A novel strategy to generatebiologically active neo-glycosaminoglycan conjugates

Jianhui Rong, Kerstin Nordling2, Ingemar Björk2 and Ulf Lindahla

Department of Medical Biochemistry and Microbiology, Box582, The Biomedical Center, Uppsala University, S-751 23 Uppsala,Sweden and 2Department of VeterinaryMedical Chemistry, Box 575, The Biomedical Center, The Swedish Universityof Agricultural Science, S-751 23 Uppsala, Sweden

Received on March 1, 1999. revisedon May 17, 1999; accepted on May 17, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Heparin and heparan sulfate are structurally related polysaccharideswith a variety of biological effects/functions. Most ofthese effects are due to interactions, of varying specificity, betweenthe negatively charged polysaccharide chains and proteins. Whilesuch interactions generally involve a single saccharide domain of decasaccharidesize or less, ternary complexes of two protein molecules bindingto separate domains on a single polysaccharide chain are known tooccur. To facilitate studies on domain organization and its importancefor biological function a strategy was developed to chemically conjugatedefined heparin oligomers in linear and chemoselective fashion.The procedure requires that the oligosaccharide to provide the reducing-terminaldomain of the conjugate is generated by lyase degradation of a parentpolysaccharide, whereas the nonreducing-terminal domain is obtainedthrough deaminative cleavage with nitrous acid. The applicabilityof the method was demonstrated by constructing a conjugate composedof two heparin 12-mers, of which the reducing-terminal componentcontained the antithrombin-binding region, whereas the nonreducing-terminaldomain did not. Contrary to any of the unconjugated oligomers, theproduct was found to efficiently promote the inactivation of thrombinby antithrombin.


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Heparin and heparan sulfate (HS) are structurally related sulfatedpolysaccharides (glycosaminoglycans; GAGs), that are composed ofrepeating disaccharide units of hexuronic (D-glucuronic,GlcA, or L-iduronic, IdoA) acid and D-glucosamine (GlcN)variably modified by N- and O- sulfation. The biological activitiesof these polymers depend on their interactions with a variety ofproteins, such as enzymes, enzyme inhibitors, growth factors, growthfactor receptors and extracellular matrix components (22GoSalmivirta et al., 1996; 4GoConrad,1998). Presumably, most of these interactions involvea single domain, or at least a single type of saccharide sequence,generally of <10-mer size, along the polysaccharide chain.However, several examples of ternary complexes involving two proteinmolecules, identical or different, binding to separate domains ona single polysaccharide chain have been described. Such multipleinteractions have been implicated in a variety of phenomena affected/modulatedby heparin/HS, including inactivation of thrombin by antithrombin(AT) (3GoBourin and Lindahl, 1993),interaction between basic fibroblast growth factor and its receptor(s)(GoGuimond et al., 1993; 29GoWalker et al., 1994; seealso 25GoSpivak-Kroizman et al.,1994), cell-surface immobilization of interleukin-8 (24GoSpillmann et al., 1998),dimerization/oligomerization of a number of proteins suchas platelet factor 4 (26GoStringer and Gallagher,1997) and transforming growth factor-ß (15GoLyon et al., 1997), andprotection of interferon-{gamma} against proteolyticdegradation (13GoLortat-Jacob etal., 1995). Similar or distinct multiple regionsof a single heparin/HS chain may be involved in proteinbinding. To facilitate studies on domain organization and its importancefor biological function a strategy was developed to chemically conjugate definedheparin oligomers in linear and chemoselective fashion.

