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Anti-Xa active heparin oligosaccharides
THROMBOSIS RESEARCH 18; Pergamon Press Ltd. (c)
BRIEF
573-578
1980.
Printed in the U.S.A. 00'+9-3848/80/100573-06$02.00/O
COMMUNICATION
ANTI-Xa ACTIVE HEPARIN OLIGOSACCHARIDES J. Choay'*, J.C. Lormeau', M. Petitou', P. Sina?"" B. Casuooo, P. Oreste"', G. TorriooO and G. Gattioooo
' Institut Choay, 46, avenue Thdophile Gautier - 75016 Paris France. O” Laboratoire de Biochimie Structurale ; HER de Sciences Fondamentales et Appliqu6es - 45045 Orleans Cedex - France. Ooo Istituto Chimica e Biochimica "G. Ronzoni" - Milan - Italy. oooo C.N.R., Istituto Chimica Macromolecole - Milan - Italy. Accepted by Editor V.V. Kekkar. (Received 11.2.1980. Received in final form by Executive Editorial Office 2.4.1980)
INTRODUCTION
The well-known anticoagulant heparin is a polysaccharide.largely consisting of the regular disaccharide sequence 2-0-sulfated-L-iduronic acid (Is)N-sulfated-6-0-sulfated-D-glucosamine (A >, with heterogeneous regions containing*D-glucuronic acid (G), non sulfa$ed L-iduronic acid (I) and N-acetylated-D-glucosamine (A,) (1). Heparin is also heterogeneous as regards the molecular weight (2). Recently Rosenberg and Lam (3) have isolated, by fractionation of crude porcine heparin, oligosaccharides with a molecular weight of about 7,000 (degree of polymerisation of about 25). Lindahl et al. also reported on oligosaccharides (degree of polymerisation s 12) obtained by partial chemical and/ or enzymatic degradation of heparin (4). Both types of oligosaccharides had affinity for At-III. It has been demonstrated that heparin anticoagulant activity is modulated by the molecular weight of the polysaccharide, low molecular weight components being more active against activated factor X (5,6). More recently it was suggested that those different activities towards factor Xa could result from the neutralization of high molecular weight species by some plasma components (7-9). While low molecular weight compounds could escape the inhibition thus enhancing only At-III effect against factor Xa. We thought of special relevance to isolcte and characterize the smallest molecular species still having anti-Xa activity, arrrj:-xg the minor constituents of commercial heparin. This is a preliF?inarv report c-7OCR t:ork. R to whom correspondence should ‘ic ndlressei Key words
: hepariu - nligos,occharides - factor Xz - antithrombin-III 573
574
ANTI-Xa
OLIGOSACCARIDES
vo1.18,No.3/4
MATERIAL AND METHODS
Preparation of the oligosaccharide fractions : Commercial (Choay) pig mucosal heparin (50 kgs) was extracted with a mixture of ethanol and water (312, v/v ; 1200 l).'The low molecular weight material obtained (fraction A ; 500 g) was further fractionated by gel filtration (ultrogel ACA 44 ; NaCl 0.5 M ; TrisHCI 0.1 M pH 7.4). The lowest molecular weight material (50 g) was collected. This product was then submitted to an affinity chromatography on agarose bound bovine At-III (10 mg of protein/ml of gel ; adsorption 0.2 M NaCl, 0.05 M Tris-HCl pH 7.5 ; elution 2 M NaCl, 0.05 M Tris-HCl pH 7.5) that yielded compounds possessing At-III affinity (fraction B ; 100 mg). Biological assays : APTT and anti-Z& assays were carried out according to the methods of Caen et al. (10) and Yin et al. (II). Electrophoreses were performed on cellulose acetate in HCl and barium acetate buffers (B. Casu. to be nublished). Gas chromatographic analyses were-performed on SE-30 (for trimethylsilylated compounds) or OV-221 (for acetylated compounds) columns with nitrogen as carrier gas. Before analysis the oligosaccharide fractions were modified by known techniques (12) (carboxyl reduction, N-desulfation, N-acetylation), in order to get compounds which-were then methanolysed and trimethylsilylated. In a second set of experiments the modified oligosaccharides were submitted to the following classical treatment (12) : periodate oxidation, borohydride reduction, hydrolysis and acetylation. Conductimetric titrations were carried out according to Casu and Gennaro (13) Nuclear magnetic resonance (NMR) spectra were recorded on a Varian(l3C, 20 MHZ) and a Bruker (lH, 270 MHz) spectrographs. Samples were dissolved in D20 (IO X for *3C ; 5 X for 1H). Chemical shifts are relative to TMS. RESULTS
