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Efficacy and safety of i.v. dermatan sulphate (DS) and heparin (H) in controlling laboratory alterations due to DIC were compared in 10 patients with acute leukaemia, in a prospective, randomised pilot study. The time courses of the coagulation and fibrinolysis markers for DIC were similar in the two treatment groups except for activated partial thromboplastin time and thrombin time, which were prolonged in the H but not in the DS group. Blood product support tended to be greater in the H than in the DS group. DS appears to be as effective as H in controlling thrombin production during leukaemic cytolysis and may represent a safer alternative to H in the management of DIC in acute leukaemia.

The aim of this study was to set up a depolymerization process which resulted in the formation of a low molecular weight dermatan sulphate (LMWDS), retaining the chemical properties possessed by native dermatan sulphate (DS), fundamental for the expression of its specific biological activity. The depolymerization of DS by a β elimination process led to the production of oligosaccharide chains having a 4,5 unsaturated uronic acid at the nonreducing end. The chemical evaluation has shown that the most important parameters (degree of sulphation, sulphate to carboxyl ratio, and specific rotation) have not undergone any particular modification compared to native DS. The biochemical results demonstrate that the LMWDS obtained retains most, if not all, of the specific biological activity. The reduction in molecular weight significantly enhanced the bioavailability of the product after subcutaneous administration.

A structure-activity relationship of low molecular weight dermatan sulfate was undertaken to understand better this new non-heparin, glycosaminoglycan-based antithrombotic agent. A dermatan sulfate prepared from bovine intestinal mucosa [average molecular weight (MWavg) 25,000], and currently in clinical trials as an antithrombotic agent, was used in this study. Dermatan sulfate was partially depolymerized using hydrogen peroxide and copper(II) as catalyst to MWavg 5600 to obtain a low molecular weight dermatan sulfate. This low molecular weight dermatan sulfate was then fractionated by gel permeation chromatography to obtain four subfractions having MWavg 7800, 5500, 4200 and 1950. The dermatan sulfate, low molecular weight dermatan sulfate and its subfractions showed substantially different optical rotations. The 1H-NMR spectroscopic analysis of dermatan sulfate samples showed some differences including increased content of GalpNAc4S6S residues and improved resolution in ring resonances for low molecular weight dermatan sulfate fractions, primarily the result of reduced molecular weight and lowered heterogeneity. Saccharide compositional analysis relied on chondroitin ABC lyase treatment followed by capillary electrophoresis. Polyacrylamide gel-based oligosaccharide mapping was also performed by treating dermatan sulfate samples with chondroitin B, AC and ABC lysases. These analyses showed increased amounts of sulfation as the MWavg decreased. In vitro bioassay showed maximum anti-Xa activity in the 4.2 kDa fraction and maximum heparin cofactor II-mediated anti-IIa activity in the 5.5 kDa fraction. The in vivo antithrombotic activity of these fractions was measured using a modified Wessler stasis thrombosis model. The 4.2 kDa fraction showed greater antithrombotic activity than the other low molecular weight dermatan sulfate fractions, dermatan sulfate, and low molecular weight dermatan sulfate. This enhanced activity may result from several structural features of the 4.2 kDa fraction including: a high content of 4,6- and 2,4-disulfated disaccharide sequences; the requirement of specific chain length; a change in the ratio of iduronic to glucuronic acid; and the presence of chondroitin ABC lyase resistant material.

Synthesis of α-L-IdoA(2-SO3)-1→3-β-D-GalNAc(4-SO3)-(1→4)-α-L-IdoA(2-SO3)-{2-(1→3)-D-GalNAc(4-SO3), was carried out for the first time in a regio- and stereocontrolled manner by use of the trichloroacetimidate technology for the carbohydrate chain extension.

The synthesis of a “non-glycosamino” glycan counterpart (i.e. compound II ) of a naturally occuring dermatan sulphate hexasaccharide that binds with high affinity to heparin cofactor II is described.

A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.