A model system was developed based on the previously establishedmechanism of thrombin inhibition by AT, in the presence of heparin.Heparin (and HS) bind to AT via a unique pentasaccharide sequence,-GlcNSO3(6-OSO3)-GlcA-Glc­NSO3(3,6-OSO3)-IdoA(2-OSO3)-GlcNSO3(6-OSO3)-,and this interaction is sufficient to greatly increase the rateof inhibition of Factor Xa (3GoBourin and Lindahl,1993). Similar potentiation of the inhibition of thrombinrequires binding of this proteinase as well as of AT, adjacent toeach other, to a saccharide sequence that must consist of at least18 monosaccharide units. Information relating to purely syntheticoligosaccharides predicted that the reducing-terminal portion ofsuch a sequence should contain the AT-binding pentasaccharide sequence, whereasthe nonreducing-terminal, thrombin-binding, portion could lack thisregion (28Govan Boeckel et al.,1994; 8GoGrootenhuis etal., 1995). This information was used to validateour novel strategy to generate regio-specific neo-GAG conjugates,starting from natural heparin oligosaccharides.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Generation of neo-heparin conjugate
The aim of the project was to develop a general procedure by whichspecific oligosaccharides, derived from heparin or HS, may be linkedtogether to form a "neo-GAG" species composedof defined domains. The procedure is based on specific functionalizationof oligosaccharides, to ascertain their conjugation in predeterminedsequence. It was applied to the formation of a heparin conjugatecapable of promoting the inactivation of thrombin by AT. An experimentalprotocol was devised to generate two precursor molecules, FragmentA lacking the AT-binding region and Fragment B containing this region,that would constitute the nonreducing-terminal and reducing-terminalportions, respectively, of the biologically active conjugate.

The formation of Fragment A (Figures 1, 2) was initiated by partial deaminative cleavageof beef lung heparin with nitrous acid essentially as describedpreviously (19GoPejler et al.,1988). This reaction resulted in the generation of variouslysized oligosaccharides with a reducing-terminal 2,5-anhydromannose unit(23GoShively and Conrad, 1976). Fractionationof these oligosaccharides by gel chromatography on Biogel P-10 yielded a10-mer fraction and a 12-mer fraction (data not shown) that wererecovered and desalted. The aldehyde group of the reducing-terminal2,5-anhydromannose residue was substituted by reductive amination(see 9GoHoffman et al., 1983)with cystamine·2HCl in the presence of NaB3H3CN,thus introducing a 3H label at the reducing end of theoligomers. The newly introduced amino group was acetylated by reactionwith acetic anhydride (Figure 2), yieldinga product that failed to react with ninhydrin (5GoDoi et al., 1981) (data not shown). The substituted12-mers were separated by affinity chromatography on AT-Sepharose(10GoHöök et al., 1976)into a major nonbound fraction, that was unretained by the immobilizedAT in 50 mM NaCl, a low-affinity (LA) fraction (~16.2 % ofthe initial oligo­saccharide mass) and a high-affinity(HA) fraction (~5.4 % of the initial oligosaccharide mass)by elution with a linear gradient from 0.05 M to 3.0 M NaCl (datanot shown).



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Fig. 1. Schematic representation ofthe chemical and enzymatic cleavage procedures utilized to produceheparin fragments with selectively modified reducing-terminal andnonreducing-terminal monosaccharide units.

 


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Fig. 2. Scheme of modification proceduresleading to regioselective conjugation of heparin fragments generatedthrough partial cleavage of the polysaccharide with nitrous acid(Oligo-A) and heparin lyase (Oligo-B). Reaction steps: a, reductiveamination with cystamine; b, N-acetylation; c, reduction with dithiothreitol;d, peracetylation; e, removal of 4,5-unsaturated hexuronic acidunit; f, iodoacetylation; g, conjugation.

 
Following reduction of the -S-S- bond in the terminal cystaminegroup of LA-12-mers by treatment with dithio­threitol,Fragment A, bearing a free sulfrylhydryl group (-SH) at the reducingend, was separated from excess dithiothreitol and N-acetylated 2-mercaptoethylamineby gel chromatography on Sephadex G-15. The predicted fraction of 3H-labeled, thiol-containing(positive to Ellman’s reagent; 6GoEllman,1959) products (Figure 3) wasrecovered and lyophilized. It should be noted that small moleculescontaining -SH group must be removed completely, to avoid problemsin the subsequent conjugation reaction.



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Fig. 3. Isolation by gel chromatographyon Sephadex G-15 of heparin-derived, 3H-labeled oligosaccharidesbearing a reducing-terminal thiol group (Fragment A). Thiol wasdetermined by Ellman’s reagent (solid squares) and radioactivityby scintillation counting (open squares).