Biological activity. The oligosaccharide fraction A had a specific activity of 200 IIJ/mgaccording to the anti-Xa assay and of 50 IU/mg according to the APTT assay. In the same assays fraction B presented activities of 1800 and 12 IU/mg respectively. The ratios between both types of activities, in tests using human plasma as source of At-III is thus 4 for fraction A and 150 for fraction B. Physicochemical study* The oligosaccharide fraction was shown electrophoretically homogeneous. No unusual sugars were detected by gas chromatography and particularly no galactosamine was found in the chemically modified oligosaccharides after methanolysis. The content of non-sulfated-L-iduronic acid (determined as peracetylated threitol in the second set of experiments described in methods) was consistently higher in our oligosaccharides than in unfractionated heparin. The sulfate to carboxyl ratio of the oligosaccharides, determined by conductimetry was 2.0 and it dropped to 1.0 after selective Ndesulfation.
x The term "oligosaccharide fraction" used in this chapter only applies to the fraction B obtained above.
ANTI-Xa
OLIGOSACCARIDES
575
onomeric hydrogen,
6.0
5.0
4.0
3.0
2.0
pm
FIG. I 1 H-NMR spectra of : a) Heparin and b) active oligosaccharide . *
*
:
:
150
100
wm
FIG. 2 13 C-NMR spectra of : a) Heparin and b) active oligosaccharides. The arrows indicate the anomeric carbons of reducing end-residues.
576
ANTI-Xa
OLIGOSACCARIDES
Vo1.18,No.3/4
The NMR spectra of the oligosaccharide fractions from different preparations were very similar to each other, and significantly different from the spectra of unfractionated heparin, especially for "new" signals that are not usually observable or are much weaker in heparin. Differences are si nificantly noticeable in the "anomeric" regions in both 1H (Fig. 1) and Y3C (Fig. 2) spectra. While unfractionated heparin is characterized by two main signals in this region (A -I and I,-I), oligosaccharides display up to eight signals. The two high-field signals in the region of the anomeric carbons (92.5 and 96.5 ppm) are clearly assigned to a reducing end-residue (14). The area of these two signals, as referred to the overall area of the anomeric carbons, suggests an average degree of polymerization of 9. Minor signals in the reducing carbons area indicate some degree of heterogeneity as regards the molecular weight. in the 55-62 ppm region, characteristic of C-2 of aminosugar residues (l), a new signal at 58 ppm is typically present in the active oligosaccharides. Signals from N-acetylated-D-glucosamine residues (C-2 at 56 ppm and CH3 at 25 ppm) (1) account for one of these residues er oligosaccharide molecule. Another straightforward information from the 15C spectra is that the hydroxyl groups at C-6 of the aminosugar residues are largely sulfated. After nitrous acid deamination, which usually splits the glycosidic bonds between a sulfated aminosugar residue and an uranic acid residue, signals corresponding to those of the regular disaccharide units Is-As (15) disappear, giving place to signals from di- and (probably) tetrasaccharides with terminal 2,5_anhydromannose residues. The above-mentioned signals at 92.5. and 96.5 PPm are substantially unaffected by this controlled chemicals degradation.
DISCUSSION
The above data suggest that the molecular size of the present oligosaccharides having high-affinity for At-III and high anti-Xa activity are octa to decasaccharides consisting of a "regular" region of a 1,-A, blocks plus an irregular block containing nonsulfated a-L-iduronate (I), B-D-glucuronate (G) and N-acetyl-u-D-glucosamine (A,) residues. These biologically active oligosaccharides are therefore smaller than those isolated by Rosenberg and Lindahl groups (3, 4). However, they contain the residues considered by the above authors as essential for binding to AtIII, and it is probable that they also contain the so-called "irregular block" that is the tetrasaccharide characterized after degradation by Rosenberg and Lam (3). Our results on biological activity show that it is possible to obtain a product which has a very high anti-Xa activity and almost no effect on the whole coagulation system. Compared to Lindahl's results on anti-Xa activity (8), our results also suggest that the part of Lindahl's oligosaccharide missing in ours would not be essential for binding to At-III and for potentiating its activity towards factor Xa.
