 mg of UF-heparin was then loaded on the column. After extensive washing with 0.02 M Tris buffer (pH 7.4) containing 1 mM CaC12, 1 mM MgC12, 1 mM MnC12 and 0.05M NaC1, a

137

fraction of heparin with low affinity for AT was eluted with the same buffer containing 0.25 M NaC1; a highaffinity (HA) fraction was obtained with 1 M NaC1. The anti-Xa activity of the HA-heparin fraction was measured in the presence of purified AT, as described below, using UF-heparin as standard and bovine F Xa. The concentration was estimated with toluidine blue, using UF-heparin as standard [33]. The specific activity of the HA-heparin used in this study was 444 anti-Xa U/rag. We used a synthetic methyl-pentaoside (batch SR90107A, Sanofi-Recherche, kindly provided by Dr. Petitou) with a molecular mass of 1714 Da and a specific activity of 823 anti-Xa U / m g in our study. Thrombin and F Xa inhibition rates. A two step-procedure was used. Human thrombin or F Xa was first allowed to react with AT in the presence or absence of heparin, at concentrations giving exponential decay of the enzyme as a function of time. Residual enzyme activity was then measured in the presence of an excess of synthetic substrate, giving a linear increase in absorbance (A). Second-order rate constants for thrombin and F Xa inhibition by the purified ATs: Human thrombin (20 nM) was incubated with a 10-fold molar excess of purified AT at 37°C in a buffer containing 0.02 M Tris, 0.5% PEG, 0.15 M NaC1 (pH 7.4). After 1 min, S 2238 was added at a final concentration of 0.2 mM. A was recorded for 30 sec at 37°C using a PCP 6121 Eppendorf photometer. The observed pseudo-first-order rate constant was calculated as (ln A 0 - I n A t ) / t , where A 0 is the absorbance in the absence of AT. The second-order rate constant was calculated as K " = k/(AT). For F Xa inhibition, the same procedure was used, except that 26 nM F Xa and the substrate CBS 31.39 (3.25 mM) were used. Second-order rate constant for thrombin and F Xa inhibition by the purified ATs in the presence of heparin: The ability of UF-heparin to accelerate thrombin inhibition by purified normal and variant ATs was tested in second-order conditions where inhibitor (10 nM) was allowed to react with enzyme for 10 s in the presence of 1 0 - 9 - 1 0 - 4 M UF-heparin. The secondorder rate constant was determined from the classical equation as described elsewhere [34]: [E] = [E0]/1 + [ E o ] . K " . t

(1)

where [E o] is the initial enzyme concentration in the absence of heparin, [E] is the concentration of free enzyme which is proportional to the residual enzymatic activity and related to the reaction time t. Both E 0 and E were calculated with a calibration obtained by plotting A values observed with increasing thrombin concentration. The ability of smaller increasing concentrations of

various heparin preparations (UF-heparin, HAheparin, pentasaccharide) to bind and stimulate the antithrombin and anti-Xa activity of the different AT variants was tested under second-order conditions by allowing equimolecular proteinase and inhibitor amounts to react for 30 s in the presence of heparin (see Figs. 4 and 6). Genomic DNA analysis. We used a technique developed by our group and published elsewhere [35,36]. Results

Characterization of the mutation responsible for defective heparin binding The three patients presented plasma assay profiles typical of a defective heparin-binding variant, with normal AT antigen concentrations, normal antithrombin activity in the absence of heparin and a 50% reduction in heparin cofactor activity, measured using methods described elsewhere [26,28]. None of the three patients had a personal or family history of thrombosis. The family pedigree suggested an autosomal dominant trait for both AT Nice and AT Bayonne, but could not be studied for AT Colombes. Dot-blot analysis of amplified exon II [35] confirmed that two of the patients were heterozygous for a P41L mutation (AT Colombes) and a R47H mutation (AT Nice) known to be responsible for defective heparin binding in several families [35]. The third was a novel mutation, Arg-129 ~ Gin, and was identified simultaneously by our group in the present patient (AT Bayonne) and in AT Geneva by direct sequencing of exon IIIa [23]. To be sure that the nucleotide substitution was the causal mutation, we sequenced all the coding domains of the AT gene [36] and found no additional mutations.

