Glycobiology Advance Access originally published online on December 22, 2007
Glycobiology 2008 18(3):225-234; doi:10.1093/glycob/cwm136
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Antithrombin activity and disaccharide composition of dermatan sulfate from different bovine tissues
2 CSIRO, Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Queensland 4067
3 CSIRO, Food Futures Flagship, 5 Julius Ave, Riverside Corporate Park, North Ryde, New South Wales 2113
4 CSIRO, Food Science Australia, 671 Sneydes Road, Werribee, Victoria 3030, Australia
1 To whom correspondence should be addressed: Tel: +617-3214-2274; Fax: +617-3214-2900; e-mail: Simone.Osborne{at}csiro.au
Received on September 21, 2007; revised on November 16, 2007; accepted on December 13, 2007
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Abstract |
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Dermatan sulfate is a glycosaminoglycan that selectively inhibits the action of thrombin through interaction with heparin cofactor II. Unlike heparin it does not interact with other coagulation factors and is able to inhibit thrombin associated with clots. This property has made dermatan sulfate an attractive candidate as an antithrombotic drug. Previous studies have showed that dermatan sulfate derived from porcine/bovine intestinal mucosa/skin or marine invertebrates is capable of stimulating heparin cofactor II-mediated thrombin inhibition in vitro. This biological activity is reported for the first time in this study using dermatan sulfate derived from mammalian tissues other than intestinal mucosa or skin. Ten different bovine tissues including the aorta, diaphragm, eyes, large and small intestine, esophagus, skin, tendon, tongue, and tongue skin were used to prepare dermatan sulfate-enriched fractions by anion exchange chromatography and acetone precipitation. Heparin cofactor II/dermatan sulfate-mediated thrombin inhibition measured in vitro revealed activity comparable to or higher than the commercial standard with 2-fold differences observed between some tissues. Analysis of the extracted dermatan sulfate using fluorophore-assisted carbohydrate electrophoresis revealed significant differences in the relative percentage of all the mono-sulfated disaccharides, in particular the predominant mammalian disaccharide uronic acid
N-acetyl-D-galactosamine-4-O-sulfate, confirming previous reports regarding variations in sulfation in dermatan sulfate from different tissues. Overall, these findings demonstrate that dermatan sulfate extracted from a range of bovine tissues exhibits in vitro antithrombin activity equivalent to or higher than that observed for porcine intestinal mucosa, identifying additional sources of dermatan sulfate as potential antithrombotic agents. Key words: Bovine / dermatan sulfate / disaccharide / heparin cofactor II / thrombin inhibition
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Introduction |
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Dermatan sulfate (DS), also known as chondroitin sulfate B (CSB), is a linear polysaccharide of variable length composed of alternating disaccharide units containing a hexuronic acid that is either D-glucuronic (GlcA) acid or L-iduronic acid (IdoA), and the hexosamine, N-acetyl-D-galactosamine (GalNAc) (reviewed in Trowbridge et al. 2002
). These alternating disaccharide units can be variably O-sulfated at the C-2 position in IdoA (IdoA2SO3) and/or the C-4 (GalNAc4SO3) and/or C-6 (GalNAc6SO3) positions in GalNAc (Halldorsdottir et al. 2006), giving rise to eight different states of disaccharide sulfation.
DS is synthesized as a glycosaminoglycan (GAG) side chain that is covalently bound through O-xylose linkages to specific serine residues in the protein core of several dermatan sulfate proteoglycans (DSPG) like decorin, biglycan, thrombomodulin, endocan, epiphycan, and versican (reviewed in Trowbridge et al. 2002). The proteoglycans, through either the DS side chain or the protein core, bind a diverse range of molecules, including matrix molecules, growth factors, protease inhibitors, cytokines, and chemokines. These binding interactions implicate DS/DSPG as having a role inhibiting coagulation, enhancing extracellular matrix stability, reducing inflammation, and stimulating cell proliferation (Trowbridge et al. 2002). Biological activities specifically assigned to DS include the promotion of Fibroblast Growth Factor-2 (Penc et al. 1998), Fibroblast Growth Factor-7 (Trowbridge et al. 2002), hepatocyte growth factor/scatter factor activity (Deakin and Lyon 1999), tenascin-X-mediated stability of a connective tissue (Elefteriou et al. 2001) and heparin cofactor II (HCII)-mediated thrombin inhibition.
