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Relationships between chemical characteristics and anticomplementary activity of fucans

Relationships between chemical characteristics and anticomplementary activity of fucans
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  ELSEVIER Eiomaterialsl7 1996)597403 0 1996 Elsevier Science Limited Printed in Great Britein. All rights reserved 0142-9612/96/ 15.00 zyxwvutsrqp Relationshks between chemical character&s and anticomplementary activitv of fucans J zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Catherine Blondin, Frederic Chaubet, Alain Nardella, Corinne Sinquin* and Jacqueline Jozefonvicz Laboratoire de Recherches SW /es MacromolBcules CNRS URA 502 IFREMER URM2 lnstitut GalilBe UniversiM Paris Nord Av. J.B. CIBment 93430 Villetaneuse France; ‘IFREMER B.P. 1049 Rue de /‘l/e d’Yeu 44037 Nantes Cedex 01 France We have shown previously that a low-molecular-weight fucan extracted from the brown seaweed Ascophyllum nodosum strongly inhibited human complement activation in Vitro and its mechanism of action was largely elucidated. We further investigated the influence of molecular weight and chemical composition of fucan on its anticomplementary activity. The capacity of 12 fragments of fucan (ranging from a molecular weight of 4100 to 214000) to prevent complement-mediated haemolysis of sheep erythrocytes (classical pathway) and of rabbit erythrocytes (alternative pathway) increased with increasing molecular weight, and reached a plateau for 40000 and 13500, respectively. The most potent fucan fractions were 40-fold more active than heparin in inhibiting the classical pathway. They were, however, as active as heparin in inhibiting the alternative pathway. In addition, we have developed a haemolytic test based on the CH50 protocol, which allows discrimination between activators and inhibitors of complement proteins. Although the mannose content within the different fucan fragments did not vary, the galactose and glucuronic acid contents increased with increasing activity, suggesting that these residues should be essential for full anticomplementary activity. Meanwhile, sulphate groups appeared to be necessary, but were clearly not a sufficient requirement for anticomplementary activity of fucans. Taken together, these data illustrate the prospects for the use of fucans as potential anti-inflammatory agents. Keywords: Fucans sulphated polysaccharides heparin-like complement Received 20 November 1994; accepted 15 May 1995 Complement activation, regulated at multiple levels by plasma and cells, leads to biological activities that depend on interactions between complement proteins or activated fragments and specific receptors on cells. Interaction of the complement proteins with polymers specifically developed for clinical use, such as extracorporeal or implanted devices, is one of the major elements of biomaterial biocompatibilityl. Complement activation by a foreign surface occurs mainly through the alternative pathway and depends on the chemical and physical properties of the surfaceszS 3. On one hand, efforts have been made in designing immunocompatible polymers based on the analysis of surface chemical composition4* or on the grafting of chemical groups on a polymer backbone7S8. On the other hand, several studies have focused on developing molecules that could inhibit complement activation. Synthetic inhibitors of both classical and alternative pathways have been described elsewhere*l’. Many of Correspondence to Dr F. Chaubet. these compounds, however, are toxic and most of them require relatively high concentrations for inhibiting complement activation both in vitro and in vkd2. Numerous natural or semi-synthetic polyanions, such as complestatin13, derivatized dextrans14, chondro’itin heparin 17-lg, sulphate, dextran sulphate15,16 and have been found to inhibit complement activation. The ability of heparin, an anticoagulant glycosaminoglycan, to prevent formation of human t alternative C3 convertase is independent of antithrombin binding activity and requires O- sulphation and N-substitution of the molecule’7. The heparin-like anticoagulant activity of fucans has been demonstrated previouslyzSz2. Recently, we have found that a low-molecular-weight fucan with a low anticoagulant activity inhibits the early steps of both classical and alternative pathway activationz3. There were many parallels between the stages of complement inhibited by fucan and heparin; however, some significant differences were observed. In particular, the molar concentration of fucan required to inhibit classical pathway activation in whole serum was 597 Biomaterials 1996, zyxwvutsrqponmlkjihgfe ol 17 No. 6  598 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA nticomplementary activity of fucans: C. Blondin et al. fivefold lower than that of