 
Fragment B was designed to provide the reducing-terminal portionof the heparin neo-conjugate, and thus required functionalizationat its nonreducing terminus prior to the conjugation reaction. Tothis end, oligosaccharides were generated from heparin by partiallyase cleavage, and the resultant nonreducing-terminal, 4,5-unsaturatedhexuronic acid residue was utilized as a large protecting grouprelative to the C-4 position of the penultimate GlcN unit (Figures 1, 2). Following partial digestionof bovine lung heparin with heparinase I (see Materialsand methods) both 10- and 12-mer fractions were isolated bygel chromatography on Biogel P-10 (data not shown) and radiolabeledby reduction with NaB3H4. The reduced oligosaccharideswere peracetylated, essentially as described (20GoPetitou,1992), and were then treated with Hg(OAc)2 toeliminate the nonreducing-terminal, unsaturated hexuronic acid unit(14GoLudwigs et al., 1987).In principle, the hydroxyl group at C-4 of the terminal GlcN residue,thus exposed, would be the only hydroxyl function available for furtherderivatization (Figure 2). The efficacyof the Hg(OAc)2 treatment of the 10-mer fraction wasdemonstrated by poly­acrylamide gel electrophoresis whichshowed, after staining with Alcian blue, the appearance of a reasonablydistinct component at a migration position between those of 8- and10-mers (Figure 4a). An iodoacetyl groupwas then introduced at the nonreducing-terminal 4-position by acylationwith iodoacetic anhydride.



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Fig. 4. Analysis by electrophoresisin 20% polyacrylamide gel of (a) heparinoligosaccharides generated by partial lyase digestion of heparin;lane 1, 9-mer formed by Hg(OAc)2 treatment of 10-merlyase product; lane 2, 10-mer; lane 3, 8-mer; (b)products formed after conjugation of LA Fragment A 12-mers and HAFragment B 11-mers. The arrows indicate the migration positionsof oligosaccharide standards obtained by deaminative cleavage ofheparin.

 
Finally, the neo-GAG conjugate was generated by reacting thethiol group at the reducing terminus of Fragment A (12-mers), LAtype, with the iodoacetyl group at the nonreducing end of FragmentB (11-mers), HA or LA type (Figure 2; see Materials and methods). Major formation of thepredicted product was demonstrated by polyacrylamide electrophoresis (Figure 4b). We conclude that the conjugation reactionis chemoselective, such that the two fragments are conjugated in alinear manner. For preparative purpose, the synthesized LA-LA andLA-HA conjugates were purified by Biogel P-10 gel chromatography(data not shown), calculations based on the distribution of 3Hradioactivity indicated coupling yields of ~30%.

Analysis of anticoagulant activity
A dissociation constant of 13 ± 0.1(range; n = 2) nM was measured for the complex of the LA-HA(nonreducing-reducing domain) neo-heparin conjugate with AT at ionicstrength 0.05 by titrations, monitored by tryptophan fluorescence,of AT with the saccharide. This ionic strength was chosen to increasethe AT affinity of the conjugate, compared with the affinity atI 0.15, because of the small amounts of conjugate available. Theinteraction thus appears somewhat weaker than that between nativeHA-heparin or the HA decasaccharide and AT, for which dissociationconstants of 4.8 ± 0.4 (SE; n = 3) and5.3 ± 1.2 (SE; n = 5) nM, respectively,were measured in parallel assays. Nevertheless, the LA-HA conjugatepromoted the inhibition of thrombin by AT about twice as efficientlyas authentic HA-heparin (Figure 5). As expected,neither the LA-LA neoconjugate nor the unconjugated HA-10-mer showed anyapparent ability to accelerate the AT-thrombin reaction. These resultsconform to the notion that the specific AT-binding pentasaccharideis essential but not sufficient for acceleration of thrombin inactivationand that an additional thrombin-binding sequence outside the pentasaccharideregion is required for such an effect.



