Is There a Unique Sequence in Heparin for ... - Semantic Scholar

Val. 263, No. 18,Issue of June 25, pp. 8686-8690.1988 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY The American Society for Biochemistry and Molecular Biology, Inc.

(9 1988 by

Is There a Unique Sequence in Heparin for Interaction with Heparin Cofactor II? STRUCTURAL AND BIOLOGICAL STUDIES OF HEPARIN-DERIVED OLIGOSACCHARIDES* (hceived for publication, May 4,

1987)

Maurice PetitouS, Jean-Claude LormeauS, Bruno Perlyj, Patrick Berthaultt, Veronique Bossennect, Pierre Sien, and JeanChoayS From the Slnstitut Choay, 46, Avenue Thiophile Gautier, 75782 Paris Cedex 16, the BDipartement de Physico-Chimie, Centre d’Etudes Nuckaires de Saclay, 91 191 Gif-sur-Yvette Cedex, and the llhboratoire d’Hemstase, Centre Hospitalier Universitaire de Toulouse Purpan, Place du Docteur-Baylac, 31052 Toulouse Ceder, France

To study the structural requirements in heparin for interaction with heparin cofactor I1 (HC 11) we have analyzed the properties of oligosaccharide fractions obtained after digestion of heparin by heparinase and gel filtration. No activation of HC I1 was detected in the presence of di-, tetra-, hexa-, octa-, deca-, or dodecasaccharides. The hexasaccharide pool was fractionated by ion-exchange chromatography,and the structure of the major species, obtained in a homogeneous state, was investigated by NMR. All the resonances wereunambiguously assigned using correlation by homonuclear andheteronuclear scalar coupling. The six monosaccharide residues of this hexasaccharide were thus easily identified. The sequence was established through two-dimensional nuclear Overhauser effect experiments. The results indicate that this productis a hexasaccharide recentlydescribed by Linhardt et d. (Linhardt, R. J., Rice, K. G., Merchant, Z. M., Kim, Y. S., and Lohse, D. L. (1986) J. Biol. Chem. 261, 14448-14464). However, we could not confirm the anticoagulant activity observed by these authors. Moreover, none of the individual components obtained after fractionation of the hexasaccharide pool was able either to activateHC I1 against thrombin or to inhibit HC I1 activation by heparin. Thus, our data led us toconclude that no unique sequence is involved in heparin for binding to HC I1 and inactivation of thrombin. Theinteraction merely resultsfrom the highly anionic character of heparin.

The well known anticoagulant properties of heparin are mainly attributed to itsability to interactwith AT 111’ through a specific pentasaccharide sequence that has been fully characterized (1-5) and chemically synthesized (6, 7). The recent discovery (8) of a second heparin cofactor (HC 11) which inhibits thrombin inthe presence of heparin raises the problem of the interaction of HC I1 and heparin. Whether this interaction requires a specific region in heparin or is merely due to the highly anionic character of this polysaccharide is an open question.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‘The abbreviations used are: AT 111, antithrombin 111; APTT, activated partial thromboplastin time; COSY, correlation spectroscopy; GPC, gel permeation chromatography; HC 11, heparin cofactor II; HPLC, high performance liquid chromatography; IEC, ion-exchange chromatography; TSP, 3-(trimethylsily1)propionic acid.

Heparin has acomplex polysaccharide structure consisting of the repetition of a disaccharide, uronic acid-glucosamine. The polysaccharide chains containso-called “regular regions” in which 2-O-sulfo-~-iduronicacid and 6-0-sulfo-N-sulfo-Dglucosamine are present exclusively and “irregular regions” in which L-iduronic acid, D-glucuronic acid, and 3-0-sulfo-Dglucosamine maybe encountered (9). Heparinase which cleaves glycosidic linkages between N-sulfo-D-glucosamine and 2-O-sulfo-~-iduronicacid (10) is particularly well suited for the degradation of regular regions and thepreparation of irregular regions. Therefore, we used this enzyme for isolating AT 111-bindingoligosaccharides which occur in irregular segments of the heparin molecules (1-3). The compounds thus obtained were analyzed by chemical and physicochemical techniques, particularly NMR (2). We also analyzed in coagulation tests the activity of heparin-derived oligosaccharides obtained after heparinase cleavage and never found significant anticoagulant activity either by the APTT assay or the USP assay in di-, tetra-, hexa-, octa-, deca-, and dodecasaccharide fractions. Such results are at variance with those previously reported by others who isolated and characterized a highly active hexasaccharide. This prompts us to publish our own data. The availability of heparin-related oligosaccharides obtained by heparinase cleavage, by chemical degradation (2, 12, 32), and by chemical synthesis (13) allowed us to accumulate NMR data on this class of compounds, thus greatly facilitating the analysis of newderivatives. These datacoupled with recent NMR methods allow the rapid determination of unknown structures. Here we report on the application of these methods to the major hexasaccharide species obtained after heparinase cleavage of heparin. Its structurecorresponds to the one proposed by Linhardt et al. (11)for a compound endowed with the ability to activate HC I1 and inhibit thrombin. However, the hexasaccharide described in the present communication is devoid of any anticoagulant activity. EXPERIMENTAL PROCEDURES