In order to increase the ratio of anti-HIV activity to anticoagulant activity, glycosaminoglycan derivatives selectively substituted at OH and/or COOH groups were prepared. Standard heparin, heparin fragments, or dermatan sulfate were converted to their tributylammonium or tetrabutylammonium salts. Their selective O-acylation to various (controlled) degrees was carried out in a homogeneous way in N,N-dimethylformamide using carboxylic acid anhydrides and 4-(dimethylamino)pyridine as catalyst. Esterification of the COOH groups was performed by the addition of alkyl halide to an N,N-dimethylformamide solution of glycosaminoglycan tetrabutylammonium salts. The in vitro anticoagulant activity, the activity against HIV-1 and HIV-2 cytopathicity, the cytotoxicity, and the activity on the induction of giant cell formation were determined. O-acylation (O-butyrylation or O-hexanoylation) of the heparin fragments obtained by periodate depolymerization (compounds 2d and 2e), and their esters (compounds 7i and 7j), yielded products with very low anticoagulant effects in vitro, yet potent activity against both HIV-1 and HIV-2 induced cytopathicity, and low, if any, cytotoxicity. As compared to other anionic polysaccharides, these acylated derivatives are more active as inhibitors of HIV-induced giant-cell formation. Their anti-HIV activity is related to the degree of O-acylation and is mainly due to the inhibition of virus adsorption to the target cells.

Heparin from beef intestinal mucosa, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their structures and physico-chemical properties were evaluated by different techniques (disaccharide patterns by specific enzymatic cleavage, relative molecular mass by high-performance size-exclusion chromatography, sulfate-to-carboxyl ratio by potentiometric determination). Heparin was fractionated into "slow moving" and "fast moving" fractions by selective precipitation as the barium salt at different temperatures. The "fast moving" and "slow moving" components of heparin, dermatan sulfate, and chondroitin sulfate were utilized to run calibration curves in agarose-gel electrophoresis. Mixtures containing different amounts of these glycosaminoglycans were made and separated by agarose-gel electrophoresis, and these were analyzed quantitatively. For analysis of relative amounts, the area of each individual component of mixtures, obtained by photodensitometric readings, was divided by the sum of the areas of all glycosaminoglycans and expressed as a percentage. For analysis of absolute amounts, the area under the curve for each component of mixtures was fitted to specific calibration curves, and the amount of each glycosaminoglycan was calculated in micrograms. The quantitative procedure performed by analysing absolute amounts was used to obtain an accurate quantitative evaluation of each component in mixtures of glycosaminoglycans utilized for pharmaceutical purposes. A sensitive method was developed for the evaluation of very small amounts (0.2% w/w) of possible glycosaminoglycans as contaminants in preparations of a single species of glycosaminoglycan. This technique requires specific enzymatic degradation by bacterial lyases, separation in agarose-gel electrophoresis, and quantitative analysis by photodensitometric analysis and specific calibration curves.

Lyophilized mid-wall left ventricular myocardial tissues of the long-tailed non-human primate, Macaca fascicularis, were examined for the presence of glycosaminoglycans and chondroitin/dermatan sulfate proteoglycans. Mean uronic acid concentration in all samples was 0.97 +/- 0.27 micrograms per mg dry weight myocardium. The distribution of the glycosaminoglycans in the myocardium, determined by cellulose acetate strip electrophoresis was 62 +/- 4% heparan sulfate, 20 +/- 6% hyaluronan, and 16 +/- 5% chondroitin/dermatan sulfate. The analysis of chondroitin/dermatan sulfate proteoglycans, done directly on the extracts of lyophilized myocardium using agarose-acrylamide gel electrophoresis and Western blotting with monoclonal antibodies to various carbohydrate epitopes and with polyclonal antibodies to the protein core, showed the presence of biglycan and decorin. That these two and no other chondroitin/dermatan sulfates were present was established by core protein analysis using SDS PAGE and Western blotting. Quantification of chondroitin/dermatan sulfate proteoglycans uncovered high individual specific variability of the chondroitin/dermatan sulfate epitopes, but only moderate variability of biglycan and decorin core proteins. The variability of the chondroitin/dermatan sulfate epitopes is most likely related to individual specific differences in chain number, iduronate content and sulfation patterns of biglycan and decorin.

The interaction of interleukin 8 (IL-8) with heparin was studied by using synthetic IL-8 analogs with C- and N-terminal truncations. Elimination of the N-terminal region preceding the first cysteine, which constitutes the IL-8 receptor binding site, did not affect the affinity to heparin-Sepharose. Affinity, however, decreased with progressive truncation at the C terminus, and no binding was observed when the C-terminal alpha-helix was eliminated. The effect of heparin and other glycosaminoglycans on IL-8 activity was also tested. When IL-8 was applied together with heparan sulfate, neutrophil chemotaxis in vitro was enhanced up to 4-fold, and the stimulus-dependent increase in cytosolic free Ca2+ increased markedly in both rate and peak value. Heparin had a similar effect on the Ca2+ response but did not enhance chemotaxis. The glycosaminoglycans by themselves did not elicit neutrophil responses. Their enhancing effect was restricted to stimulation with IL-8 and was not observed when the unrelated chemoattractant fMet-Ile-Phe-Leu was used as the stimulus. Elastase released from stimulated neutrophils was inhibited by heparin, heparan sulfate, and, to a lesser extent, chondroitin sulfate B, confirming previous observations. Taken together, these results suggest that heparan sulfate, which is present on the endothelial cell surface and in the basement membrane, may have a dual function in diapedesis, promotion of IL-8-dependent transmigration of neutrophils, and protection of the tissue microenvironment from damage by lytic enzymes released from the migrating cells.