Heparin cofactor II is one of the serine protease inhibitors (or serpins) involved in the blood coagulation cascade that regulates blood clotting and fibrinolysis. HCII selectively inhibits the action of the serine protease, thrombin, in the presence of different polyanions, including the glycosaminoglycans DS, heparan sulfate and heparin, and other polysulfates, polyphosphates, and polycarboxylates (reviewed in Pike et al. 2005). In the presence of glycosaminoglycans, HCII-mediated inhibition of thrombin occurs via the formation of a HCII/thrombin/glycosaminoglycan complex whereby the glycosaminoglycan binds to a basic (GAG-binding) domain within HCII that displaces the HCII N-terminal acidic peptide sequence causing it to unfold from the GAG binding domain and interact with thrombin (Baglin et al. 2002; Casu et al. 2004). Formation of this ternary complex generally occurs at the cell surface; however, if the thrombin is bound to fibrin or at the surface of an injured blood vessel, this complex forms more effectively with DS than with any other glycosaminoglycan including heparin (Bendayan et al. 1994). This biological activity, along with the selective inhibition of thrombin through HCII without interaction with other coagulation factors, deems DS an attractive candidate as an antithrombotic drug (Nenci 2002) and highlights the importance of revealing alternative sources of DS capable of stimulating HCII-mediated thrombin inhibition.
DS/heparin cofactor II-mediated thrombin inhibition has been demonstrated with DS from porcine intestinal mucosa (Halldorsdottir et al. 2006), porcine skin (Maimone and Tollefsen 1990), and bovine lung (Ofosu et al. 1987). These studies have also revealed that the binding sites within DS exhibiting a high affinity for HCII contain repeating units of disulfated disaccharides. Variations in O-sulfation and in hexuronic acid content have also been found to significantly impact on the ability of DS to mediate HCII thrombin inhibition.
Highly and specifically sulfated DS from ascidians exhibits potent anticoagulation activities through HCII-mediated thrombin inhibition. Early studies by Ofosu et al. (1987) increased bovine lung DS sulfation 2-fold revealing that these over sulfated DS molecules significantly decreased the activity of thrombin in plasma. In these studies the precise locations of the sulfate groups were unknown; however, more recent studies investigating positional relevance revealed an increase in sulfation at specific positions enhances thrombin inhibition by DS more than an overall nonspecific increase in the level of sulfation. In vitro assays, measuring activated partial thromboplastin time (APTT) and HCII-mediated thrombin inhibition, showed that highly 2-O and 4-O (IdoA2SO3GalNAc4SO3) sulfated DS from Styela plicata and Halocynthia pyriforms were potent anticoagulants in comparison to DS from Ascidian nigra that was also highly sulfated but at positions 2-O and 6-O (IdoA2SO3GalNAc6SO3) (Pavao et al. 1998). DS from A. nigra had no measurable anticoagulant activity in the APTT assay and required concentrations 100-fold higher to inhibit thrombin activity via HCII, highlighting the importance of 2-O and 4-O sulfation in the DS catalysis of HCII-mediated thrombin inhibition.
Numerous other studies also confirmed the importance of 2-O and 4-O sulfation whilst further refining the sulfation requirements to 4-O and 6-O sulfation. HCII-Sepharose column affinity studies involving partially degraded DS from porcine intestinal mucosa showed that DS oligosaccharides ranging from hexa- to dodecasaccharides with an increasing GalNAc4,6SO3 content, bound with increasing affinity (Halldorsdottir et al. 2006). Additionally, deca- and dodecasaccharides with elevated GalNAc4,6SO3 contents displayed enhanced anticoagulant activities by increasing in vitro HCII-mediated thrombin inhibition and prolonging the activated partial thrombplastin time of normal pooled plasma (APTT assay). Overall, these studies indicate a requirement for repeating disaccharide units of either IdoA2SO3 GalNAc4SO3 or GlcA/ IdoA GalNAc4,6SO3 to be present within DS molecules capable of HCII-mediated thrombin inhibition (Halldorsdottir et al. 2006).
In this study, glycosaminoglycan fractions enriched with DS were prepared from bovine aorta, diaphragm, eyes, large and small intestine, esophagus, skin, tendon, tongue, and tongue skin, in order to identify mammalian sources of DS other than the commercial and commonly used bovine/porcine intestinal mucosa, capable of facilitating in vitro HCII-mediated thrombin inhibition. In vitro measurements revealed antithrombin activity from all of the various bovine DS preparations comparable to or higher than the commonly sourced porcine intestinal mucosa DS, subsequently identifying new bovine tissue sources of DS for potential commercial production and antithrombotic research.