ACKNOWLEDGMENTS
The assistance of Mr. Zuber in gas chromatography experiments is gratefully acknowledged.
ANTI-Xa
OLIGOSACCARIDES
577
REFERENCES
1.
PERLIN, A.S., Recent structural studies on heparin. In: Heparin : Structure, cellular functions and clinical applications. N.M. McDuffie (Ed) New-York-London : Academic Press, 1979 pp. 25-37
2.
LAURENT, T;C;, TENGBLAD, A., THUNBERG, L., HOOK, M. and LINDAHL, U. The molecular weight dependence of the anticoagulant activity of heparin. Biochem. J., 175, 691-701, 1978
3.
ROSENBERG, R.D. and LAM, L. Correlation between structure and function of heparin. Proc. Natl. Acad. Sci. USA. 76, 1218-1222,.1979
4.
LINDAHL, U. BACKSTROM, G. HOOK, M., THUNBERG, L., FRANSSON, L.A. and LINKER, A. Structure of the antithrombin-binding site in heparin. Proc. Natl. Acad. Sci. USA, -76, 3198-3202, 1979
5.
ANDERSSON, L.O., BARROWCLIFFE, T.W., HOLMER, E., JOHNSON, E.A. and SIMS, G.E.C. Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration Thrombosis Res., 2, 575-583, 1976
6.
BARROWCLIFFE, T.W., JOHNSON, E.A., EGGLETON, E.A., and THOMAS, D.P. Anticoagulant activities of lung and mucous heparins. Thrombosis Res. -12, 27-36, 1978
7.
ANDERSSON, L.O., BARROWCLIFFE, T.W. HOLMER, E., JOHNSON, E.A. and SODERSTROM, G. Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma. Thrombosis Res., -15, 531-541, 1979
8.
THUNBERG, L., LINDAHL, U., TENGBIAD, A., LAURENT, T.C. and JACKSON, C.M. On the molecular-weight-dependence of the anticoagulant activity of heparin. Biochem. J. 181, 241-243, 1979
9.
YIN E.T., SALSGIVER, W.J., TANGEN, 0. A hitherto undescribed naturally occuring plasma antagonist of activated factor X inhibitor (Antithrombin III) VIIth International Congress on Thrombosis and Haemostasis London 1979, F.K. Schattauer Verlag, Stuttgart-New-York 1979, p.122 abstr.0282; Thrombosis Haemostasis 42 122, 1979
10.
CAEN, J. LARRIEU, M.J. and SAMAMA, M. In : L'hemostase. Paris : Expansion Scientifique Francaise, 1968, pp. 133-135.
11.
YIN, E.T., WESSLER, S. and BUTLER, J.V. Plasma heparin : a unique practical, submicrogram-sensitive assay. J. Lab. Clin..Med., 8'1, 298-310 1973 '12. LINDBERG, B. LONNGREN, J. Methylation analysis of complex carbohydrates : general procedure and application for sequence analysis. In : "Methods in Enzymology 50 V. Ginsburg (Ed.) New-York-San Francisco-London : Academic Press,'1978 pp.3-33
578
ANTI-Xa
OLIGOSACCARIDES
13.
CASU, B. and GENNARO, U. A conductimetric method for the determination of sulphate and carboxyl groups in heparin and other mucopolysaccharides. Carbohyd. Res., 39, 168-176, 1975
14.
PERLIN A.S. Carbon-13 N.M.R. spectroscopy of carbohydrates In : Organic Chemistry Series.Two (International Review of Science) vol. 7 Carbohydrates , G.O. Aspinall (Ed.) London-Boston : Butterworths, 1976 pp. I-34
15.
GATTI, G., FASU, B. and PERLIN, A.S. Conformations of the major residues in heparin H-NMR spectroscopic studies. Biochem. Biophys. Res. Commun., 85, 14-20, 1978.
BRIEF
573-578
1980.