Lipoprotein lipase (lipase), a key enzyme in lipoprotein triglyceride metabolism, has been shown to markedly increase low density lipoprotein (LDL) retention by subendothelial matrix. In the present study we assessed the role that lipoprotein and matrix components play in retention of LDL by lipase anchored to the subendothelial matrix. Lipase addition to subendothelial matrix increased LDL retention by 66-fold. Scatchard analysis of LDL binding to lipase-containing matrix yielded an association constant of 12 nM. Exogenous addition of the matrix components, heparan sulfate and dermatan sulfate (i.e. chondroitin sulfate B), reduced LDL retention by greater than 90%. These glycosaminoglycans (GAGs) also reduced lipolytic activity associated with the matrix, suggesting that lipase was released from its binding sites on the matrix. In contrast, other matrix components (collagen, fibronectin, vitronectin, and chondroitin sulfate A) neither affected LDL release nor matrix lipolytic activity. Thus, heparan sulfate and dermatan sulfate function to anchor lipase to the subendothelial cell matrix. The effects of apolipoprotein E (apoE) and apoA-I were also examined. Preincubation of the subendothelial matrix with apoE, followed by washing, did not affect subsequent lipase binding to the matrix nor its ability to retain LDL. However, the direct addition of apoE alone or in combination with phospholipid liposomes decreased lipase-mediated LDL retention in a concentration-dependent fashion. Addition of apoA-I had no effect. Thus, in these studies apoE functions to displace LDL bound to lipase, but not lipase anchored to the matrix. To further examine the physiologic implications of this process, we assessed the ability of human apoE-rich and apoE-poor high density lipoproteins (HDL) to displace LDL from matrix-anchored lipase. ApoE-rich HDL reduced LDL retention dramatically (86% at 2.5 micrograms/ml). In contrast, apoE-poor HDL, at the highest concentration evaluated (400 micrograms/ml), decreased LDL retention by only 32%. Overall, these data suggest apoE and specifically apoE-containing HDL reduce the lipase-mediated retention of LDL by subendothelial matrix. This observation, in part could explain the protective effects of apoE and apoE-containing HDL against atherosclerosis.

Transplacental passage of the low molecular weight dermatan sulphate Desmin 370 was investigated in pregnant sheep, using 125I-labelled Desmin 370 to optimize the sensitivity of the study. Chronically catheterized cross-bred pregnant ewes at approximately 120 d gestation received 3700 kBq 125I-labelled Desmin 370 with 1 mg/kg carrier unlabelled Desmin 370 intravenously. Early clearance from the maternal circulation was biexponential, and the volume of distribution corresponded closely with the theoretical value for distribution in total body water. Soon after injection low levels of radioactivity were detected in the fetal circulation and accumulated over the next 2 h, so that as the concentration of 125I-Desmin 370 in the maternal circulation declined with time the fetal level of radiolabel rose to represent a significant concentration in relation to that in the mother. Radioactivity was also excreted into the fetal urine. However, while 50% of the radiolabelled material present in maternal plasma 150 min post-injection was intact Desmin 370 and the remaining 50% represented degradation products, fetal urine contained only these fragments. By contrast, intact Desmin 370 was readily excreted into fetal urine after direct introduction to the fetal circulation. Thus molecules of intact Desmin 370 with anticoagulant activity cannot cross the ovine placenta, and low molecular weight dermatan sulphates may be valuable for prophylaxis and treatment of thrombotic disease during pregnancy.

