Department of Biologia Animale, University of Modena and Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy
Received on May 6, 2004; revised on July 6, 2004; accepted on July 7, 2004
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Abstract |
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Key words: anticoagulant drug / glycosaminoglycans / heparin / molluscs / Tapes phylippinarum
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Introduction |
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Heparin is well known for its ability to prevent the coagulation of blood (Bjork and Lindahl, 1982). It has been used clinically as an anticoagulant for over half a century, but this natural polymer also has an antiviral activity, binds to a variety of growth factors, inhibits complement activation, and regulates angiogenic activity (Casu, 1985
; Folkman et al., 1983
; Jackson et al., 1991
; Weiler et al., 1992
). Commercial manufacture of heparin relies on either porcine or bovine intestinal or bovine lung tissue as raw material. The appearance of bovine spongiform encephalopathy, commonly called mad cow disease, and its apparent link to the similar prion-based Creutzfeldt-Jakob disease in humans (Schonberger, 1998
), has limited the use of bovine heparin. Moreover, it is not easy to distinguish bovine and porcine heparins, thus making it difficult to ensure the species source of heparin (Linhardt and Gunay, 1999
). Furthermore, porcine heparin also has problems associated with religious restrictions on its use. Nonanimal sources of heparin, such as chemically synthesized, enzymatically synthesized, or recombinant heparins, are currently not available for pharmaceutical purposes. These concerns have motivated us to look for alternative nonmammalian sources of heparin.
An enzyme-based oligosaccharide mapping has been developed (Linhardt et al., 1992a) capable of qualitatively and quantitatively detecting part of the ATIII-binding sites and ATIII-binding site precursors in heparin. In this article we describe the structural characterization of a heparin possessing high anticoagulant activity isolated from a species of marine clam, Tapes phylippinarum, a bivalve mollusc normally cultivated as a commercial food souce (Cima et al., 1998
). By using the methodology developed by Linhardt and co-workers (1992a)
, the content of the oligosaccharide sequences bearing part of the ATIII-binding region,
UA2S(1
4)-
-D-GlcN2S6S(1
4)-ß-D-GlcA(1
4)-
-D-GlcN2S3S6S and
UA2S (1
4)-
-D-GlcN2S6S (1
4)-
-L-IdoA (1
4)-
-D-GlcNAc6S (1
4)-ß-D-GlcA (1
4)-
-D-GlcN2S3S6S, was determined. Furthermore, to our knowledge, this is the first work describing a clam heparin having the ATIII-binding site mainly identical to those of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin, but different from that found in beef lung heparin.
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Results |
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Figure 1A illustrates the agarose gel electrophoresis of the mollusc heparin showing the two components, the slow moving species having high molecular mass and charge density and the fast-moving heparin, possessing a lower molecular mass and sulfate groups amount (Volpi, 1993; Volpi and Maccari, 2002
). Figure 1B illustrates the densitometric scanning of T. phylippinarum heparin showing 22 ± 6.8% of the slow-moving component and 78 ± 5.4% of the fast-moving species.
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Discussion |
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Linhardt and co-workers (1992a) developed an oligosaccharide mapping strategy able to correlate the anticoagulant properties of heparins with the quantitative presence of oligosaccharides bearing part of the ATIII-binding region. When porcine mucosal heparin is treated with heparin lyase I, six major oligosaccharides (2, 36, 8) are observed on SAX-HPLC, accounting for 8590 mol % of the oligosaccharide products (Linhardt et al., 1988
, 1992a
; Rice and Linhardt, 1989
). The structures of additional minor oligosaccharides (1, 3a, 3b, 4a, 6a, and 7) have also been reported (Linhardt et al., 1990
, 1992a
; Loganathan et al., 1990
). Furthermore, specific oligosaccharides 3-O-sulfated corresponding to part of the ATIII-binding site have been identified as the tetrasaccharide 7 [
UA2S(1
4)-
-D-GlcN2S6S(1
4)-ß-D-GlcA(1
4)-
-D-GlcN2S3S6S] and the hexasaccharide 8 [
UA2S(1
4)-
-D-GlcN2S6S(1
4)-
-L-IdoA(1
4)-
-D-GlcNAc6S (1
4)-ß-D-GlcA(1
4)-
-D-GlcN2S3S6S] (Linhardt et al., 1992a
). By using the same methodological approach used by Linhardt et al. (1992a)
to characterize the oligosaccharide pattern of porcine mucosal heparin, we analyzed the structure of a heparin sample purified from the clam T. phylippinarum in comparison with bovine mucosal heparin. T. phylippinarum heparin was found to be composed of a greater mol % (5.1) of the hexasaccharide 8 than bovine mucosal heparin (2.2) and porcine mucosal heparin (2.2) (Linhardt et al., 1992a
) whereas the mol % of the tetrasaccharide 7 was calculated to be lower (1.1) in comparison with bovine heparin (1.9) but greater than porcine heparin (0.6) (Linhardt et al., 1992a
).