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Results |
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Preparation of DS-enriched precipitates from bovine tissues
Total glycosaminoglycans were obtained from homogenized adult bovine tissues using papain/bromelain protease digestion, filtration, and anion exchange chromatography using Q Sepharose Big Beads. To establish the conditions for isolating DS, sequential acetone precipitations of the glycosaminoglycans from the aorta, diaphragm, large and small intestine, and tongue were separated using a modified 0.5% agarose/50 mM diaminopropane gel electrophoresis method (Figure 1) (Dietrich et al. 1977
). Comparisons with commercially obtained chondroitin sulfate A, B, and C and heparin standards revealed that the glycosaminoglycans precipitated out of the tissue extracts in a similar pattern to that described by Volpi in 1994 (Volpi 1994b) with heparin precipitating first, followed by DS and chondroitin sulfate A and C. DS precipitated from the 2% NaCl/GAG solution with 0.7 to 1.0 volumes of acetone with all of the 0.7 volume acetone precipitates of DS containing other sulfated carbohydrates that migrated similarly to the commercial heparin standard on the agarose gels.
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Samples of the 0.8 to 1.0 acetone volume DS precipitates were digested with chondroitin ABC and ACII lyase and their composition visualized again by agarose gel electrophoresis (Figure 2). DS extracted from the large intestine and tongue displayed the highest amount of precipitation at 0.9 volumes of acetone with minimal digestion by chondroitin ACII lyase and complete digestion with chondroitin ABC lyase, confirming a DS-enriched precipitate. DS extracted from the aorta, small intestine, and diaphragm displayed similar trends with DS precipitation occurring with 0.8 to 1.0 volumes of acetone. This precipitated DS was completely digested with chondroitin ABC lyase and partially digested with chondroitin ACII lyase, possibly indicating an increased content of glucuronic acid.
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From the results shown in Figures 1 and 2, extractions with 0.5 to 1.5 acetone volumes were performed on the remaining total GAG extracts from the eyes, esophagus, skin, tendon, and tongue skin. Precipitates from the 0.7–1.2 acetone volumes were selected for digestion with chondroitin ACII and ABC lyase and visualized using agarose gel electrophoresis (Figure 3). Precipitation of DS was observed from the eyes, tongue skin, tendon, and esophagus glycosaminoglycan extracts at 0.9 volumes of acetone whereas precipitation of DS from the skin total glycosaminoglycans occurred at 0.8 volumes of acetone. Overall, precipitation using 0.9 volumes of acetone ensured the precipitation of DS-enriched precipitates from all tissue total glycosaminoglycans with the only exception being the skin extracts. Where precipitation of DS occurred over multiple volumes of acetone (the aorta, diaphragm, and small intestine, Figure 2), the 0.9 volume precipitation was selected for further investigation as this protocol resulted in the bulk of the DS precipitation and would ensure the comparison of DS-enriched precipitates from the different tissues that would display similar properties, like charge density (Volpi 1994b) resulting from comparable sulfation states.
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In vitro heparin cofactor II-mediated thrombin inhibition from DS-enriched bovine tissue precipitates
Following preparation of the DS-enriched fractions from each tissue, the amount of sulfated glycosaminoglycan in the DS-enriched material was estimated using the BlyscanTM sulfated glycosaminoglycan assay and diluted to 1250 ng/mL, 125 ng/mL, and 12.5 ng/mL in order to determine the in vitro HCII-mediated thrombin inhibition from each DS-enriched tissue fraction. The kinetic assay (Dupouy et al. 1988
) revealed that HCII-mediated thrombin inhibition for all DS derived from bovine tissues was comparable with the commercial DS standard at all three concentrations (data not shown). In vitro activity from all tissue-derived DS (aorta, eyes, large and small intestine, skin, tendon, tongue, and tongue skin) is summarized in Table I.
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The highest activity for all sources was observed at the 1250 ng/mL concentration exhibiting between 94.2% and 97.3% inhibition in thrombin activity. The largest variations in activity were achieved at 125 ng/mL for all DS samples ranging from 41.5% to 88.8% thrombin inhibition. At this concentration, DS derived from the esophagus and the small intestine potentiated the largest inhibition of thrombin activity at 88.8% and 85.6% respectively, whilst the skin, tendon, and aorta tissue sources exhibited the lowest inhibition, ranging from 41.5% to 48.1%. The remaining tissues (tongue skin, eye, tongue, diaphragm, and large intestine) inhibited thrombin activity by 59.6% to 71.9%. Antithrombin activity at 12.5 ng/mL also displayed similar patterns ranging from 10.6% to 28.0% (Table I).
Inhibition of thrombin activity by the commercial standard from porcine intestinal mucosa at 1250 ng/mL and 125 ng/mL was 92.8% and 30.4% respectively (Table I). This demonstrated that all DS-enriched preparations from the alternative bovine tissues sources were capable of mediating in vitro HCII thrombin inhibition comparable to the commercial standard at 1250 ng/mL, and significantly higher than the standard at 125 ng/mL. At a DS concentration of 125 ng/mL, inhibition of thrombin activity from the DS-enriched preparations was up to 3-fold higher when compared to the commercial standard (ANOVA P < 0.0001). Inhibition of thrombin activity by DS-enriched preparations from the aorta, diaphragm, eyes, small and large intestine, esophagus, tongue, and tongue skin were significantly higher than the commercial DS standard (Tukey's multiple comparison test P < 0.05).