Characterization of purified ATs obtained from patient plasma The addition of dextran sulfate to patient plasma [27] precipitated most of the protein binding to heparin-Sepharose, with the notable exception of AT. The plasma supernatant, containing both variant and normal molecules, was then dialyzed against 0.02 M Tris buffer (pH 7.4), centrifuged and applied to the heparin-Sepharose column. There was no detectable AT antigen in the break-through from the three plasmas, showing that the three variant ATs retained some affinity for heparin at this low ionic strength. The AT from the three patients' plasma eluted in two separate peaks, one at the normal AT position (between 0.6 and 0.8 M NaCI), and the other prior to normal AT (between 0.10 and 0.40 M NaCI). We assumed that the low-affinity ATs were variant molecules bearing the P41L, R47H and R129Q mutations, respectively. Each variant or normal AT was further purified, yielding products homogeneous in SDS-PAGE (see Fig. 1). The

138

MM(kDa)

1

2

3

4

1

2

97~

45--

31--

Fig. 1. S D S - P A G E of the purified ATs. N, A T purified from normal plasma; R129Q, low-affinity A T purified from patient Bayonne; R47H, low-affinity A T purified from patient Nice; P41L, low-affinity A T purified from patient Colombes A T (1.25 /zM). A T s were incubated for 10 min at 37°C with the same concentration of purified h u m a n thrombin ( + ) or buffer ( - ).

migration as a 58-kDa compound was identical for the normal and each of the three variant ATs. The antigen concentrations of the purified P41L, R47H and R129Q variants were 97, 76 and 11%, respectively, of the concentration expected from the protein assay. Each preparation was also characterized by measuring specific heparin cofactor activity and the second-order rate constant (K") for thrombin and F Xa inhibition (Table I). The specific heparin cofactor activities were less than 0.5 U / m g for all three variant ATs and in the same range as standard A T for the normal molecules, except for the patient with AT

TABLE I

Specific activity and second-order rate constant values (K") for thrombin and factor Xa inhibition by each purified antithrombin (AT) Standard A T was purified from the plasma of a healthy subject and tested at least 6-times in three different experiments. A T s purified from patients were tested in duplicate. Heparin cofactor

K " ( × 104M-X s - I )

activity ( U / m g )

Thrombin

Factor Xa

A T Colombes Pro-41 ~ Leu Normal A T

0.5 3.8

1.16 1.40

0.130 0.150

A T Nice Arg-47 --* His Normal A T

0 5.5

0.50 3.00

0.113 0.316

A T Bayonne Arg-129 ~ Gln Normal A T

0 8.0

0.03 1.20

0.021 0.176

Standard A T Mean S.D.

8.4 2.62

1.28 0.40

0.181 0.035

AT

HCI]

AT

Fig. 2. Immunoblots of purified R129Q variant, tor he parin cofactor II (HC II) and for A T Left panel: Blot was made to react with polyclonal anti-HC II antibodies. Lanes 1-3: decreasing amount of H C II purified according to Toulon [29]. Lane 1, 200 ng; lane 2, 20 ng; lane 3, 2 ng; lane 4, purified R I 2 9 Q variant, 300 ng. Right panel: Blot was made to react with polyclonal anti-AT antibodies. Lane l, purified normal AT, 300 ng: Lane 2, purified R I 2 9 Q variant, 300 ng.