Printed in the U.S.A. 00'+9-3848/80/100573-06$02.00/O
COMMUNICATION
ANTI-Xa ACTIVE HEPARIN OLIGOSACCHARIDES J. Choay'*, J.C. Lormeau', M. Petitou', P. Sina?"" B. Casuooo, P. Oreste"', G. TorriooO and G. Gattioooo
' Institut Choay, 46, avenue Thdophile Gautier - 75016 Paris France. O” Laboratoire de Biochimie Structurale ; HER de Sciences Fondamentales et Appliqu6es - 45045 Orleans Cedex - France. Ooo Istituto Chimica e Biochimica "G. Ronzoni" - Milan - Italy. oooo C.N.R., Istituto Chimica Macromolecole - Milan - Italy. Accepted by Editor V.V. Kekkar. (Received 11.2.1980. Received in final form by Executive Editorial Office 2.4.1980)
INTRODUCTION
The well-known anticoagulant heparin is a polysaccharide.largely consisting of the regular disaccharide sequence 2-0-sulfated-L-iduronic acid (Is)N-sulfated-6-0-sulfated-D-glucosamine (A >, with heterogeneous regions containing*D-glucuronic acid (G), non sulfa$ed L-iduronic acid (I) and N-acetylated-D-glucosamine (A,) (1). Heparin is also heterogeneous as regards the molecular weight (2). Recently Rosenberg and Lam (3) have isolated, by fractionation of crude porcine heparin, oligosaccharides with a molecular weight of about 7,000 (degree of polymerisation of about 25). Lindahl et al. also reported on oligosaccharides (degree of polymerisation s 12) obtained by partial chemical and/ or enzymatic degradation of heparin (4). Both types of oligosaccharides had affinity for At-III. It has been demonstrated that heparin anticoagulant activity is modulated by the molecular weight of the polysaccharide, low molecular weight components being more active against activated factor X (5,6). More recently it was suggested that those different activities towards factor Xa could result from the neutralization of high molecular weight species by some plasma components (7-9). While low molecular weight compounds could escape the inhibition thus enhancing only At-III effect against factor Xa. We thought of special relevance to isolcte and characterize the smallest molecular species still having anti-Xa activity, arrrj:-xg the minor constituents of commercial heparin. This is a preliF?inarv report c-7OCR t:ork. R to whom correspondence should ‘ic ndlressei Key words
: hepariu - nligos,occharides - factor Xz - antithrombin-III 573
574
ANTI-Xa
OLIGOSACCARIDES
vo1.18,No.3/4
MATERIAL AND METHODS
Preparation of the oligosaccharide fractions : Commercial (Choay) pig mucosal heparin (50 kgs) was extracted with a mixture of ethanol and water (312, v/v ; 1200 l).'The low molecular weight material obtained (fraction A ; 500 g) was further fractionated by gel filtration (ultrogel ACA 44 ; NaCl 0.5 M ; TrisHCI 0.1 M pH 7.4). The lowest molecular weight material (50 g) was collected. This product was then submitted to an affinity chromatography on agarose bound bovine At-III (10 mg of protein/ml of gel ; adsorption 0.2 M NaCl, 0.05 M Tris-HCl pH 7.5 ; elution 2 M NaCl, 0.05 M Tris-HCl pH 7.5) that yielded compounds possessing At-III affinity (fraction B ; 100 mg). Biological assays : APTT and anti-Z& assays were carried out according to the methods of Caen et al. (10) and Yin et al. (II). Electrophoreses were performed on cellulose acetate in HCl and barium acetate buffers (B. Casu. to be nublished). Gas chromatographic analyses were-performed on SE-30 (for trimethylsilylated compounds) or OV-221 (for acetylated compounds) columns with nitrogen as carrier gas. Before analysis the oligosaccharide fractions were modified by known techniques (12) (carboxyl reduction, N-desulfation, N-acetylation), in order to get compounds which-were then methanolysed and trimethylsilylated. In a second set of experiments the modified oligosaccharides were submitted to the following classical treatment (12) : periodate oxidation, borohydride reduction, hydrolysis and acetylation. Conductimetric titrations were carried out according to Casu and Gennaro (13) Nuclear magnetic resonance (NMR) spectra were recorded on a Varian(l3C, 20 MHZ) and a Bruker (lH, 270 MHz) spectrographs. Samples were dissolved in D20 (IO X for *3C ; 5 X for 1H). Chemical shifts are relative to TMS. RESULTS
Biological activity. The oligosaccharide fraction A had a specific activity of 200 IIJ/mgaccording to the anti-Xa assay and of 50 IU/mg according to the APTT assay. In the same assays fraction B presented activities of 1800 and 12 IU/mg respectively. The ratios between both types of activities, in tests using human plasma as source of At-III is thus 4 for fraction A and 150 for fraction B. Physicochemical study* The oligosaccharide fraction was shown electrophoretically homogeneous. No unusual sugars were detected by gas chromatography and particularly no galactosamine was found in the chemically modified oligosaccharides after methanolysis. The content of non-sulfated-L-iduronic acid (determined as peracetylated threitol in the second set of experiments described in methods) was consistently higher in our oligosaccharides than in unfractionated heparin. The sulfate to carboxyl ratio of the oligosaccharides, determined by conductimetry was 2.0 and it dropped to 1.0 after selective Ndesulfation.