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Fig. 5. Acceleration of antithrombininhibition of thrombin by LA-HA (nonreducing-reducing domains) neo-GAGconjugate (open circles); LA-LA neo-GAG conjugate (solid squares);native HA-heparin (solid circles); HA 10-mer (open squares); noadded saccharide (solid triangle).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
A number of functionally important interactions between proteinsand heparin/HS chains have been shown to involve more thanone protein-binding domain along the polymer (see Introduction),and this number is likely to increase in future studies. The presentreport describes a novel approach to the study of such interactions,by creating neo-GAG conjugates composed of defined oligosaccharideregions that are arranged in predetermined sequence and togetherexpress a desired biological activity. The novel key feature ofthis approach is the selective C-4 functionalization of a nonreducing-terminalGlcN unit, that is exposed following the removal of a 4,5-unsaturated uronicacid protecting group. The method should be equally applicable toheparin and to HS, thus enabling the combination of domains of highlydifferent structures, including products of selective chemical desulfation.Also galactosaminoglycans (chondroitin sulfate, dermatan sulfate)should be amenable to this principle, since these polysaccharidesare similarly cleaved by lyases yielding the required terminal 4,5-unsaturateduronic acid unit (12GoLinhardt etal., 1990). Fragments containing reducing-terminalaldehyde groups (here provided by 2,5-anhydro-D-mannoseunits) could be generated by partial N-deacetylation through hydrazinolysis,followed by deaminative cleavage at elevated pH (23GoShivelyand Conrad, 1976). Finally, it should be noted that theprocess is adaptable to microscale performance, of particular importancein projects dealing with small samples of polysaccharides isolatedfrom selected organs or even from cultured cells.

The group protection strategy applied involved blocking of thefree hydroxyl groups of Fragment B through peracetylation (Figure 2). The O-acetyl groups could not be readilyremoved following formation of the conjugate, since deesterification wouldbe expected to break up the newly formed linkage between FragmentsA and B. However, previous studies indicated that the weakeningof the interaction between heparin and AT caused by the presenceof O-acetyl groups should be relatively modest (1GoBarzu et al., 1993). This expectation is verifiedby the only moderately lower observed affinity of the conjugateas compared to that of HA-heparin or the HA-decasaccharide for AT.Still, we were surprised to find that the ability of the LA-HA conjugateto accelerate AT inhibition of thrombin was about twice as high,on a molar basis, as that of authentic HA-heparin (Figure 5). We ascribe this finding to the optimal positioningof the AT- and thrombin-binding domains of the LA-HA conjugate,as opposed to the random location of the AT-binding region in theHA-heparin chain (18GoOscarsson et al., 1989) which thus could accommodate thrombinon the "wrong" side of the AT molecule. Nevertheless,deacetylation may be prerequisite to studies of interactions involvingother proteins. To ensure general applicability the method shouldbe modified to allow removal of the protecting groups without the riskof cleaving the conjugate linkage.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Materials
Heparin from bovine lung (Upjohn, Kalamazoo, MI) was purifiedas described previously (11GoLindahl et al., 1965). Heparin with high affinity for AT(10GoHöök et al., 1976),having an average molecular weight of 8000 and reduced polydispersity (17GoOlson et al., 1993), wasa gift from Dr. S.T.Olson, University of Illinois at Chicago. ATwas purified from human plasma by heparin affinity and ion-exchangechromatography (17GoOlson et al.,1993). Human {alpha}-thrombin, >90% activeby active-site titration (7GoErsdal et al., 1995), was a gift from Dr. John Fenton, NewYork State Department of Health, Albany, NY. Heparinase I (200–600U/mg protein) was obtained from Sigma. Bio-Rad Laboratories(Stockholm, Sweden) supplied Biogel P-10 gel, AG 50W-X4 cation exchangeresin (200–400 mesh, H+ form), the Mini-ProteanII Dual Slab Cell and the Bio-Rad Gel Doc 1000 system. SephadexG-15 and Sephadex G-25, prepacked in PD-10 columns,were obtained from Pharmarcia Fine Chemicals (Uppsala, Sweden).AT-Sepharose affinity matrix was prepared as described previously(10GoHöök et al., 1976).Sodium borotritide (NaB3H4, 29 Ci/mmol)and sodium cyanoborotritide (NaB3H3CN, 5–10Ci/mmol) were purchased from Amersham International (Stockholm,Sweden). Sigma Chemical Co. (Stockholm, Sweden) supplied sodiumboro­hydride, sodium cyanoborohydride, cystamine, sodiumnitrite, dithiothreitol, iodoacetic anhydride, acetic anhydride,N,N-dimethylaminopyridine, N,N-dimethylformamide, and Ellman’sreagent. Ultra pure Protogel 30% (w/v) acrylamide stocksolution containing 0.8% (w/v) bisacrylamide was obtainedfrom C. R. Hintze AB, Sweden.