To control for possible contaminants in the DS-enriched preparations that may influence the observed in vitro HCII-mediated thrombin inhibition, equivalent amounts of sulfated GAG from each DS-enriched fraction were digested with chondroitin ABC lyase and assayed for in vitro activity. At 12.5 ng/mL and 125 ng/mL GAG concentration, inhibition in thrombin activity was below 20% and showed only significant variation in activity from the DS-enriched preparation obtained from the tongue and tongue skin (P < 0.05) as revealed by Tukey's multiple comparison test. However at the 1250 ng/mL concentration, antithrombin activity ranging from 20% to 35% was observed from the DS-enriched preparations obtained from the large and small intestine, skin, and tongue, and also from the commercial DS standard (ANOVA P < 0.0001). This observed residual activity from the chondroitin ABC lyase digested material indicates the presence of a contaminant other than chondroitin sulfate A, B (DS), or C. As shown in Figures 2 and 3, agarose gel electrophoresis of the DS-enriched fractions digested with chondroitin ABC lyase removed all visible glycosaminoglycan species that migrated similarly to the commercial chondroitin sulfate A, B, and C standards, however a slower moving species comparable to the commercial heparin standard is observable in the small and large intestine. With porcine intestinal mucosa a common source for heparin, the likely contaminants contributing to the unexplained thrombin inhibition are heparin or heparan sulfate. The presence of these apparent contaminants in the DS-enriched preparations from the small and large intestine, skin, and tongue, and the commercial DS are not thought to contribute significantly to the GAG concentration estimated in the DS-enriched fractions. However, it is important to note that the in vitro thrombin inhibition measured from these samples may not be entirely mediated by DS.
Determination of disaccharide composition in the DS-enriched precipitates using FACE
To determine the disaccharide composition in the DS-enriched precipitates from the various bovine tissues, fluorophore-assisted carbohydrate electrophoresis (FACE) was performed by modified methods as described (Calabro et al. 2000; Lehrman and Gao 2003). Chondroitin ABC lyase digested samples from each DS-enriched tissue fraction, along with the commercial unsaturated chondro/dermato/hyaluro-disaccharides, were derivatized with 2-aminoacridone, separated using 35% acrylamide gel electrophoresis, and visualized under UV light (Figure 4). The disaccharide composition was measured as the relative percentage of each detectable disaccharide within a particular sample and expressed as the mean of triplicate analyses ± the standard error (Table II).
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By far the most abundant disaccharide was the 4-O-monosulfated disaccharide (uronic acid
N-acetyl-D-galactosamine 4-O-sulfate) that was present in all of the prepared DS and ranged in relative percentage from 64.6% to 93.2% in the DS derived from the eye and the skin, respectively. The next most abundant disaccharide, also detectable in all DS preparations, was the 6-O-monosulfated disaccharide (uronic acidN-acetyl-D-galactosamine 6-O-sulfate) that ranged in relative percentage from 21.5% to 6.5% in the DS derived from the aorta and the tongue, with trace amounts (denoted as a relative percentage less than 4%) measured in the esophagus and skin. The remaining mono-sulfated disaccharide, iduronic acid-2-O-sulfateN-acetyl-D-galactosamine, was below detectable levels (comparable to background intensity) in all of the DS-enriched precipitates. The trisulfated disaccharide, iduronic acid-2-O-sulfateN-acetyl-D-galactosamine-4,6-O-sulfate, was also below detectable levels in all samples analyzed.
The disulfated disaccharide, iduronic acid-2-O-sulfateN-acetyl-D-galactosamine 4-O-sulfate, was also detected in all samples ranging from trace levels (<4%) up to 8.0% as measured in the DS-enriched precipitate from the esophagus. Trace levels of the remaining disulfated disaccharides, uronic acidN-acetyl-D-galactosamine 4,6-O-sulfate and iduronic acid-2-O-sulfateN-acetyl-D-galactosamine 6-O-sulfate, were only detected in the tendon and the esophagus-derived DS-enriched precipitates, respectively. These disaccharides were below detectable levels in all other DS-enriched precipitates.