Porcine mucosal heparin samples with high (antifactor Xa activity of 457 U/mg) and low (antifactor Xa activity of 24 U/mg) ATIII affinity were prepared by Linhardt (Linhardt et al., 1992a). The high-ATIII-affinity sample yielded a high mol % of the hexasaccharide 8 (7.2 mol %) after treatment with heparin lyase I, whereas low-ATIII-affinity heparin showed little or no 3-O-sulfation. T. phylippinarum heparin is enriched in this ATIII-binding site portion showing 130% more ATIII-binding sites than bovine mucosal and porcine mucosal heparin (Linhardt et al., 1992a
) calculated from the mole % of the hexasaccharide 8. This increased number of ATIII-binding sites in clam heparin is accompanied by increased anticoagulant activity. The anticoagulant activity of T. phylippinarum heparin expressed as APTT is
140% more than that of bovine mucosal heparin (this study and Dietrich et al., 1985
). By considering anti-Xa activity, T. phylippinarum heparin (317 U/mg) had 120150% greater potency than did porcine (145 U/mg) (Linhardt et al., 1992b
) and bovine heparin (125 U/mg), and it showed
30% lower activity than a high-ATIII-affinity heparin sample (Linhardt et al., 1992a
).
As defined by Linhardt and colleagues (1992a), oligosaccharide 5 has the same structure as 7 except for the lack of the 3-O-sulfate group, and hexasaccharide 6a shows the same sequence of 8 without possessing the 3-O-sulfate group. For this reason, oligosaccharides 5 and 6a represent the precursors of the ATIII-binding site. Porcine mucosal heparin possesses a more abundant precursor oligosaccharide 5 (6.9 mol %) than the 3-O-sulfated parent tetrasaccharide 7 (0.6 mol %), whereas the precursor 6a is present in lower amounts (6.9 mol %) than the hexasaccharide 8 containing the ATIII-binding site (2.2 mol %) (Linhardt et al., 1992a
). Interestingly, the same trend of the oligosaccharide precursors and the 3-O-sulfated parent oligosaccharides have been found in this study for bovine mucosal and T. phylippinarum heparins, confirming that the 3-O-sulfation of precursor 5, contrary to precursor 7, may be limiting not only in mammalian species but also in molluscs.