Colombes in whom normal A T specific activity was bordeline. The K " were all in the normal range, except for the R129Q variant. This confirmed that the P41L and R47H ATs had normal activity in the absence of heparin [28,37]. The R129Q variant showed a 40-fold reduced ability to inhibit thrombin and F Xa after purification, suggesting a modification of the reactive site during the experimental procedure. The ability of the P41L, R47H and R129Q variants to form complexes with thrombin was further evaluated by SDS-PAGE after incubation with thrombin (molar ratio 1 : 1). High-molecular-mass complexes were formed with the P41L and R47H variants, but not with the R129Q variant, confirming the inactivated state of the latter (Fig. 1). The reduced ability of the molecules purified from the patient with A T Bayonne to inhibit thrombin and F Xa could be explained by the isolation of another protein with reduced affinity for heparin. Using immunoblot analysis, we provide further evidence that the purified preparation is not contaminated by the presence of HC II, a thrombin inhibitor which binds weakly to heparin. We also confirm that the purified protein reacts with anti-AT antibodies and migrates like normal A T in immunoblot (Fig. 2). The loss of reactivity was partial after heparin-Sepharose chromatography. The inactivated state was achieved after chromatography at pH 6, as evidenced by the absence of high-molecular-mass complexes in the presence of thrombin (not shown). The heat stability of the purified AT preparations was studied by incubation for 2 h at 50, 60, 80 and 100°C. The ATs were all stable up to 50°C, but antigen

139 % initial antigen 105

84

63

42

21

45

60

76

90

105

Temperature(°C) Fig. 3. Heat stability of purified ATs from patient Bayonne plasma. Samples (100/zl) of each normal and variant AT (100 ~1 of 1 # M ) were heated for 2 h. At the end of the incubation period, each sample was centrifuged and the concentration of AT in the supematant was measured by electro-immunoassay using rabbit polyclonal antiserum (Diagnostica Stago). The residual concentrations (%) after heating are expressed as the mean of six determinations. (©, o), Normal AT and R129Q AT purified from patient Bayonne's plasma; ( + ), AT purified from normal plasma.

recognition fell above this temperature. The heat-stability curves were similar for the normal ATs and those purified from Nice and Colombes plasma. The R129Q variant molecule had a higher melting temperature, suggesting a more stable conformation (Fig. 3). The purified preparation were submitted to a reducing agent in order to detect AT cleaved at the reactive site (Fig. 4), which normaly presents a reduced molecu-

Fig. 4. SDS-PAGE of the purified ATs in reducing conditions. ATs (1.25 ~M) were reduced by incubation with /3-mercaptoethanol. Normal AT and R129Q AT incubated with thrombin ( + ) or buffer ( - ) prior to reduction, under the conditions as described in Fig. 1.

lar mass after cleavage of the disulfide bound that link the C-terminus fragment after the reactive site cleavage. As a control normal AT was incubated with thrombin prior to electrophoresis to generate small amounts of cleaved thrombin migrating as a compound of 50 kDa. Most R129Q AT migrated as a compound presenting a higher molecular mass (63 kDa). This shift in molecular mass suggests an abnormal conformation of the R129Q variant. The absence of the 50-kDa compound shows that the R129Q purified AT was not cleaved during the purification process. This excluded the possibility that the thermostability of the purified

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UF-heparin M Fig. 5. Effect of saturating concentrations of UF-heparin on the inactivation of thrombin by AT. The observed second-order rate constant (Kob s) was plotted against the logarithm of the heparin concentration. Each point is the mean of four determinations performed in two different experiments. Assays were performed as described in Materials and Methods, with the same concentration for AT and for thrombin (10 -8 M). (D, • ), Normal AT and P41L AT purified from patient Colombes; (©, e), normal AT and R129Q AT purified from patient Bayonne; (+), AT purified from nomal plasma.