Nonsulfated disaccharides were also detected in all samples, except in the DS-enriched precipitate from the tendon, and ranged in relative percentages from 4.5% to 14.4% as observed in the DS-enriched precipitates prepared from the tongue and diaphragm, respectively. Only trace amounts (<4%) were detected in the esophagus and skin-derived DS-enriched precipitates.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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Heparin cofactor II/dermatan sulfate-mediated thrombin inhibition in vitro has been demonstrated using mammalian DS isolated from multiple tissues and animal sources, including porcine intestinal mucosa (Halldorsdottir et al. 2006
), porcine skin (Maimone and Tollefsen 1990), and bovine lung (Ofosu et al. 1987). In terms of antithrombotic research, DS isolated from porcine intestinal mucosa tends to provide the commonly used DS (Nenci 2002; Casu et al. 2004; Vicente et al. 2004). It has been demonstrated that the structural characteristics of DS required for high affinity binding to HCII are repeating disaccharide units of either IdoA2SO3GalNAc4SO3 (Maimone and Tollefsen 1990) or GlcA/IdoAGalNAc4,6SO3 (Halldorsdottir et al. 2006). In an attempt to identify alternative mammalian sources of DS (other than porcine skin or intestinal mucosa) capable of in vitro HCII-mediated thrombin inhibition, we obtained DS-enriched preparations from bovine aorta, diaphragm, eye, large and small intestine, esophagus, skin, tendon, tongue, and tongue skin tissue using anion exchange chromatography and acetone precipitation. Agarose gel electrophoresis stained with toluidine blue (Figure 1) confirmed enrichment of DS using 0.8 to 1.0 volumes of acetone from the glycosaminoglycan samples prepared from the bovine tissues. The presence of DS was further confirmed by digestion with chondroitin ABC and chondroitin ACII lyase (Figures 2 and 3).
An in vitro kinetic assay (Dupouy et al. 1988) using the chromogenic substrate chromozym TH, allowed HCII/DS-mediated thrombin inhibition to be measured from all DS-enriched precipitates. The ability of the DS samples to reduce thrombin activity was expressed as the percentage decrease in thrombin activity compared to uninhibited thrombin activity (Table I). Almost all thrombin activity was inhibited at a glycosaminoglycan concentration of 1250 ng/mL for all samples including commercially obtained DS; however, the most significant variations were observed at 125 ng/mL where a 1.4- to 3-fold increase in thrombin inhibition was measured comparative to a commercial porcine intestinal mucosa DS. This indicated that all of the DS-enriched preparations from the different bovine tissues mediated HCII thrombin inhibition in vitro with efficacy comparable to or higher than the commercial DS at 1250 ng/mL and 125 ng/mL.
The antithrombin assay was also used to measure any detectable inhibition from the DS-enriched preparations and commercial DS, following digestion with chondroitin ABC lyase (Figures 2 and 3), revealing the presence of contaminants (other than chondroitin sulfate A, B, or C) with antithrombin activity. Activity was measured in all chondroitin ABC lyase digested material, particularly at the highest glycosaminoglycan concentration (1250 ng/mL) from the commercial DS sample, and from the DS-enriched preparations obtained from the small and large intestine, skin, and tongue. Heparin and heparan sulfate also mediate HCII thrombin inhibition (reviewed in Pike et al. 2005), and with heparin known to precipitate prior to DS during sequential acetone precipitation (Volpi 1994b), the contaminant is most likely to be heparin/heparan sulfate. Trace amounts of a slower moving glycosaminoglycan that migrated similarly to the heparin standard were also observed in the small and large intestine DS samples using agarose gel electrophoresis (Figure 2).
Following the observed differences in HCII-mediated thrombin inhibition by the DS-enriched preparations, investigations regarding the disaccharide composition were performed using FACE (Figure 4) to determine which disaccharides contributed to the observed in vitro activity. Significant variations in the predominant mammalian 4-O-sulfated disaccharide (GlcA/IdoAGalNAc4SO3) ranging from 65% to 95% were observed in the DS derived from all of the different tissues. FACE analysis also revealed that approximately 0–15% of the disaccharides were nonsulfated (GlcA/IdoAGalNAc), 5–20% were 6-O-sulfated (GlcA/IdoAGalNAc6SO3), and <4% (trace) to 8% were 2-O- and 4-O-sulfated (IdoA2SO3GalNAc4SO3) (Table II). Hence all of the DS-enriched precipitates contained up to 8% of the IdoA2SO3GalNAc4SO3 disaccharide, a disaccharide that represents approximately 5% of all disaccharides in DS prepared from porcine skin (Maimone and Tollefsen 1990) known to function as a high affinity HCII binding site (Halldorsdottir et al. 2006).