A structural variability in heparins from various species within and immediately adjacent to the ATIII-binding site has been reported (Loganathan et al., 1990). The ATIII-binding sites in human and porcine intestinal mucosal heparins (Linhardt et al., 1992b
) and in bovine intestinal mucosal heparin (this study) are identical having the predominant structure
4)-
-L-IdoA2S(1
4)-
-D-GlcN2 S6S(1
4)-
-L-IdoA(1
4)-
-D-GlcNAc6S(1
4)-ß-D-GlcA(1
4)-
-D-GlcN2S3S6S(1
4)-
-L-IdoA2S(1
4)-
-D-GlcN2S6S(1
but different from that found in beef lung heparin (Loganathan et al., 1990
) possessing the main structure
4)-
-L-IdoA2S(1
4)-
-D-GlcN2S6S(1
4)-
-L-IdoA2S(1
4)-
-D-GlcN2S6S(1
4)-ß-D-GlcA(1
4)-
-D-GlcN2S3S6S(1
4)-
-L-IdoA2S(1
4)-
-D-GlcN2S6S(1
. The oligosaccharide 8 contains a portion of the ATIII-binding site as it is missing of the
4)-
-L-IdoA2S(1
4)-
-D-GlcN2S6S(1
residue at its reducing end as the result of the heparinase cleavage through the ATIII binding region (Shriver et al., 2000
). As a consequence, the presence of this oligosaccharide is suggestive of the presence of the same ATIII-binding site in the heparin preparations. The results of this study, in particular the high mol % of oligosaccharide 8, enable us to affirm that the prevalent structure of the ATIII-binding sites in T. phylippinarum and in bovine intestinal mucosal heparins are identical to those of human and porcine intestinal mucosal heparin samples.
The biological function of the clam heparins and their apparently specifically ATIII-binding regions is unclear at the moment. Molluscs do not possess any blood coagulation system similar to that of mammals, yet their heparins are capable of dramatically accelerating the inactivation of mammalian coagulation enzymes by the mammalian protease inhibitor ATIII. It is possible that clam heparin is designed to interact with an endogenous antithrombin-like protease inhibitor acting on serine protease target enzymes. The existence and function of such an enzyme system remain to be established. Heparin is released from the mast cells in response to specific inflammatory agents, such as IgE antibodies or complement fragments (anaphylatoxins). Indeed, a series of observations suggest that heparin may serve as a modulator of cellular immunological reactions or other defense mechanisms. In mammals, the heparin-containing mast cells are accumulated in lymphoid organs and in tissues exposed to the external milieu (skin, lungs, intestine), and one suggested role for this polysaccharide in mammalian is to fight external parasites (Straus et al., 1982). The findings that heparin is present in molluscs, which apparently have no immune response, leads us to support the hypothesis that this macromolecule could function as a mechanism for the surveillance of these organisms against certain pathogens.
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Materials and methods |
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Preparation of T. phylippinarum heparin
Adult specimens of the bivalve mollusc T. phylippinarum were procured from a local market and immediately killed and the shell removed. The body (112 g) was defatted by grinding with 10 volumes of acetone, filtration, and drying at 60°C for 24 h. The pellet (
14 g) was solubilized (1 g/20 ml) in 100 mM Na-acetate buffer, pH 5.5, containing 5 mM ethylenediamine tetra-acetic acid and 5 mM cysteine. Papain (100 mg per g of tissue) was added and the solution incubated for 24 h at 60°C in a stirrer. After boiling for 10 min, the mixture was centrifuged at 5000 x g for 15 min, and three volumes of ethanol saturated with sodium acetate were added to the supernatant and stored at 4°C for 24 h. The precipitate was recovered by centrifugation at 5000 x g for 15 min and dried at 60°C for 6 h. The dried precipitate was dissolved in 10 ml of 50 mM NaCl. After centrifugation at 10,000 x g for 10 min, the supernatant was applied to a column (2 cm x 40 cm) packed with QAE Sephadex A-25 anion-exchange resin equilibrated with the same NaCl solution.
Glycosaminoglycans were eluted with a linear gradient of NaCl from 50 mM to 1.2 M from 0 to 150 min using low-pressure liquid chromatography (Biologic LP chromatography system from BioRad, Hercules, CA) at a flow of 1 ml/min. Two volumes of ethanol were added to the collected fractions corresponding to single species of polysaccharides evaluated by uronic acid (Bitter and Muir, 1962) assay and agarose gel electrophoresis (Volpi, 1993
; Volpi and Maccari, 2002
) and polysaccharides precipitated at 4°C. After centrifugation at 10,000 x g for 10 min, the pellet was dried at 60°C and solubilized in 20 mM Tris-Cl buffer, pH 8.0, containing 2 mM MgCl2 and treated with DNAse I (750 mg) at 37°C for 12 h. After boiling for 5 min, NaCl concentration was brought to 16%, and the glycosaminoglycans were precipitated by adding 80% methanol. The dried precipitate was dissolved in 50 mM Tris-Cl buffer, pH 7.9, and treated with 1 U chondroitinase ABC at 37°C for 12 h. After boiling for 5 min, NaCl concentration was brought to 16%, and the glycosaminoglycans were precipitated by adding 80% methanol. The recovered precipitate was solubilized in 20 ml bidistilled water, dialyzed overnight at 4°C, and freeze-dried. Approximately 30 mg of heparin were recovered.