heparin. Additionally, in Elemental analyses of sulphur and nitrogen were contrast to heparin, fncan did not inhibit the terminal performed according to the Kjeldahl method by the components of complement. CNRS (Vernaison, France). This paper investigates the influence of molecular weight and chemical composition on the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA n vitro anticomplementary potency of fucan fractions. We present evidence that the capacity of fucan to prevent activation of both classical and alternative pathways in whole serum is correlated to the molecular weight of fucan fragments. The molar ratios of xylose, galactose and glucuronic acid increase with increasing anticomplementary activity, suggesting that these residues should be essential for full anticomplementary capacity. However, the presence of sulphate groups is not a sufficient condition for anticomplementary activity of fucans, in contrast to the anticoagulant activity of fucans. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Gas chromatographic analysis of monosaccharide derivatives MATEXIALS AND METHODS Reagents and materials Veronal-buffered saline (VBS) containing 0.15 mM Cazf and 0.5mM Mg2+ (VBS’+), VBS containing 0.1 gelatin (GVB) and GVB containing 2mM Mgzf and 8mM EGTA (GVB-Mg-EGTA) were prepared as described previouslyz4. Heparin HlO8 (sodium salt, from hog intestin mucosa, specific anticoagulant activity 173 IUmg-‘) was obtained from SANOFI (Suresnes, France) and T40 dextran (batch 32202, A4, 43 900, M, 26 200, average M, 40000) from Pharmacia Fine Chemical (Saint Quentin, France). Sephadex G-25-80 medium was purchased horn Sigma Chemical Company (St Louis, MO, USA). Normal human serum (NHS) was prepared from a pool of freshly collected blood horn healthy volunteers. Antibody-sensitized sheep erythrocytes (EAs) were prepared by incubating sheep erythrocytes with haemolysin (Cordis Laboratories, Miami, FL) as described previouslyz5. Rabbit erythrocytes (Ers) were obtained horn blood drawn from the ear vein of New Zealand White rabbits into cold Alsevers’ buffer containing 0.01 sodium azide. Collection and purification of fucan fractions Crude fucan was extracted from brown seaweed Ascophyllum nodosum and was hydrolysed as reported previously”. Hydrolysed fucan was fractionated on a 25.2cm x 55 cm column (271) of Sephacryl S-300HR (Pharmacia Fine Chemical). The void volume and the total volume of the column were determined by chromatography of dextran (MW = 5 x 105) and potassium phthalate (MW = 204), respectively. Approximately 25 g of hydrolysed fucan, dissolved in 500ml of 0.2 M NaCl, was applied to the column. Twelve fractions were collected according to differential refractive index variation, desalinated by extensive diafiltration against bidistilled water and freeze-dried. Chromatographic molecular weights M,s of fucan fractions were determined by high- performance steric exclusion chromatography (HPSEC) as described previously” using pullulans (Polymers Laboratories, Interchim, Paris, France) as standards. Contents in fucose, mannose, xylose, galactose and gluruconic acid units were determined by gas chromatography. Each fucan fraction (1Omg) was hydrolysed in a sealed tube in 2N trifluoroacetic acid (TFA). Ten milligrams of BaClz were added to induce total desulphation of polysaccharides. Hydrolysis was first performed for 3 h at 12O”C, then for 1 h at 100°C. The latter gave a fucoselxylose ratio which corrected the sugar composition obtained with the former. TFA was evaporated to dryness under vacuum and the residue was dissolved in 2ml of distilled water. To convert the uranic residues into their barium salts, the solution was treated with solid BaC03 at 65°C for 10min. The suspension was filtered, the solid was rinsed with 3 ml of distilled water, 50mg of solid NaBH4 were added and the solution was stirred for 3 h. The excess of NaBH4 was neutralized with a few drops of glacial acetic acid. The final solution was eluted on a short column (6cm x 0.6cm) of strong cation exchanger (Amberlite IR 120 H+). The combined eluate and washings from the column were evaporated to dryness under reduced pressure and the boric acid was removed as volatile methyl borate by several distillations with dry methanol. The dry residue was dissolved in 0.5 ml of concentrated HCl and the solution was quickly dried. The final residue was dissolved in 0.5 ml of dry pyridine, 50~1 of HMDS and 25~1 of TMCS. The silylated alditols obtained were analysed on a Fison GC8000 gas chromatograph. Separation was carried out on a 25m DB-1 bonded phase fused silica capillary column (0.25 pm coating thickness, 0.245 mm diameter) with the following temperature program: after injection of sample (0.1 pl), the oven temperature was kept at 90°C for 2min, then steadily increased to 160°C at a rate of 12°C min-l, from 160 to 200°C at 2°C