The association between low-density lipoprotein (LDL) and a series of well characterized dermatan and chondroitin sulfates has been investigated by means of the fluorescence anisotropy technique with competition experiments using a fluorescein-labeled high LDL-affinity heparin fraction as a reference. Preparations of glycosaminoglycan (GAG) with sulfation degrees varying over a wide range, as obtained by fractionation or by chemical modification, were chosen for this study. The influence of chain length, which had been found sizeable in a former study of heparin affinity for LDL, was taken into account with an empirical correction of dissociation constants. After this correction, a linear relationship was found between the logarithm of dissociation constants and the number of sulfate groups per disaccharide unit, ns, both for dermatan and chondroitin sulfates, and for heparins. At comparable ns values, however, dermatan sulfates and heparins, which contain L-iduronic acid in their backbone, show higher LDL-affinity than chondroitin sulfates, which contain only D-glucuronic acid. Though confirming a non-specific, predominantly electrostatic interaction between GAGs and LDL, these results indicate modulation of LDL affinity by the polysaccharide backbone.

The proton nuclear magnetic resonance (1H-NMR) spectra of chondroitin sulfates (chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate) have been obtained at 500MHz and 333K in deuterium oxide. The J connectivities of sugar ring protons of chondroitin sulfates are revealed by the uses of homonuclear two-dimensional (2D) multiple-relayed-chemical-shift-correlated spectroscopy (multiple-relayed COSY), homonuclear Hartman-Hahn spectroscopy (2D HOHAHA), and multiple-quantum-filtered COSY. Formation of sulfate ester on N-acetylgalactosamine hydroxy group causes predictable 0.4-0.6ppm deshielding of the proton directly attached to the sulfation site. Furthermore, the partial structure of low-sulfated chondroitin sulfate chain isolated from human urinary trypsin inhibitor was determined from the obtained 1H-NMR data.

SummaryThe aim of this study was to investigate the mechanism by which the anticoagulant activity of dermatan sulfate (DS) is increased by low molecular weight heparin (LMWH). In platelet poor plasma, LMWH enhances the effect of DS on thrombin (IIa) inhibition as determined by thrombin clotting times and with a chromogenic substrate assay. Analysis of the results of the chromogenic assays using either the algebraic fractional or the graphic isobole method suggests that LMWH has an additive effect on the anti-IIa activity of DS. This additive effect was lost when the experiments were repeated in plasma immunodepleted of antithrombin III (ATIII), indicating that the anti-IIa activity of LMWH is ATIII-dependent. To further explore the mechanism of the interaction between LMWH and DS, 125I-labeled IIa was added to plasma in the presence or absence of DS and/or LMWH and the formation of IIa-inhibitor complexes was assessed using SDS-PAGE followed by autoradiography. DS addition selectively increases the formation of heparin cofactor II (HCII)-IIa complexes, whereas LMWH enhances ATIII-IIa complex generation. Compared to plasma containing DS alone, the formation of ATIII-IIa complexes also is increased when the combination of DS and LMWH is added. These findings suggest that the additive effect of LMWH on the anti-IIa activity of DS reflects their different modes of IIa inhibition; DS potentiates IIa inhibition by HCII, while LMWH catalyses ATIII-dependent IIa inactivation. The potential clinical significance of these findings requires further investigation.

Single i.v. bolus doses of dermatan sulphate MF701 were administered before the onset of haemodialysis to patients with chronic renal failure, to prevent clotting in the extracorporeal circuit. Six patients received 2 mg kg-1; six were given 2.5 and 3 mg kg-1; 13 received 4.5 and 6 mg kg-1. Plasma MF701 concentrations (chromogenic assay), activated partial thromboplastin time (APTT) and plasma markers of coagulation and platelet activation (TAT and beta-TG) were measured over 4 or 8 h from the onset of dialysis. The disposition of MF701 was described by a monoexponential function. C(0) and AUC values increased proportionally with dose. Volumes of distribution (approximately 4 l) were dose-independent. Half-lives showed a non significant increase with dose (from 2.2 to 3.1 h) and were 2.5-3 times longer than those reported for healthy subjects. There was a significant correlation between plasma MF701 concentration and its effects in prolonging APTT and suppressing TAT and beta-TG generation during dialysis.