Chemical and spectroscopic characterization
The percentage of slow-moving and fast-moving heparin species was determined by agarose-gel electrophoresis (Volpi, 1993; Volpi and Maccari, 2002
). The molecular mass and polydispersity of the heparin were determined by PAGE (Edens et al., 1992
) and HPSEC by using heparin of known molecular mass (Volpi and Bolognani, 1993
).
The 1H-NMR spectra of heparin were recorded with a Bruker AMX400 Wb spectrometer operating at 400.13 MHz. The sample was previously lyophilized three times with D2O (99.96 atom %). Heparin samples were at 1.0 mM, and all spectra were obtained at 29°C. The 1H chemical shifts (d) were quoted with respect to external sodium 4,4-dimethyl-4-silapentene-1-sulfonate (0.0 ppm). The assignment of the signals was made according to Casu et al. (1996) and Linhardt et al. (1992b).
The optical rotation ([]D) of T. phylippinarum heparin was measured by polarimetry at c = 1 mg of heparin/ml of water and l = 10 cm.
Oligosaccharide mapping
T. phylippinarum heparin (0.1 mg in 100 µl of 50 mM pH 7.3 acetate buffer containing 25 mmol calcium acetate) was treated for 12 h at 37°C with 15 mU heparinase, after which it was frozen and stored at 70°C.
Oligosaccharides defined 18 according to Linhardt et al. (1992a) produced by the action of heparinase were separated and quantified by SAX-HPLC separation at 232 nm using a 5-µm Spherisorb SAX column (150 x 4.6 mm from Phase Separations, Deeside Industrial Park, Deeside Clwyd, UK). Isocratic separation was run from 0 to 5 min with 50 mM NaCl, pH 4.00, and linear gradient separation was from 5 to 90 min with 100% 50 mM NaCl, pH 4.00, to 100% 1.2 M NaCl, pH 4.00, at a flow of 1.2 ml/min, as previously reported (Volpi, 2003
). The structures of 18 have been fully established (Linhardt et al., 1989
, 1992a
; Loganathan et al., 1990
; Rice and Linhardt, 1989
) and are as follows:
Major species were identified and quantified on the basis of their comigration with oligosaccharide standards prepared according to Linhardt (Linhardt et al., 1989, 1992a
; Loganathan et al., 1990
; Rice and Linhardt, 1989
) and the structure established by comparison with disaccharide standards after treatment with heparin lyase I (EC 4.2.2.7), heparin lyase II (no EC number), and heparan sulfate lyase (EC 4.2.2.8) and SAX-HPLC separation. The major peaks were first assigned by the coinjection of oligosaccharide standards, and the peaks were integrated to obtain the oligosaccharide composition. The disaccharide composition was calculated from the oligosaccharide map with
UA2S being assigned to
-L-IdoAp2S consistent with the known specificity of heparinase (Linhardt et al., 1990
).
Anticoagulant properties
The APTT and the amidolytic anti-factor Xa assay of T. phylippinarum heparin in comparison with bovine mucosal heparin were determined by methods previously described (Bianchini et al., 1982; Dietrich et al., 1985
). A standard curve was prepared for each test by using the 3rd International Heparin Standard, and the heparin samples to be tested were diluted so that their activities fell within the standard curve range. Specific activities were calculated as units per milligram. The concentration of heparin used in these bioassays was estimated by carbazole (Bitter and Muir, 1962
), agarose-gel electrophoresis (Volpi and Maccari, 2002
), and HPSEC (Volpi and Bolognani, 1993
) assays.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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