rnin~l and from 200 to 250°C at 10°C min-‘. The program ended after 4min at 250°C. Injector and detector (FID) areas were kept at 280°C. The retention times of unknown persilylated alditols were correlated with those of standards run under identical conditions. Complement assays Anticomplementary activity of fucan was first assessed in the presence of Sephadex. One hundred microlitres of human serum were preincubated with 0, 50, 60 or 70 pg of fucan fraction S8 and with a sufficient amount of Sephadex particles, 7mg and 15 mg (obtained by crushing Sephadex beads), which activate the alternative pathway by 50 and 100 respectively, for 30min at 37°C under gentle agitation. Sephadex particles were spun by centrifugation and the residual CH50 units of supernatants were immediately measured by CH50 assay. During this second assay, fucan which remains in the supernatant continues to react with complement proteins. This activity was then measured in a control test without Sephadex Biomaterials 1996, Vol. 17 No. 6  Anticomplementary activity of fucans: C. Blondin et al. 599 zyxwvutsrq particles and was subtracted from the values obtained in the Sephadex-CH50 assay. The final anticomplementary activity was the result of the SephadexXH50 assay, from which the value of anticomplementary activity measured without Sephadex particles (control) was subtracted. i i The capacity of NHS to lyse EAs through the classical pathway and to lyse Ers through the alternative pathway was assessed in the absence or presence of fucan. For classical pathway activation (CH51) assay), 400 ~1 of VBS’+ containing 0-2O/*g of fucan were added to 400~1 of VBS2+ containing 2.5 ~1 of NHS. Two hundred microlitres of EAs at lo8 cellsml-’ were then added to initiate the activation and the mixture was incubated for 45 min at 37°C. The amount of NHS used in the experiment was sufficient to lyse 50-60 of 2 x lo7 EAs in the absence of fucan. The reaction was stopped by addition of 2ml of cold zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA .15~ NaCl. After centrifugation at 2200g for 5min, the optical density of the supernatant was measured spectro- photometrically at 414nm and is directly correlated to haemolysis. For alternative pathway activation (AP50 assay), fucans (0-1Opg) were added to 12~1 of NHS diluted in GVB-Mg-EGTA (238~1). This amount of NHS has been determined to lyse 60 of lo7 Ers in experimental conditions. One hundred microlitres of Ers at lOa cellsml-’ were then added and the mixtures were incubated for 1 h at 37°C. The reaction was stopped and the lysis of Ers was measured as described above. Elution volume (ml) vt zyxwvutsrqponml Figure 1 Steric exclusion chromatography of fucan. Column size: 25.2 cm x 55cm. The sample amount was 27 g and it was eluted with 0.2~ NaCl at a flow rate of 9750ml h-‘. The crude extract of fucan was fractionated in 12 fractions as indicated in the figure. per fucose unit. There was no significant variation in the molar ratios of mannose and 0-sulphate. As the zyxwvutsrqp C of fucan fractions increased, the molar ratios of xylose increased steadily from 0.15 to about 0.50. As for galactose and glucuronic acid residues, the molar ratios increased from 0.04 and 0.03 to about 0.15 and 0.10, respectively, and remained constant for MC above approximately 30 000. zyxwvutsrqponmlkjihgfedcbaZY RESULTS CH50 in the presence of Sephadex Preparative steric exclusion chromatography and gas chromatographic analysis of fucans To determine whether fucans are activators or inhibitors of the complement system, CH50 assay Twelve fractions of I& ranging from 4100 to 214000 was performed in the presence of an insoluble were obtained (Figure 1). The amount of polymer in activator of the alternative pathway, Sephadex the last two fractions (SlO and Sll) was not sufficient particleP. Fucan fraction S8 was found to inhibit to complete chemical analysis, presumably due to Sephadex-induced complement activation in a dose- losses during the diafiltration step. The results of gas dependent manner (Figure 2). This result showed chromatographic analysis (Table Z) have been fucans as true inhibitors of the complement system expressed as the molar ratio of xylose, galactose, in the sense that they prevent consumption of mannose, glucuronic acid and 0-sulphate groups complement proteins. Table 1 Chemical analysis of fucan fractions (molar ratios per fucose units) Fraction M,’ Fucose Mannose Xylose Galactose Glucuronic acid 0-Sulphate s2 120200 s3 95 000 s4 76 000 s5 46 800 S6 38 000 S7 22 600 S8 16600 s9 13500 SlO 12500 Sll 10000 s12 4100 s.d. 1000 1 oo - 1.00 1.00 1 oo 1.00 1.00 1.00 1.00 - 0.01 0.1 0.56 0.12 0.07 0.95 nd P nd nd nd nd 0.08 0.48 0.12 0.08 0.88 0.08 0.65 0.22 0.13 1.06 0.10 0.50 0.17 0.10 0.98 0.10 0.42 0.13 0.07 0.98 0.09 0.30 0.07 0.06 1.14 0.09 0.30 0.05 0.05 1.26 0.09 0.15 0.04 0.03 0.77 nd nd nd nd nd nd nd nd nd nd 0.01 0.01 0.01 <O.Ol 0.03 ‘MC, chromatographic molecular weight determined by HPSEC t nd, not determined. Biomaterials 1996, Vol. 17 No. 6  600 Anticomplementary activity of fucans: C. Blondin et a/. 