The aim of the present study was to achieve the immobilization of dermatan sulfate (DS) on polyethylene terephthalate (PET) surfaces and to evaluate its biocompatibility. DS obtained from the skin of Scyliorhinus canicula shark was immobilized via carbodiimide on knitted PET fabrics, modified with carboxyl groups. PET-DS characterization was performed by SEM, ATR-FTIR and contact angle measurements. Biocompatibility was evaluated by investigating plasma protein adsorption and endothelial cell proliferation, as well as by subcutaneous implantations in rats. The results indicated that DS immobilization on PET was achieved at ∼8 μg/cm2. ATR-FTIR evidenced the presence of sulfate groups on the PET surface. In turn, contact angle measurements indicated an increase in the surface wettability. DS immobilization increased albumin adsorption on the PET surface, whereas it decreased that of fibrinogen. In vitro cell culture revealed that endothelial cell proliferation was also enhanced on PET-DS. Histological results after 15 days of subcutaneous implantation showed a better integration of PET-DS samples in comparison to those of nonmodified PET. In summary, DS was successfully grafted onto the surface of PET, providing it new physicochemical characteristics and biological properties for PET, thus enhancing its biointegration. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

In 2008, some 900 cases of adverse events associated with the use of heparin were reported to the Food and Drug Administration of USA and the Federal Institute of Drugs and Medical Devices in Germany. 238 patients died from heparin in the USA. In March 2008, oversulfated chondroitin sulfate (OSCS) was identified to be responsible for these cases. NMR spectroscopic evaluation of heparin samples revealed OSCS, dermatan sulfate (DS), chondroitin sulfate A and C as well as various residual solvents to be present in heparin batches, which could not be identified by means of conventional methods described in various pharmacopoeias at that time. In order to evaluate the situation on the German market, 145 representative samples were collected in 2008 and analyzed by means of 1H NMR spectroscopy, water determination, optical rotation and sheep plasma clotting assay. 66 samples were found to contain pure heparin, 51 samples heparin plus DS, 5 samples heparin plus OSCS, and 23 samples heparin, DS and OSCS, each in varying amounts. In 94 out of 145 batches especially ethanol was found in strongly varying amounts up to about 9.5%. Traces of acetone and formic acid were found with concentrations up to 0.04%, as well as sodium acetate and methanol up to 0.5%. Additionally, in many batches the content of water was found to be relatively high. Whereas the optical rotation was able to identify samples with a high contamination of OCSC, all samples tested fulfilled the requirements of the anticoagulation potency assay of the European Pharmacopoeia 6.0. The presented analysis of a representative set of heparin samples proves the suitability of 1H NMR spectroscopy for the quality control of heparin of both glycosaminoglycans and residual solvents.

DS is a natural, complex polysaccharide which plays an important role in cell growth, differentiation, morphogenesis, cell migration, and bacterial/viral infections. Although its clinical use is limited, DS performs interesting biological activities, which should help in the development of DS-based therapeutics, such as drugs for parasitic and viral infections, regenerative medicine, anti-tumor drugs, or simply as a marker of significant disease progression. Biological activities of DS chains are likely to involve various growth factors, and specific DS chains having distinctive structures and properties are able to recruit factors and/or potentiate their activities, suggesting that minute amounts of functional DS chains can be utilized as active biomolecules. To this aim, new bioactive sources of DS may represent potential drugs for future research and development, as well as for a better understanding of the structure–function relationship, and would enable the production of this polysaccharide with its distinctive composition, structure and biological activities.

Capillary electrophoresis (CE) was applied to the quantitation of dermatan sulfate (DS) and chondroitin sulfate (CS) as related substances in sodium heparin. The method is based on the selective digestion of either CS and DS contained in the main drug heparin, by using chondroitinase ABC (specific for both DS and CS) and chondroitinase AC (specific for only CS). The unsaturated disaccharides released after exhaustive digestion, can be separated by CE using a 110mM phosphate buffer, pH 3.5 as the background electrolyte in a fused silica capillary (64.5cmx50mum i.d.) at 40 degrees C and -30kV. Since the level of each disaccharide released upon enzymatic digestion corresponds to its content in the native glycosaminoglycan, the amount of CS and DS was determined by proportion with the released disaccharides. In particular, DeltaUA--GalNAc-4S Na(2) and DeltaUA--GalNAc-6S Na(2) were selected for quantitation of CS and DS because of their significant response and short migration time (less than 7min).The method was validated for linearity, accuracy, precision and it showed to be able in detecting selectively, DS and CS at impurity level (LOD 0.01%, w/w). The proposed CE approach was finally applied to real samples. The results obtained were found in excellent correlation with those achieved by the analysis of the same samples using the official USP method based on high performance anion exchange chromatography (HPAEC) with pulsed amperometric detector.

1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmstrom, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.