Functionalization at the reducing end of heparin-derived oligosaccharides(type A fragment)
Bovine lung heparin (100 mg) was subjected to partial deaminativecleavage, essentially as described previously (19GoPejler et al., 1988). The sample was dissolvedin 10 ml of deionized water at +4°C,and the solution was acidified to pH 1.5 by dropwise addition of1.0 M H2SO4. Following the addition of 2 mgsodium nitrite in 50 µl deionized water,under vigorous stirring, the reaction mixture was maintained atpH 1.5, in an ice bath, for 3 h. The sample was then adjusted topH 7.0 with 1 M sodium bicarbonate, concentrated to 1 ml, and thenfractionated by gel chromatography on a column (1 x 150cm) of Biogel P-10 in 0.5 M NaCl. Effluent fractions of 1.0 ml were collectedat a rate of 1.4 ml/h and analyzed for hexuronic acid bythe carbazole reaction (2GoBitter and Muir,1962). Both 10-mer and 12-mer fractions, correspondingto 8.4% and 5.2% of the starting material, respectively,were recovered, dialyzed in dialysis bags with molecular weightcut-off 1000 (Spectra) at +4°Cagainst deionized water, and then lyophilized to dryness.

A sample (2.5 mg) of 12-mer heparin deamination product in 1ml of 0.5 M phosphate buffer (pH 7.0) was mixed with 83 mg of cystamine{bullet}2HClin 300 µl of the same buffer, and themixture was vortexed and was then incubated at room temperature. After30 min, 0.5 mCi of NaB3H3CN (10 mCi/mmol)was added in the fume hood, and incubation was continued for another2 h. To ensure complete reductive amination, excess unlabeled NaBH3CN(67 mg) in 100 µl of 0.5 M phosphate buffer(pH 7.0) was added, and incubation was continued at room temperatureovernight. The pH was then adjusted to 4.0 by addition of 4 M aceticacid to eliminate any excess NaBH3CN, after which thesolution was concentrated to ~1 ml and passed through a 1 x 180cm column of Sephadex G-15 in 0.2 M ammonium bicarbonate. A partof the resultant oligosaccharide fraction (840 µg;11,100 d.p.m. 3H/µg),dissolved in 100 µl 0.1 M sodium bicarbonate,was cooled in an ice-bath and N-acetylated by treatment with aceticanhydride (100 µl). After incubationat +4°C for 30 min (no freeprimary amine detectable by the ninhydrin reaction (5GoDoi et al., 1981)) the sample was again desaltedby gel chromatography on Sephadex G-15, and was then lyophilized.

The cystamine-substituted, N-acetylated oligosaccharides werefractionated with regard to affinity for AT by chromatography onAT-Sepharose, essentially as described previously (10GoHöök et al., 1976). The sample (~2 mg) wasapplied to a 5 ml column of the affinity matrix, equilibrated with50 mM NaCl. The NA (unretained) and LA (weakly bound) fractions(subsequently combined into one "LA" pool) andthe HA component were recovered following elution with a lineargradient from 0.05 M to 3.0 M NaCl and were then desalted. The LA12-mers (300 µg) were reduced by treatmentwith 200 µl of 0.1 M dithiothreitolcontaining 1 mM EDTA at room temperature overnight. 3H-Labeledoligosaccharides positive to Ellman’s reagent were recoveredafter passage through a column (1 x 100cm) of Sephadex G-15 in 40 mM NaCl containing 1 mM EDTA.