15 mg Sephadex + Fucan zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA classical pathway by half under the same experimental conditions was 4.2 mg ml-‘. For the AP50 assay, lcso of the fucan fractions ranged from 0.56 per ml-’ of undiluted serum to 5.4 mgml-l. ICY,,, once again, decreased with increasing zyxwvutsrqponmlkjihgfedcbaZYX C and a plateau was reached for MC above 15 000 (Figure 4). The IC,, of heparin (0.6mgml-‘) was identical to that of the most active fucan fractions. The T40 dextran (average molecular weight 40000) was tested in the same experimental conditions, and no inhibitory effect was observed in both classical and alternative pathway haemolytic assays. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ Figure 2 Inhibition of Sephadex-induced complement activation by fucan fraction S8 (M, = 16000). The first bar on the left represents the percentage of CH50 units spontaneously consumed during experiments in the absence of Sephadex and fucan. The second bar corresponds to maximal activation of complement achieved in the presence of 15mg Sephadex particles. The third, fourth and fifth bars represent the haemolytic activity of serum (%) following its incubation with a fixed amount of Sephadex particles (15mg) and different concentrations of S8 fucan. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Effect of fucan on classical and alternative pathway activation We have investigated the influence of molecular weight on the capacity of fucan to inhibit classical (CH50 assay) and alternative (AP50 assay) pathway activation. Incubation of human serum with increasing concentrations of fucan resulted in a dose-dependent reduction of the complement-mediated lytic activity of serum, as measured by haemolysis of EAs (classical pathway; Figure 3A) or Ers (alternative pathway; Figure 3B). The anticomplementary activity of each fucan fragment was expressed as the concentration required to reduce the haemolytic complement activity by 50 IC,,). or the CH50 assay, the ~cso of the fucan fraction ranged horn 0.07mg per ml of undiluted serum to more than 25 mgml-‘. ~cso decreased with increasing MC and remained constant for MC above approximately 50000 Figure 4). The concentration of heparin HlO8 MC 14000) required to inhibit the 0 2 4 6 8 10 12 Concentration mg/ml of undiluted serum) DISCUSSION The present study investigated the influence of molecular weight and chemical composition on the in vitro anticomplementary activity of 12 fucan fractions of MC ranging from 4100 to 214000. We have first determined the capacity of fucans to prevent activation of the classical and alternative pathways in whole serum. Fucans were found to reduce complement- mediated lysis of EAs in NHS-VBS’+ (CH50 assay) and lysis of Ers in NHS-Mg-EGTA (AP50 assay) in a dose- dependent manner Figure 3). The effect of fucan could not be based on the alterations in osmolarity of the reaction mixture, since the dextran T~CI had no effect on complement-mediated haemolysis. The concentrations by which heparin inhibited 50 of EA lysis (4.2 mgml-‘) and Er lysis (0.60mgml-‘) are in agreement with data from the literaturez7. CH50 assay is a routine test commonly used to estimate the potency of compounds with respect to the complement system. Since these compounds are preincubated with serum before addition of cells, the consumption of CH50 units can result from: (i) activation of complement proteins and subsequent decrease in the level of native components able to react with cells, or (ii) inhibition of protein activation on the cell surface, leading to cell protection against complement-mediated lysis. In order to distinguish between these two possibilities for fucan activity, we have developed a test based on the CH50 protocol. zyxwvutsrqpon 0 2 4 6 8 Concentralion (mg/ml oP undiluted serum) Figure 3 Dose-dependent inhibition of the classical (A) and alternative B) pathway-mediated haemolysis by heparin H108 (A), T40 dextran (0) and fucan fractions of M, respectively equal to 22600 (A), 12500 (O), 10000 (m) and 4100 (0). Each point represents the mean of duplicate measurements. Biomaterials 1996. Vol. 17 No. 6  Anticomplementary activity of fucans: C. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA londin t /. 