We performed different “in vivo” investigations to study the pharmacological, properties of a native DS: antithrombosis by the stasis model, bleeding potential by tail transection bleeding time and template bleeding time, and profibrinolysis by a growing thrombus model and by an established thrombus model. The results suggest that DS is a safe antithrombotic drug by i.v. administration without bleeding potential, even at very high doses (up to 16 mg/Kg). DS has shown a protective index of at least 4 in contrast to heparin that has shown a protective index of 1. The profibrinolytic models so far studied did not evidence a clear profibrinolytic contribution to the antithrombotic properties of DS, but showed a prolonged antithrombotic action that cannot be explained only by the heparin cofactor II potentiation.

Corneal transparency is dependent upon the development of an organized extracellular matrix containing small diameter collagen fibrils with regular spacing, organized as orthogonal lamellae. Proteoglycan-collagen interactions have been implicated in the regulation of collagen fibrillogenesis and matrix assembly. To determine the role of dermatan sulfate proteoglycan in the development and organization of the secondary corneal stroma, its synthesis was disrupted using β-D xyloside. The secondary corneal stroma contains two different proteoglycans, dermatan sulfate and keratan sulfate proteoglycan. β-D xyloside interferes with xylose-me-diated O-linked proteoglycan synthesis, and thus disrupts dermatan sulfate proteoglycan synthesis. Corneal keratan sulfate proteoglycan, a mannose-mediated N-linked proteoglycan, should not be altered. Biochemical analysis of corneas treated both in vitro and in ovo revealed a reduced synthesis of normally glycosylated dermatan sulfate proteoglycans and an increased synthesis of free xyloside-dermatan sulfate glycosaminoglycans. Keratan sulfate proteoglycan synthesis was unaltered in both cases. Corneal stromas were studied using histochemistry and electron microscopy after in ovo treatment with β-D xyloside. The observed biochemical alterations in dermatan sulfate proteoglycans translated into disruptions in the organization of β-D xyloside-treated stromas. There was a reduction in the histochemical staining of proteoglycans, but no alteration in collagen fibril diameter. In addition, focal alterations in collagen fibril packing, and a disruption of lamellar organization were observed in β-D xyloside-treated corneas. These data suggest that dermatan sulfate proteoglycans are not involved in the regulation of corneal collagen fibril diameter, but are important in the fibril-fibril spacing as well as in lamellar organization, and cohesiveness.

Summary This paper compares the pharmacological properties of a new succinyl dermatan sulphate derivative (Suc-DS) to those of the natural dermatan sulphate (DS). Suc-DS was on average 2–3 times more potent than DS in catalysing the inhibition of thrombin by heparin cofactor II and in prolonging the activated partial thromboplastin time and the thrombin clotting time. After bolus injection. Suc-DS was also 2–3 times more potent than DS to prevent experimental venous thrombosis in a Wessler model. Thromboplastin or human serum were used as the thrombogenic stimulus. In contrast, the bleeding effect assessed by rat tail transection technique was comparable. After bolus intravenous injection, the pharmacodynamics of Suc-DS indicated a lower volume of distribution, which was close to the plasma volume, and a slightly lower clearance of elimination. Therefore this chemical alteration of natural DS yields a new compound with an improved antithrombotic benefit/haemorrhagic risk ratio.

We investigated the effect of plasmin on the release of sulfated glycosaminoglycans (GAG) from cultured vascular smooth muscle cells. Confluent cultures of vascular smooth muscle cells from bovine aorta were labelled with [ 35 S]sulfate and incubated at 37 °C for principally 60 min in a serum-free medium in the presence of plasmin. Plasmin at 10 mU/ml (approximately 2.7 μg/ml) and above significantly increased the release of [ 35 S]sulfate-labeled GAG ( 35 S-GAG) from the cell layer after a 60 min incubation. A time course study showed that plasmin at 10 mU/ml significantly increased the 35 S-GAG release after 20 min and longer. However, plasminogen at 100 mU/ml and below did not cause a significant change of the 35 S-GAG release after a 90 min incubation. A characterization of 35 S-GAG revealed that plasmin increased both dermatan sulfate and the other 35 S-GAG in the medium. Plasmin at 100 mU/ml enhanced the cell detachment significantly but only slightly. From these results, it was suggested that a endogenous thrombin inhibitor heparin cofactor II may be activated in the liquid phase by dermatan sulfate released from plasmin-stimulated vascular smooth muscle cells when the vascular is disrupted and plasma is exposed to extravessel.

To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated with p-hydroxyphenyl-beta-D-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After 125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment delta HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments delta HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on the N-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-A, Havsmark B, Silverberg I (1990) Biochem J 269:381-8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.