Functionalization at the nonreducing end of heparin-derived oligosaccharides(type B fragment)
For partial lyase cleavage, bovine lung heparin (100 mg) was dissolvedin 100 ml of 100 mM sodium acetate buffer (pH 6.5), 10 mM CaCl2,0.1 mg/ml bovine serum albumin at 30°C,and heparinase I (12 units) was added. Following incubation at 30°C for 24 h the products were fractionatedby gel chromatography on a column (1 x 180cm) of Biogel P-10 in 0.5 M sodium chloride. Effluent fractionscorresponding to 10-mers and 12-mers were combined separately anddesalted by extensive dialysis in dialysis bags with molecular weightcut-off 1000 (Spectra) against deionized water. A sample (5 mg)of the product was reduced with 0.25 mCi NaB3H4 (29Ci/mmol) in 200 µl of water,adjusted to pH 8.0 with Na2CO3. After 30 min atroom temperature 5 mg of unlabeled NaBH4 was added, and incubationwas continued for another 5 h. The mixture was acidified to pH 4by adding 4 M acetic acid, and was then adjusted to pH 8 with Na2CO3.Labeled oligosaccharides were recovered following desalting by passagethrough Sephadex G-15, and were then lyophilized.

Before O-peracetylation (21GoPetitou et al., 1992), the reduced oligosaccharides(either 10-mers or 12-mers) were converted to the free acid formby passage through a column (1 x 10cm) of AG 50W-X4 (H+ form) at +4°C, and the effluent was immediatelyneutralized with n-tributylamine. The product was lyophilized, dissolvedin 3 ml dry N,N'-dimethylformamide, evaporatedto dryness and redissolved in 1.4 ml dry N,N'-dimethylformamide.N,N'-Dimethylpyridine (25 mg), acetic anhydride(100 µl), and n-tributylamine (200 µl) were added, and the mixture wasincubated at 37°C overnight. The reaction wasinterrupted by the addition of 100 µlH2O, and the peracetylated oligosaccharides were desaltedby passage through a PD-10 column.

Removal of the 4,5-unsaturated, nonreducing-terminal hexuronicacid unit was effected by treating the peracetylated oligo­saccharide(1 mg) with 75 mM mercuric acetate in 400 µl acetatebuffer (pH 5.0) (14GoLudwigs etal., 1987). After incubation at room temperaturefor 2 h, the reaction mixture was passed through a column (1 x 10 cm) of AG 50W-X4 (H+ form),and effluent fractions were neutralized with Na2CO3. Theoligosaccharides were extensively dialyzed against deionized water.

The modified heparin 11-mers were affinity fractionated on AT-Sepharose,as described for the Fragment A preparation, and the resultant LAand HA fractions (4.4% and 3.3% of the initial oligosaccharidemass, respectively) were recovered separately and desalted. Finally,the modified heparin 11-mers were acylated by reaction with iodoaceticanhydride. Samples (200 µg)of each species were converted to the free acid form, convertedto the n-tributylamine salts and evaporated to dryness in the presenceof dry N,N'-dimethylformamide. The residues wereredissolved in 1.2 ml of dry N,N'-dimethyl­formamide/n-tributylamine(v/v, 5/1). p-N,N'-Dimethylaminopyridine (100 µg) and iodoacetic anhydride (10 µg) were added, and the reaction mixtureswere left at room temperature with shaking for 36 h. Following theaddition of 100 µl deionized water thederivatized oligosaccharides were recovered by passage through PD-10 columns.

Conjugation
A solution of Fragment A (12-mers, ~200 µg,550 x 103 d.p.m. 3H)mixed with Fragment B (11-mers, ~100 µg,160x103 d.p.m.) in a 1.5ml microfuge tube was evaporated to dryness. The residue was dissolvedin 100 µl 10 mM EDTA, pH 7.8, and thesolution was kept at room temperature overnight. The reaction productswere separated by gel chromatography on a column (1 x 150cm) of Biogel P-10 in 0.5 M ammonium bicarbonate. Effluent fractionswere analyzed for 3H radioactivity. Fractions correspondingto the putative neo-GAG conjugate were combined and lyophilized.