x The term "oligosaccharide fraction" used in this chapter only applies to the fraction B obtained above.
ANTI-Xa
OLIGOSACCARIDES
575
onomeric hydrogen,
6.0
5.0
4.0
3.0
2.0
pm
FIG. I 1 H-NMR spectra of : a) Heparin and b) active oligosaccharide . *
*
:
:
150
100
wm
FIG. 2 13 C-NMR spectra of : a) Heparin and b) active oligosaccharides. The arrows indicate the anomeric carbons of reducing end-residues.
576
ANTI-Xa
OLIGOSACCARIDES
Vo1.18,No.3/4
The NMR spectra of the oligosaccharide fractions from different preparations were very similar to each other, and significantly different from the spectra of unfractionated heparin, especially for "new" signals that are not usually observable or are much weaker in heparin. Differences are si nificantly noticeable in the "anomeric" regions in both 1H (Fig. 1) and Y3C (Fig. 2) spectra. While unfractionated heparin is characterized by two main signals in this region (A -I and I,-I), oligosaccharides display up to eight signals. The two high-field signals in the region of the anomeric carbons (92.5 and 96.5 ppm) are clearly assigned to a reducing end-residue (14). The area of these two signals, as referred to the overall area of the anomeric carbons, suggests an average degree of polymerization of 9. Minor signals in the reducing carbons area indicate some degree of heterogeneity as regards the molecular weight. in the 55-62 ppm region, characteristic of C-2 of aminosugar residues (l), a new signal at 58 ppm is typically present in the active oligosaccharides. Signals from N-acetylated-D-glucosamine residues (C-2 at 56 ppm and CH3 at 25 ppm) (1) account for one of these residues er oligosaccharide molecule. Another straightforward information from the 15C spectra is that the hydroxyl groups at C-6 of the aminosugar residues are largely sulfated. After nitrous acid deamination, which usually splits the glycosidic bonds between a sulfated aminosugar residue and an uranic acid residue, signals corresponding to those of the regular disaccharide units Is-As (15) disappear, giving place to signals from di- and (probably) tetrasaccharides with terminal 2,5_anhydromannose residues. The above-mentioned signals at 92.5. and 96.5 PPm are substantially unaffected by this controlled chemicals degradation.
DISCUSSION
The above data suggest that the molecular size of the present oligosaccharides having high-affinity for At-III and high anti-Xa activity are octa to decasaccharides consisting of a "regular" region of a 1,-A, blocks plus an irregular block containing nonsulfated a-L-iduronate (I), B-D-glucuronate (G) and N-acetyl-u-D-glucosamine (A,) residues. These biologically active oligosaccharides are therefore smaller than those isolated by Rosenberg and Lindahl groups (3, 4). However, they contain the residues considered by the above authors as essential for binding to AtIII, and it is probable that they also contain the so-called "irregular block" that is the tetrasaccharide characterized after degradation by Rosenberg and Lam (3). Our results on biological activity show that it is possible to obtain a product which has a very high anti-Xa activity and almost no effect on the whole coagulation system. Compared to Lindahl's results on anti-Xa activity (8), our results also suggest that the part of Lindahl's oligosaccharide missing in ours would not be essential for binding to At-III and for potentiating its activity towards factor Xa.