Glycosaminoglycan contents were evaluated in plasma and urine samples from volunteers treated intravenously with a mixture of dermatan sulphate and heparin, combining a novel liquid chromatographic-mass spectrometric technique for the determination of oligosaccharides from glycosaminoglycans with a classical technique for the extraction of glycosaminoglycans from biological samples (precipitation with cetylpyridinium chloride). In plasma samples dermatan sulphate and heparin can be measured for 2 h after treatment; urine excretion was detectable for 24 h. These results suggest that this novel approach is promising for future studies on the pharmacokinetics of glycosaminoglycans, although some technical aspects need further improvement, mainly regarding the procedures for sample clean-up; cetylpiridinium precipitation is a complex procedure and the recovery is limited.

Glycyl-L-histidyl-L-lysine-copper (II) complex (GHK-Cu) is a naturally occurring tripeptide with potential healing properties. We studied the effect of GHK-Cu on the synthesis of glycosaminoglycans (GAGs) by normal human fibroblasts in culture. Cells were incubated with 3H glucosamine and 35S sulfate and the radioactivity of isolated GAGs was determined. GHK-Cu induced a dose-dependent increase of the synthesis of total GAGs secreted into the culture medium and those associated with the cell layer. The effect of GHK-Cu was biphasic with a maximal stimulation at 10(-9) to 10(-8) M. At higher concentrations, the rate of synthesis returned progressively to that of control cultures. Electrophoretic analysis of the different GAG populations showed that GHK-Cu preferentially stimulated the synthesis of extracellular dermatan sulfate and cell layer associated heparan sulfate. No influence of GHK-Cu on the synthesis of hyaluronic acid was observed. GHK-Cu stimulation of GAG synthesis may be one of the phenomenons implicated in the wound healing properties of the peptide.

There is a controversy about whether or not histidine-rich glycoprotein (HRG), the most abundant plasma protein with glycosaminoglycans-neutralizing capacity, is able to prevent the inhibition of human thrombin by heparin cofactor II (HC II) in the presence of dermatan sulfate (DS). The authors studied the interaction of DS and low molecular weight DS, in a purified system with HRG, platelet factor 4 (PF 4), and with HC II. Their results show that HRG, like PF 4, has an affinity, not only for heparin, but also for DS. However, this affinity seems very weak. In fact, HRG is 10 times less effective than PF 4 in neutralizing the 50% antithrombin activity of HC II in the presence of DS.

We previously published a strong-anion-exchange-high performance liquid chromatography (SAX-HPLC) method for the detection of the contaminant over sulfated chondroitin sulfate (OSCS) in heparin sodium active pharmaceutical ingredient (API). While APIs have been processed to remove impurities, crude heparins contain insoluble material, chondroitin sulfates, heparan sulfate, and proteins that may interfere with the recovery and measurement of OSCS. We examined 500MHz (1)H NMR, capillary electrophoresis (CE), and SAX-HPLC to quantify OSCS in crude heparin. Using our standard API protocol on OSCS spiked crude heparin samples; we observed a weight percent LOD and LOQ for the NMR approach of 0.1% and 0.3%, respectively, while the SAX-HPLC method gave values of 0.03% and 0.09%, respectively. CE data was not amenable to quantitative measurement of OSCS in crude heparin. We developed a modified HPLC sample preparation protocol using crude dissolved at the 100mg/mL level with a 2.5M NaCl solution. This SAX-HPLC approach gave a weight percent LOD of 0.02% and a LOQ of 0.07% and had better performance characteristics than that of the protocol used for APIs.

Purpose To report an unusual ophthalmic presentation of a case of Hunter's syndrome/MPS II. Methods A sixteen-year-old girl presented to us with total loss of vision and forward protrusion OU since early childhood. Detailed examination, including slit lamp biomicroscopy, Intra ocular pressure (IOP) and fundoscopy was carried out. Thorough systemic evaluation including Computed Tomography (CT), metabolic and genetic analysis was undertaken in collaboration with internists. Results Characteristic facies, detection of glycosaminoglycan (GAG) variants in urine (chondroitin sulfate B and heparin sulfate) and iduronate-2-sulphatase activity in fibroblasts/leucocytes confirmed the diagnosis of MPS II. Child had severe photophobia but with no perception of light OU. OU buphthalmos with Haab's striae was noted, making a clear view of the fundus difficult. IOP OU was elevated, and 90D slit lamp biomicroscopy revealed a total glaucomatous optic atrophy in both eyes.On CT there was thickening and edema of preseptal and periorbital soft tissue with marked thinning of the optic nerves with prominent perineural CSF sleeves, indicative of marked optic atrophy. Conclusion Glaucoma is a known association of Hurler's, Scheie's and Maroteaux-Lamy syndromes but not Hunter's. In fact, there is only one report of suspected angle closure glaucoma in MPS II. Buphthalmos is not a likely presentation as the sclera in these patients is known to be thickened due to deposition of GAG. To the best of our knowledge, this is the first case report of buphthalmos in association with MPS II. The importance of a meticulous examination in this subset of patients cannot be overemphasised. An appropriate and timely intervention may result in a better quality of life for them.