140 K"obs (10 9 M-I rain -1 )

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Fig. 6. Thrombin inhibition in the presence of purified AT and low concentrations of UF-heparin and HA-heparin. The rate of thrombin inhibition Kobs by normal and variant AT in the presence of UF-heparin and HA-heparin were measured under second-order conditions with a proteinase and inhibitor concentration of 2 . 1 0 - 8 M. ( t~, • ) , Normal AT and P41L AT purified from patient Colombes; ( z~, • ), normal AT and R47H AT purified from patient Nice; (©, e), normal AT and RI29Q AT purified from patient Bayonne; (+), AT purified from normal plasma.

R129Q AT and the loss of proteinase binding ability resulted from cleavage of the reactive loop. Kinetic studies Thrombin inhibition. The purified AT preparations were tested for thrombin inhibition in second-order conditions in the presence of increasing concentrations of UF-heparin. The concentration of heparin required to obtain maximal thrombin inhibition with normal AT (10 - 7 M) was consistent with values obtained by other groups [15,38]. Only P41L and R129Q variant AT could be tested in this experiment. When compared to normal, the optimal rate of inhibition obtained for the variants were 3-times lower for P41L AT and 20-times lower for R129Q AT, respectively. The concentration of heparin required to obtain maximal inhibition was one order of magnitude higher for P41L AT than for

the normal ATs and was obtained at a similar concentration for normal ATs and R129Q variant AT. This experiment showed that the two studied variants could be activated by high concentrations of heparin. In addition, the thrombin inhibition second-order rate constant (Kob s) vs. the log heparin concentration gave a typical bell-shaped curve with the mutant ATs, as well as with normal AT (Fig. 5). In a second set of experiments, the rate of thrombin inhibition by normal and variant AT in the presence of UF-heparin and HA-heparin were measured in second-order conditions with proteinase and inhibitor concentrations of 2 . 1 0 -8 M. The amount of proteinase inhibited in the absence of heparin after 30 s was negligeable and the rate of inhibition (Kobs) increased as a function of the heparin concentration. The Kobs vs. UF-heparin or HA-heparin curves are

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HA-heparin 10-a M pentasaccharide 10-SM Fig. 7. F Xa inhibition in the presence of purified AT and low concentrations of HA-heparin and pentasaccharide. Same legend as Fig. 6. The rate of F Xa inhibition was measured under second-order conditions with respective concentrations of AT and F Xa of 4.10 -8 M and 4.10 -8 M.

141

shown in Fig. 6. The curves clearly show a decreased ability of the variant molecules to be activated by both heparins. The R129Q variant presented no detectable activation by UF-heparin in the conditions used in this experiment. However, we obtained similar activation of R47H variant molecules and of R129Q molecules in the presence of HA-heparin, confirming the ability of the latter to be activated. F Xa inhibition. The respective effects of increasing concentrations of pentasaccharide and HA-heparin on F Xa inhibition by normal and variant AT molecules are shown in Fig, 7. AT and F Xa were allowed to react for 30 s at the same concentration of 4.10 -8 M. Under these conditions, F Xa inhibition was only measurable in the presence of HA-heparin and pentasaccharide. UFheparin was not tested because insufficient amounts of the purified variant were available. KobS values were decreased by about 10-fold for the P41L and R47H variants relative to normal ATs. No F Xa inhibition was observed with the R129Q variant in the presence of HA-heparin or pentasaccharide. Thus, R129Q AT molecules, whose reaction with thrombin could be weakly catalyzed by HA-heparin, were not detectably activated by pentasaccharide or HA-heparin. Discussion