Materials Porcine mucosal heparin (injectable-grade) was from Choay and pig skin dermatan sulfate from Sigma. Preparative GPC was performed on Ultrogel AcA 202 (IBF). Preparative IEC was done on QSepharose Fast Flow (Pharmacia LKB Biotechnology Inc.) and GPCHPLC on I 60 columns (Waters Associates). IEC-HPLC was performed using a Mono Q HR 515 column (Pharmacia LKB Biotechnology Inc.). APTT reagents were from General Diagnostics, Stago, and Ortho Diagnostic Systems Inc. Human thrombin was obtained from Sigma, Polybrene from Aldrich, and the substrate CBS 34-47 from Stago. Clotting times were recorded using a KC 10 instrument

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Heparin and Heparin Cofactor 11

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(AHS-Dade). Heparinase (heparin lyase, EC 4.2.2.7) was extracted from Flavobacterium heparinurn and purified successively by chromatography on CM-Sepharose, gel filtration, and chromatography on hydroxylapatite. The preparation was homogeneous as judged by gel electrophoresis and free of heparitinase (EC 4.2.2.8). Its specific activity determined according to Yang et al. (14) was 30 IU/mg. HC I1 was prepared as already described (15). Plasma depleted in HC 11, AT 111, or both was prepared and characterized as already described (16). TSP was from Aldrich and DzO (99.95% deuterium) fromCEA. All other chemicals were reagent-grade. Methods Heparin Cleavage by Heparime-The sodium salt of porcine mucosal heparin (injectable-grade) was dissolved a t a final concentration of 10 mg/ml in 0.1 M sodium acetate, pH 7.0, containing 0.03 M calcium chloride. Heparinase a t a final concentrationof 10 pg/ml was added, and the mixture was incubated a t 30 "C. The reaction was monitored by measuring the absorbance at 232 nm. After 12 h, the absorbance reached a plateau and remained unchanged on further addition of heparinase. The reaction products were recovered by precipitation with 10 volumes of ethanol, centrifuged, and dried under vacuum. Preparative GPC of Oligosaccharides-20 g of the oligosaccharide mixture in 400ml of 0.5 M NaCl were loaded on a 20 X 100-cm column packed with Ultrogel ACA 202. Elution was carried out with 0.5 M sodium chloride at a flow rate of 1700 ml/h and monitored by recording the absorbance a t 232 nm. Appropriate fractions were pooled, and theoligosaccharideswere recovered as above after ethanol precipitation. Preparative ZEC of Hexasaccharides-1 g of the hexasaccharide mixture was applied to a column (2.5 X 30 cm) of Q-Sepharose Fast Flow equilibrated with 0.3 M NaCl and eluted with a linear 0.3-1.5 M sodium chloride gradient. The separation was monitored by recording the absorbance at 232 nm. Fractions corresponding to single peaks were pooled and theproducts recovered as described above. Analytical GPC of Oligosaccharides-GPC-HPLC was performed on an I 60 (0.78 X 30 cm) column eluted with 1 M sodium chloride a t a flow rate of 0.5 ml/min. 200 pg of the compound dissolved in 50 pl were injected. Elution was monitored at 232 nm. Analytical ZEC of Hexasaccharides-IEC-HPLC was performed on a Mono-Q HR 515 column (0.5 X 15 cm) equilibrated with 0.5 M NaCl and eluted with a linear 0.5-1.5 M sodium chloride gradient. 200 pgof the compound in 200 pl were injected. Elution was monitored a t 232 nm. Whole Blood Clotting Assays-The APTT assay was carried out using classical methods (17), the endpoint being determined with an automatic clot timer. Thrombin clotting timeswere done using commercial thrombin diluted to clot normal plasma in 18-20 s (17). AntiXa activity was determined according to Yin et al. (18). Thrombin Inhibition Assay-The thrombin inhibition assay, in a purified system in the presence of HC 11, was performed as already described (16). NMR Metho&-The hexasaccharide sodium salt was dissolved in DzO, adjusted to pH 7.4 (meter reading), and lyophilized twice from 99.95% DzO (CEA, France). 20 mM solutions were used for proton and carbon 13 NMR experiments. All proton NMR experiments were performed using a Bruker WM500 spectrometer at 500 MHz upgraded with an Aspect 3000 computer, pulse programmer, and array processor. The sample temperature was295 K. Carbon 13spectra and heteronuclear correlation experiments were performed a t 300 K on a Bruker MSL300 spectrometer operating at 75.47 MHz for carbon 13. Spectrum calibration was achieved using internal TSPfor proton and internal methanol for carbon (51.60 ppm from internal TSP). RESULTS AND DISCUSSION

The oligosaccharides resulting from heparinase cleavage of pig mucosal heparin were first sized byGPC on Ultrogel ACA 202. After precipitation with ethanol, the di-, tetra-, hexa-, octa-, deca-, and dodecasaccharide fractions were collected. They werehomogeneous when analyzed by GPC-HPLC. Yields are reported in Table I. The different fractions were then assayed for their anticoagulant properties in the APTT assay (17) (whole plasma clotting) and in theYin et al. assay (18)(anti-factor Xa activity). Results are reported in TableI. Anti-factor Xa activity was present in the hexasaccharide

TABLE I Amount and anticoagulant properties of oligosaccharides obtained after heparinase depolymerization and GPC The amount is expressed as the percentage in weight of each fraction in the whole mixture. Results of coagulation assays are expressed in IU of heparin. APTT and anti-factor Xa tests were performed according to Refs. 17 and18, resmctively. Di Tetra Hexa Octa Deca Dodeca 36 35 14 6.5 4.5 2.5 Amount (%)