Interestingly the other disaccharide, GlcA/IdoA GalNAc4,6SO3, known to contribute to the HCII-mediated thrombin inhibition by DS isolated from porcine (Halldorsdottir et al. 2006) and bovine intestinal mucosa DS (Linhardt et al. 1994) was detected in only one of the bovine tissue extracts. The GlcA/IdoAGalNAc4,6SO3 disaccharide was found only in trace amounts (<4%) in the DS-enriched precipitate from the bovine tendon and in the commercially obtained DS (porcine intestinal mucosa) at a relative percentage of 6.4 ± 0.89% (data not shown). The relative percentage of this disaccharide in the remaining tissue-derived DS-enriched precipitates may be below the detectable level of the FACE analysis utilized in this study. Alternatively, the IdoA2SO3GalNAc4SO3 disaccharides may provide the high affinity binding sites in the bovine-derived DS for HCII, as the presence of the GlcA/IdoAGalNAc4,6SO3 disaccharide in the tendon-derived DS did not appear to enhance the HCII-mediated thrombin inhibition as measured in the in vitro assay. At 125 ng/mL, the tendon-derived DS decreased thrombin activity by 43% comparable only to the aorta, skin, and commercially derived DS, but significantly lower than the thrombin inhibition mediated by the remaining tissue-derived DS.
Surprisingly the variation in the relative percentage of the IdoA2SO3GalNAc4SO3 disaccharide within the different tissue-derived DS did not appear to correlate directly with the observed HCII-mediated thrombin inhibition. The IdoA2SO3GalNAc4SO3 disaccharide was present in the small and large intestine, esophagus, skin, and tongue-derived DS at comparable relative percentages (Table II); however, the HCII-mediated thrombin inhibition was significantly higher in the esophagus-derived DS compared to the large intestine, skin, and tongue-derived DS. This could infer that the placement of the IdoA2SO3GalNAc4SO3 (or GlcA/IdoAGalNAc4,6SO3) disaccharides within the DS chain may also contribute to the HCII-mediated thrombin inhibition by DS. Other studies have also indicated that the sequence of disaccharides (Halldorsdottir et al. 2006), as well as the iduronic/glucuronic acid content (reviewed in Casu et al. 2004) may also contribute significantly to the ability of a DS molecule to facilitate HCII-mediated thrombin inhibition.
Numerous studies have investigated the structure of high affinity HCII binding sites in porcine skin and bovine and porcine intestinal mucosa-derived DS; however, it remains to be clarified which disulfated disaccharide (IdoA2SO3 GalNAc4SO3 or GlcA/IdoAGalNAc4,6SO3 or both) con- tributes most to HCII-mediated thrombin inhibition. Various studies implicate the IdoA2SO3GalNAc4SO3 disaccharide (Maimone and Tollefsen 1990), both disaccharides (Mascellani et al. 1993; Linhardt et al. 1994; Volpi 1994a; Halldorsdottir et al. 2006) or either disaccharide (Mascellani et al. 1994; Fabiana Alberto et al. 2007) as contributing to the HCII-mediated thrombin inhibition by DS. Hence, further fractionation of the bovine tissue-derived DS on the basis of size and charge would be required in order to elucidate the precise disaccharides and sequences responsible for the HCII-mediated thrombin inhibition.
Overall these studies demonstrate that a variety of bovine tissues provide bioactive sources of DS capable of stimulating HCII-mediated thrombin inhibition in vitro in a manner comparable to or higher than commercially obtained DS. These newly identified bovine tissue sources of DS may represent potential antithrombotic drug candidates for future research and development. Careful preparation of DS from these bovine tissues, by monitoring the presence of contaminant glycosaminoglycans, would enable the production of DS with a defined disaccharide composition and biological activity.
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Materials and methods |
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Materials
Unsaturated chondro/dermato/hyaluro-disaccharides (
Di-0S, Di-4S, Di-6S, Di-UA2S, Di-diSB, Di-diSD, Di-diSE, and Di-triS) and chondroitin ACII lyase (Arthrobacter aurescens) were from Seikagaku Corporation (Tokyo City, Japan). Chondroitin sulfate A (bovine trachea), chondroitin sulfate B (porcine intestinal mucosa), chondroitin sulfate C (shark cartilage), heparin (porcine intestinal mucosa), 2-Amino-9(10 H)-acridinone (AMAC), sodium cyanoborohydride, 1,3-diaminopropane, toluidine blue O, acetic acid (glacial), dimethyl sulfoxide, and chondroitin ABC lyase (Proteus vulgaris) were from Sigma-Aldrich (St Louis, MO). Bromelain and papain were from Enzyme Solutions (Croydon South, Australia). Q Sepharose Big Beads and agarose NA were from Amersham/GE Healthcare Life Sciences (England). Cetyl trimethyl ammonium bromide (CTAB) was from Ajax Chemicals (Sydney, Australia). Heparin cofactor II (human) and thrombin (alpha, human) were from USA Biological (Swampscott, MA). Chromzym TH was from Roche Diagnostics (Indianapolis). N,N,N',N'-tetra-methyl-ethylenediamine, ammonium persulfate and 40% acrylamide/bis solution 37.5:1 (2.6% C) were from Bio-Rad (Hercules, CA). The BlyscanTM-sulfated glycosaminoglycan assay was from Biocolor Ltd (Newtonabbey, Northern Ireland). All other reagents were of analytical grade and were obtained from local suppliers.