The natural mutations P41, R47 and R129 are known to produce AT variants with reduced heparin-enhanced proteinase inhibition, and their location in the three-dimensional molecular structure has led to the proposal that the two domains corresponding to helix A and helix D are involved in the interaction of AT with polysaccharides [6]. The reduced affinity of these mutant ATs for heparin was confirmed here by the lower ionic strength required to dissociate them from a heparin-Sepharose column. We used this property to isolate the mutant proteins from the plasma of three heterozygous patients. The reduced ability of the purified R129Q variant to inhibit thrombin and F Xa in the absence of heparin and its poor recognition by polyclonal antibodies confirmed that it had been modified during the purification process [23]. The replacement of the R129 by Q seems to affect the three-dimensional structure of the molecule, giving a more stable conformation during the purification process. According to Carrell [1], this serpin can adopt various conformations: the native stressed (S) conformation, the relaxed (R) form, obtained after cleavage of the reactive center, and the locked (L) conformation on exposure to low-concentration denaturant. The Lform AT results from insertion of the uncleaved reactive-center loop into the A-sheet. The purified R129Q variant showed strong similarities with the L-form, since it was uncleaved, inactive and had a higher melt-

ing temperature than native AT. It also resembled the latent form of another serpin, PAI, which undergoes a transition to an inactive form without cleavage and under physiologic conditions [2]. The P41L and R47H variants, purified using the same procedure, possessed all the characteristics of the native S-form in terms of antiproteinase activity, thermal stability and recognition by polyclonal antibodies. The R129Q mutation could thus play a specific role in the S-to-L transition that apparently occurred during purification. The putative site of AT interaction with heparin has been studied by inducing chemical modifications [3941], in terms of the heparin binding activity of AT peptides corresponding to the D helix [24,42], and by analysis of the effect of natural mutations on heparinenhanced antiproteinase activity (for reviews, see Refs. 22 and 23). The latter approach allows the role of a single amino-acid substitution to be tested in an enzymatic assay, i.e., in the presence of clotting proteinases and heparin or heparin derivatives. As recently suggested, serpin-type inhibitors require activation by the target enzyme to become inhibitors [8,43]. Cleavage of the reactive loop leads to a conformational change [44] and to the insertion of the aminoterminal peptide into the A-sheet, thereby permitting cognate enzymes to bind [8]. Heparin binds to AT and also induces a conformational change, implying increased loop exposure that might promote the association of the inhibitor with clotting proteinases [1]. Incorporation of the loop into the A-sheet involves movements of the D helix that might explain the release of heparin upon the formation of AT-proteinase complexes. Both cleavage of the reactive site and heparin binding modify the conformation of AT and their effects on proteinase inhibition are linked; this implies that the interaction with heparin cannot be dissociated from the interaction with a given proteinase. We first analysed the effect of UF-heparin on thrombin inhibition by normal and mutant ATs, P41L and R47H. The plots of the rate constant as a function of log heparin levels gave an ascending limb, a plateau and a descending limb with both normal and variant ATs. Two explanations have been forwarded for these bell-shaped curves: (1), ternary AT-heparin-thrombin complexes in which heparin binds both the inhibitor and the proteinase [13,14,45]; (2), competition between AT and thrombin for heparin binding [38], the formation of heparin-AT complexes being predominant at lower heparin concentrations (ascending limb), while competition between AT and thrombin for heparin binding occurs at higher heparin concentrations (descending limb). These two AT variants, whose binding affinities for insolubilized heparin were low, presented kinetic profiles similar to those observed with normal AT, which favours the first hypothesis. The lower affinities of the variant ATs for heparin would have