601 o,_p,,,,., -I--I---m-e- 0 25 50 75 100 125 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Chromatographic molecular weight (g/molx 10s3) Figure 4 Relationship between chromatographic molecular weight of the fucan fractions and inhibitory capacity (IC+,~) towards activation of classical (0) and alternative (m) pathways. Heparin H106 was tested in the same experimental conditions (0, 0). This test consists of preincubation of serum with both fucan and Sephadex particles (an activator of the alternative pathway)26, followed by elimination of Sephadex by centrifugation and CH50 analysis of the supernatant. When the results obtained are compared to a control containing Sephadex but no fucan, an increase in Sephadex-induced haemolysis would indicate that fucans act in the same way as does Sephadex. On the contrary, a decrease in Sephadex- induced haemolysis would result from the inhibition by fucan of Sephadex-induced haemolysis, indicating an inhibitory role of fucan on complement activation (Figure 2). The hypothesis of a direct interaction between fucan and Sephadex beads can be eliminated, since Sephadex is commonly used as a chromatographic support for separation of fucan and other anionic polysaccharides. The ‘Sephadex-CH50’ protocol presented here allows a qualitative but not quantitative estimation, and can permit one to distinguish between activators and inhibitors of the complement system. However, like the CH50 and Al’50 assays, this method imposes limits in the understanding of the compounds’ mechanism, since their molecular targets cannot be identified. Fucan fragments of molecular weight ranging from 4100 to 214000 were compared for their ability to inhibit lysis of EAs and Ers in whole serum (Table 2). For EA lysis, the inhibitory activity increased exponentially with molecular weight to become constant above 46600 (S5 fraction). The same was also observed for the inhibition of Er lysis, but the plateau was reached for molecular weight above 13 500. The S6 fragment, having a molecular weight equivalent to that of heparin on a molar basis (13 LOO), was more potent than heparin in inhibiting EA lysis (3.2 versus 4.2 mgml-‘) but was less efficient than heparin in inhibiting Er lysis (0.83 versus 0.60 mgml-‘). Furthermore, the S12 fragment (4100) although inhibiting the alternative pathway Table 2 Inhibition of classical (EA lysis) and alternative (Er lysis) complement pathways by fucans Sample M,’ Anticomplementary activity ICsO (mg per ml of undiluted NHS) EA lysis Er lysis Sl 214000 0.07 f 0.01 0.56 f 0.02 s2 120 200 0.12 * 0.02 0.56 f 0.02 s3 95 000 0.12 f 0.02 0.60 zt 0.02 S4 76 000 0.10 f 0.02 0.66 * 0.02 55 46 800 0.45 zt 0.03 0.66 f 0.02 S6 38 000 0.73 f 0.05 0.50 f 0.02 s7 22 600 0.88 f 0.04 0.50 f 0.02 S8 16600 1.20 I z 0.10 0.50 zt 0.02 s9 13500 3.20 zk 0.25 0.83 zt 0.05 SlO 12500 7.50 * 0.90 1.83 f 0.15 zyxwvutsrqpo Sll 10000 12.2 f 1.50 3.50 f 0.20 s12 4100 >25 5.40 f 0.60 Heparin H108 14000 4.2 f 0.30 0.60 i 0.02 ‘M chromatographic molecular weight determined by HPSEC. ICKY 5.4 mgml-l) was not able to inhibit classical pathway activation (I , > 25 mg ml-‘). The inhibitory activity of fucan suggests the existence of glycosidic sequences which are implicated in specific interactions between fucan and complement proteins. Based on the results observed in this study, having a low molecular weight fucan inhibiting the alternative pathway and not the classical pathway, we can postulate that the glycosidic regions of fucan involved in interactions with alternative and classical proteins may not be the same. Relationships between molecular weight and anticomplementary activity were also observed for dextrans substituted with benzylamine and benzyl sulphonate groups,‘* and for heparin27V28 as well. The influence of the molecular weight has also been implicated in the mechanism by which oligosaccharides inhibit the binding of factor H to C3b, leading to discrimination between complement- activating or non-activating surfaceszg. Sulphate groups had been reported to be an absolute requirement for anticomplementary17’27*30 as well as anticoagulant31 activity of heparin. The anticoagulant activity of fucan was shown to depend on the sulphate content of the molecule20332. Interestingly, our results showed that the sulphate content remained constant, while the capacity of fucan to inhibit EA and Er lysis increased (Tables z and 2). The presence of sulphate groups is not, however, a sufficient requirement for anticomplementary activity of fucan. Despite the increase in their anticomplementary activity, all fucan fragments had equivalent content of mannose residues. On the contrary, galactose and glucuronic acid molar ratios increased with increasing activity. Since molecular weight increases, it was not possible to determine which element is involved in the variation of anticomplementary activity. Analysis of fucan fractions, having only one variable parameter, would clarify this point. Among the anionic polysaccharides of vegetal srcin12, 33, 34 reported as ‘anticomplementary’ agents, a great number induced activation of complement proteins and consequently would deprive the patient Biomaterials 1996, Vol. 17 No. 6
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