Methods
Isolation of total glycosaminoglycans from bovine tissues.
Adult bovine tissues were removed post-slaughter using Australian Quarantine and Inspection Service (AQIS) approved procedures at Food Science Australia, Cannon Hill, Australia. The tissue samples were homogenized and suspended at 250 mg/mL (on the basis of wet weight) in 50 mM sodium phosphate buffer pH 8.0, and digested for 16 h at 55°C using papain and bromelain each at a final concentration of 10 mg/mL. Serial filtration was performed using 10 µM to 0.45 µM to remove any debris prior to anion exchange chromatography using Q Sepharose Big Beads. Approximately 200 mL of digested material (equivalent to approximately 45 g) was applied to the Q Sepharose column using 50 mM sodium phosphate pH 8.0 buffer. Anion exchange chromatography was achieved using a 2 M NaCl gradient. Fractions containing sulfated glycosaminoglycans were collected and pooled on the basis of their interactions with dimethylmethylene blue (Farndale et al. 1986) as determined by absorption at 525 nm. Pooled fractions were dialyzed using 12–14 kDa dialysis tubing to remove salt/buffer ions. A final glycosaminoglycan concentration was estimated using the BlyscanTM sulfated glycosaminoglycan assay. Aliquots of the pooled GAG extracts equivalent to 2 mg were lyophilized in preparation for acetone precipitation.
Acetone precipitation of individual glycosaminoglycans from total glycosaminoglycans isolated from bovine tissue.
Individual glycosaminoglycans were sequentially extracted from the freeze-dried material using acetone precipitation as previously described (Volpi 1994b). Briefly, aliquots equivalent to 2 mg of total glycosaminoglycans were lyophilized and reconstituted in 1 mL 2% NaCl. Increasing volumes of acetone (ranging from 0.2 to 1.8 volumes) were added to each solution to individually fractionate the different glycosaminoglycan species. For each acetone volume precipitation, the mixtures were placed at 4°C for 2 h prior to centrifugation at 9000 x g for 10 min. Both the precipitated glycosaminoglycan pellet and the supernatant were retained. The supernatant had additional acetone added for the following sequential precipitations and each pellet was resuspended in 100 µL of sterile deionized water.
Identification of individual glycosaminoglycans using chondroitin ABC/ACII lyase treatments and agarose gel electrophoresis.
To identify the glycosaminoglycans in the acetone precipitates and to observe the relative abundance of the different glycosaminoglycan species from each tissue, 20 µL of each resuspended acetone precipitate was digested with 10 mU chondroitinase ACII/ABC lyase in 50 mM Tris–HCl pH 8/60 mM sodium acetate/0.02% w/v BSA (final volume 45 µL) at 37°C for 16–18 h. The glycosaminoglycans (with/without lyase treatment) were visualized by diaminopropane agarose gel electrophoresis as previously described (Dietrich et al. 1977) with modifications. Briefly, 10 µg of standard chondroitin sulfate A, B, C, and heparin, 15 µL of ABC/ACII lyase-digested material, and 6.7 µL of undigested material were applied to a 0.5% agarose/50 mM diaminopropane gel (adhered to GelBond Film, Cambrex Bio Science Rockland Inc., Rockland, ME) in 50 mM diaminopropane for 90 min at 80 V. The samples were fixed 16–18 h in 0.1% w/v CTAB before the agarose gel was dried, stained with 0.1% toluidine blue/50% (v/v) ethanol/1% (v/v) acetic acid solution and the stained gel scanned on a desktop scanner (hp scanjet 3670).
Heparin cofactor II-mediated thrombin inhibition by DS-enriched precipitates.
Heparin cofactor II (HCII)-mediated thrombin inhibition was measured in a 96 well plate kinetic assay as previously described (Dupouy et al. 1988), with modifications. Briefly, 3 µL 680 nM HCII, 21 µL 0.02 M Tris–HCl pH 7.4/0.15 M NaCl/PEG 1 mg/mL, and 3 µL of 0.075–0.00075 mg/mL (final concentration 1250–12.5 ng/mL) DS-enriched preparations or commercial standard (concentration determined using the BlyscanTM sulfated glycosaminoglycan assay) were mixed and incubated at room temperature for 2 min before 3 µL of 150 nM thrombin was added. This solution was mixed gently for 1 min prior to the addition of 150 µL of 1.9 mM Chromozym TH. The assay was incubated at 37°C for 40 min with the absorbance measured at 405 nm (Spectra max PLUS 384) at 2 min intervals. All samples with and without digestion with chondroitin ABC lyase were assayed twice in triplicate. The HCII-mediated thrombin inhibition by the DS-enriched precipitates/commercial standard was expressed as the percentage decrease in thrombin activity up to mid-log phase (0–10 min) of the kinetic assay. Each sample was assayed twice in triplicate and expressed as the mean ± the standard error.