Interaction of AT with neo-GAG conjugate
Stoichiometries and affinities of heparin, HA-decasaccharide orneo-GAG conjugate binding to AT at 25°Cwere measured by titrations, monitored by the increase of AT tryptophanfluorescence induced by the interaction, as in previous work (17GoOlson et al., 1993; 27GoTurk et al., 1997). Thebuffer was 0.02 M sodium phosphate, 0.1 mM EDTA, 0.1 % (w/v)poly(ethyleneglycol), pH 7.4.

The ability of heparin, HA-decasaccharide or neo-GAG conjugateto potentiate the AT-dependent inhibition of thrombin was assessedat 25°C in 0.02 M sodium phosphate,0.1 M NaCl, 0.1 mM EDTA, 0.1% (w/v) poly(ethyleneglycol),pH 7.4, essentially as described previously (27GoTurk et al., 1997). AT and saccharide, at finalconcentrations of 100 nM and 0–0.5 nM, respectively, weremixed with thrombin at a final concentration of 10 nM. The concentrationsof HA-heparin, HA-decasaccharide, and LA-HA neo-GAG conjugate werethose calculated from the measured stoichiometries of binding to AT,whereas concentrations measured by carbazole analyses were usedfor the LA-LA neo-GAG conjugate. After varying times, portions ofthe reaction mixtures were diluted tenfold into a cuvette, containingthe chromogenic thrombin substrate, D-Phe-Pip-Arg-p-nitroanilinide (Chromogenix, Mölndal,Sweden) at a final concentration of 100 mM, and the residual enzymeactivity was obtained from the linear rate of the absorbance increaseat 405 nm. The time-dependent loss of enzyme activity was fittedby nonlinear regression to a single exponential function with anendpoint of complete inactivation to give the observed pseudo-first-orderrate constant, kobs (17GoOlson et al., 1993). The accelerating effect ofthe saccharides on the AT-thrombin reaction was evaluated by plottingkobs vs. saccharide concentration.

Additional analytical procedures
Uronic acid was determined by the carbazole method (2GoBitter and Muir, 1962). A standard curveof absorbance at 530 nm vs. µg of heparinwas made using a standard heparin (from bovine lung) solution. Radioactivitywas measured by liquid scintillation counting using a Beckman modelLS 3800 liquid scintillation spectrometer. Free sulfrylhydryl groupswere determined by a microscale assay as described previously (6GoEllman, 1959).

Analysis of heparin oligosaccharides by polyacrylamidegel electrophoresis
Polyacrylamide gel electrophoresis was done in a solution of 0.89M Tris base, 0.89 M boric acid, and 20 mM EDTA, pH 8.3, on 20% acrylamidecontaining 0.53% bisacrylamide. A 10 ml volume of monomersolution was poured onto glass plates and allowed to polymerizefor 1 h. The minigel was pre-run at 120 V for 1 h, to achieve aconstant current. Oligosaccharide samples (1 to 30 µgof oligosaccharides in 5 µl deionized water)were mixed with 0.5 µl 2.0 M sucrosein electrophoresis buffer. After electrophoresis was performed at100 V for 2–3 h, the gel was stained with 5% Alcianblue, or further by silver staining essentially as described (16GoMöller et al., 1993).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We thank Dr. Dorothe Spillmann for valuable discussions. Thiswork was supported by Grants 2309 and 4212 from the Swedish MedicalResearch Council, European Commission Grants BMH4-CT96-O937 andB104-CT95-0026, and by Polysackaridforskning AB, Uppsala, Sweden.


    Abbreviations
 
AT, human antithrombin; HS, heparan sulfate; GAG, glycosaminoglycan;GlcA, D-glucuronic acid; IdoA, L-iduronic acid;GlcN, D-glucosamine; EDTA, ethylenediaminetetraacetic acid.


    Footnotes
 
a Towhom correspondence should be addressed. Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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