ACKNOWLEDGMENTS
The assistance of Mr. Zuber in gas chromatography experiments is gratefully acknowledged.
ANTI-Xa
OLIGOSACCARIDES
577
REFERENCES
1.
PERLIN, A.S., Recent structural studies on heparin. In: Heparin : Structure, cellular functions and clinical applications. N.M. McDuffie (Ed) New-York-London : Academic Press, 1979 pp. 25-37
2.
LAURENT, T;C;, TENGBLAD, A., THUNBERG, L., HOOK, M. and LINDAHL, U. The molecular weight dependence of the anticoagulant activity of heparin. Biochem. J., 175, 691-701, 1978
3.
ROSENBERG, R.D. and LAM, L. Correlation between structure and function of heparin. Proc. Natl. Acad. Sci. USA. 76, 1218-1222,.1979
4.
LINDAHL, U. BACKSTROM, G. HOOK, M., THUNBERG, L., FRANSSON, L.A. and LINKER, A. Structure of the antithrombin-binding site in heparin. Proc. Natl. Acad. Sci. USA, -76, 3198-3202, 1979
5.
ANDERSSON, L.O., BARROWCLIFFE, T.W., HOLMER, E., JOHNSON, E.A. and SIMS, G.E.C. Anticoagulant properties of heparin fractionated by affinity chromatography on matrix-bound antithrombin III and by gel filtration Thrombosis Res., 2, 575-583, 1976
6.
BARROWCLIFFE, T.W., JOHNSON, E.A., EGGLETON, E.A., and THOMAS, D.P. Anticoagulant activities of lung and mucous heparins. Thrombosis Res. -12, 27-36, 1978
7.
ANDERSSON, L.O., BARROWCLIFFE, T.W. HOLMER, E., JOHNSON, E.A. and SODERSTROM, G. Molecular weight dependency of the heparin potentiated inhibition of thrombin and activated factor X. Effect of heparin neutralization in plasma. Thrombosis Res., -15, 531-541, 1979
8.
THUNBERG, L., LINDAHL, U., TENGBIAD, A., LAURENT, T.C. and JACKSON, C.M. On the molecular-weight-dependence of the anticoagulant activity of heparin. Biochem. J. 181, 241-243, 1979
9.
YIN E.T., SALSGIVER, W.J., TANGEN, 0. A hitherto undescribed naturally occuring plasma antagonist of activated factor X inhibitor (Antithrombin III) VIIth International Congress on Thrombosis and Haemostasis London 1979, F.K. Schattauer Verlag, Stuttgart-New-York 1979, p.122 abstr.0282; Thrombosis Haemostasis 42 122, 1979
10.
CAEN, J. LARRIEU, M.J. and SAMAMA, M. In : L'hemostase. Paris : Expansion Scientifique Francaise, 1968, pp. 133-135.
11.
YIN, E.T., WESSLER, S. and BUTLER, J.V. Plasma heparin : a unique practical, submicrogram-sensitive assay. J. Lab. Clin..Med., 8'1, 298-310 1973 '12. LINDBERG, B. LONNGREN, J. Methylation analysis of complex carbohydrates : general procedure and application for sequence analysis. In : "Methods in Enzymology 50 V. Ginsburg (Ed.) New-York-San Francisco-London : Academic Press,'1978 pp.3-33
578
ANTI-Xa
OLIGOSACCARIDES
13.
CASU, B. and GENNARO, U. A conductimetric method for the determination of sulphate and carboxyl groups in heparin and other mucopolysaccharides. Carbohyd. Res., 39, 168-176, 1975
14.
PERLIN A.S. Carbon-13 N.M.R. spectroscopy of carbohydrates In : Organic Chemistry Series.Two (International Review of Science) vol. 7 Carbohydrates , G.O. Aspinall (Ed.) London-Boston : Butterworths, 1976 pp. I-34
15.
GATTI, G., FASU, B. and PERLIN, A.S. Conformations of the major residues in heparin H-NMR spectroscopic studies. Biochem. Biophys. Res. Commun., 85, 14-20, 1978.