Determination of disaccharide composition within DS-enriched precipitates using FACE.
The disaccharide composition of the DS-enriched precipitates from the various bovine tissues was determined using FACE as described by Calabro et al. (2000) and Lehrman and Gao (2003), with modifications. Disaccharides were prepared by digesting the DS-enriched preparations to completion using chondroitin ABC lyase (as confirmed by agarose gel electrophoresis). Approximately 20 µg (or 48 nmol) of chondroitin ABC lyase-digested DS or a mixture of all unsaturated chondro/dermato/hyaluro-disaccharides (6 nmol of each unsaturated disaccharide) were lyophilized until dry in a vacuum dessicator. The samples were derivatized in 20 µL of 12.5 mM (240 nmol) 2-aminoacridone in 85% DMSO (v/v)/15% (v/v) acetic acid at room temperature for 15 min followed by the addition of 20 µL of 1.25 M sodium cyanoborohydride (25 µmol) for 16–18 h at 37°C (in the dark). All derivatized samples were analyzed immediately by acrylamide electrophoresis or stored in the dark at –80°C until required. Derivatized disaccharides (4 µL or 5 nmol) were analyzed by electrophoresis on gels comprising a 4% acrylamide/bis solution (37.5:1)/0.07 M Tris–HCl pH 6.7 stacking gel and a 35% acrylamide–bis (37.5:1)/0.25 M Tris–HCl pH 8.5 resolving gel. Electrophoretic separation was performed in the dark at 4°C using 0.025 M Tris/0.192 M glycine running buffer at 25 mA (500 V) for approximately 2 h. Electrophoresis was terminated when 0.05% bromophenol blue, an anionic dye that migrated similarly to the fastest moving disaccharide standard (Di-triS), reached the bottom of the gel. The FACE gels were visualized under UV light (230 nm) using the Gel Doc 2000 (Quantity One-4.0.3, Bio-Rad). The migration distance and intensity of the fluorescently labeled disaccharides was measured using Diversity Database 2.1.0 software (Bio-Rad). Each bovine-extracted disaccharide was compared with the commercial unsaturated chondro/dermato/hyaluro-disaccharide standards. The disaccharide composition was expressed as the relative percentage of each detectable disaccharide within a particular DS-enriched precipitate. Each sample was analyzed by FACE in triplicate and expressed as the mean relative percentage ± the standard error. Relative percentages less than 4% are denoted in the text as trace amounts. These trace levels represented the limits of disaccharide detection that were difficult to distinguish above background intensity. Relative percentages expressed as zero represented disaccharide levels that were indistinguishable from the background levels of intensity measured in the gels.
Statistical analysis.
All statistical analyses were conducted using a one-way ANOVA followed by post hoc comparisons using Tukey's multiple comparison test. These calculations were performed using GraphPad Prism 5 Software for Windows (GraphPad Software, San Diego CA, www.graphpad.com).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We would like to acknowledge the laboratory staff at Food Science Australia (Cannon Hill, Queensland, Australia) for their expertise and technical assistance. We would also like to acknowledge Gregory S. Harper for his inspiration and knowledge. This work was supported by the Food Futures Flagship (a CSIRO initiative) and the Division of Livestock Industries (CSIRO).
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Abbreviations |
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APTT, activated partial thromboplastin time; CSA, Chondroitin sulfate A; CSB, Chondroitin sulfate B; CSC, Chondroitin sulfate C; CTAB, Cetyl trimethyl ammonium bromide; DS, dermatan sulfate; DSPG, dermatan sulfate proteoglycans; FACE, fluorophore-assisted carbohydrate electrophoresis; GalNac, N-acetyl-D-galactosamine; GlcA, D-glucuronic acid; HCII, heparin cofactor II; Ido, L-iduronic acid;
Di-0S, GlcA/IdoAGalNAc; Di-4S, GlcA/IdoA GalNAc4SO3; Di-6S, GlcA/IdoAGalNAc6SO3; Di-UA2S, IdoA2SO3GalNAc; Di-diSB, IdoA2SO3 GalNAc4SO3; Di-diSD, IdoA2SO3GalNAc6SO3; Di-diSE, GlcA/IdoAGalNAc4,6SO3; Di-triS, IdoA2SO3 GalNAc4,6SO3 |
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