142 r e d u c e d the d i f f e r e n c e in affinity b e t w e e n A T and t h r o m b i n ; c o m p e t i t i o n for h e p a r i n b i n d i n g is, in this case, a less p r o b a b l e e x p l a n a t i o n for the d e s c e n d i n g limb of the curve. O t h e r s e r p i n inhibitors with lower affinities for h e p a r i n , such as h e p a r i n cofactor, p r o t e i n C i n h i b i t o r [15] a n d p r o t e i n a s e - n e x i n [31], w h o s e antit h r o m b i n activity e n h a n c e m e n t d o e s not rely on t h e p r e s e n c e o f the p e n t a s a c c h a r i d e , also p r e s e n t this p a r ticular kinetic profile reflecting the f o r m a t i o n of t e r n a r y complexes. T h e c o n c e n t r a t i o n r e q u i r e d to o b t a i n the o p t i m a l activation w e r e 10 - 7 M for n o r m a l A T s a n d R 1 2 9 Q A T , a n d 10 - 6 M for P 4 1 L A T , respectively. T h e maximal r a t e o f i n h i b i t i o n was less t h a n o n e o r d e r o f m a g n i t u d e d e c r e a s e d with t h e R 4 7 H v a r i a n t a n d was two o r d e r s of m a g n i t u d e d e c r e a s e d with t h e R 1 2 9 Q variant. This shows that, a l t h o u g h the R 1 2 9 Q A T m o l e c u l e s can be a c t i v a t e d by h e p a r i n , t h e s t i m u l a t i o n is w e a k e r t h a n t h a t o b t a i n e d with the P 4 1 L variant. O t h e r e x p e r i m e n t s w e r e p e r f o r m e d with U F - h e p a r i n a n d H A - h e p a r i n at i n c r e a s i n g c o n c e n t r a t i o n s r a n g i n g b e t w e e n 10 - s a n d 1 0 - 7 M . This c o n f i r m e d t h e dec r e a s e d activation of P 4 1 L a n d R 4 7 H v a r i a n t m o l e c u l e s ( a b o u t o n e o r d e r o f m a g n i t u d e ) . Similar results w e r e o b s e r v e d w h e n F X a i n h i b i t i o n was m e a s u r e d in the p r e s e n c e of H A - h e p a r i n a n d of t h e p e n t a s a c c h a r i d e . W i t h R I 2 9 Q variant, t h r o m b i n i n h i b i t i o n c o u l d b e s t i m u l a t e d by H A - h e p a r i n at a b o u t the s a m e level as R 4 7 H variant. In contrast, no s t i m u l a t i o n of F X a inhibition of R 1 2 9 Q A T by H A - h e p a r i n or p e n t a s a c c h a r i d e was o b s e r v e d . T h e d a t a from this study, d e s i g n e d to d e t e r m i n e t h e relative i m p o r t a n c e of R129 a n d o t h e r r e s i d u e s k n o w n to play a role in A T s t i m u l a t i o n by h e p a r i n , l e a d to the conclusion t h a t the R 1 2 9 Q m u t a t i o n m o d i f i e s t h e folding of t h e m o l e c u l e , which s u b s e q u e n t l y a d o p t s an inactive, stable c o n f o r m a t i o n r e s s e m b l i n g t h e L f o r m r e c e n t l y d e s c r i b e d by C a r r e l l [1]. This a b n o r m a l conform a t i o n was r e a c t i v a t e d by h e p a r i n to b i n d t h r o m b i n b u t did n o t i n t e r a c t with the A T - s p e c i f i c p e n t a s a c c h a ride s e q u e n c e , as shown by t h e a b s e n c e o f F X a inhibition. O u r d a t a a r e c o n s i s t e n t with t h e h y p o t h e s i s in which t h e d o m a i n e n c o m p a s s i n g R129 plays a specific role in b o t h t h e c o n f o r m a t i o n of t h e i n h i b i t o r a n d its i n t e r a c t i o n with t h e p e n t a s a c c h a r i d e . F u r t h e r m o r e , they suggest t h a t t h r o m b i n a n d F X a inhibition enh a n c e m e n t a r e g o v e r n e d by d i f f e r e n t m e c h a n i s m s .

Acknowledgements W e wish to t h a n k Dr. P i e r r e Si6 for p r o v i d i n g D N A a n d p l a s m a f r o m t h e p a t i e n t with A T B a y o n n e . W e also t h a n k M a r i e - J o s 6 D e g u i n g a n d for excellent secret a r i a l assistance. This w o r k was s u p p o r t e d by I N S E R M a n d by a g r a n t f r o m the Sanofi R e s e a r c h F o u n d a t i o n .

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