Chick embryonic skeletal muscle synthesizes three major types of proteoglycans: large chondroitin sulfate proteoglycans, small dermatan sulfate proteoglycans and small heparan sulfate proteoglycans. A monoclonal antibody has been raised which recognizes the small dermatan sulfate proteoglycan. Immunoblot analysis of a partially purified preparation of skeletal muscle proteoglycans indicates that the antibody reacts with a molecule which migrates with an estimated Mr of 100,000. Prior treatment of the proteoglycans with chondroitinase results in immunostaining of a species of estimated Mr 45,000. These values for the intact proteoglycan and its core protein suggest that the antibody is directed against a proteoglycan of the PG-II or decorin class. Immunohistochemistry indicates a widespread distribution of the proteoglycan, which is localized in connective tissue septa of skeletal and cardiac muscle, dermis, tendon, bone, perichondrium and cornea. Immunoblot analysis of the proteoglycan core proteins from these tissues demonstrates that the antibody recognizes the same 45,000-dalton band in each tissue. The widespread tissue distribution is also consistent with the antibody being directed against an epitope of PG-II. Neither the glycosaminoglycan chains nor N-linked oligosaccharides are required for reactivity and the antibody cross-reacts with other avian material, but not mammalian. This antibody, which has been designated CB-1, reveals developmental stage-specific changes in the deposition of PG-II in embryonic limb bud and skeletal muscle.

The sulfation of dermatan sulfate by SO3-trimethylamine in N,N-dimethylformamide led to substitution initially at HO-6 of residues of 2-acetamido-2-deoxy-beta-D-galactopyranosyl 4-sulfate (1), to produce the 4,6-disulfate (6). When this step reached a level of greater than 50%, sulfation occurred with equal facility at HO-2 and HO-3 of residues of alpha-L-idopyranosyluronic acid (2), giving rise to a mixture of 2-,3-, and 2,3-disulfates. An analogous substitution pattern was observed for HO-2 and -3 of a simpler idopyranose unit, in the sulfation of methyl 4,6-O-benzylidene-alpha-D-idopyranoside (12). This lack of regioselectivity in the reaction of 2 (and 12) contrasts markedly with the high affinity of the reagent for HO-3 of residues of alpha-L-idopyranosyluronic acid present in a modified form of heparin. It is attributed to a difference between the two polymers in the relative orientation of their neighboring amino sugar residues, whereby there is an unobstructed access of the reagent in one instance, and hindrance of HO-2 selectively in the other. Enzymolysis by chondroitinase ABC was found to yield unsaturated disaccharide containing residues of 4,6-disulfate, as well as larger fragments containing unsaturated glycosyl groups derived from L-idopyranosyluronic acid 2-sulfate, evidence of a relatively broad enzyme specificity. The presence of extra sulfate groups in dermatan sulfate did not enhance its weak antithrombotic activity, as measured by anti Xa assay, in disagreement with earlier reports.

Eight healthy volunteers were given single subcutaneous doses of dermatan sulfate (DS, 100, 200 and 400 mg), heparin (5,000 IU) and placebo in random order. Wash-out between treatments was greater than or equal to 10 days. Serial blood samples were taken before and up to 24 hours after treatment to measure coagulation and fibrinolytic parameters. Thrombin generation was significantly inhibited by DS and heparin as compared to placebo. The effect of DS was dose-dependent. Peak inhibition after 200 mg DS was comparable to that of 5,000 IU heparin, but lasted longer. A small, bordeline significant prolongation of APTT was observed after 400 mg DS and heparin. The changes in PAI and fibrinolytic activities were those of the circadian variation. No changes were seen in the other parameters tested. In conclusion, single s.c. doses of DS (200, or 400 mg) inhibit ex vivo thrombin generation equally or more than 5,000 IU heparin and for a longer time. The effect of both treatments on fibrinolysis is negligible.

Cytophaga columnaris produces, in culture, an enzyme that degrades chondroitin sulfates A and C and hyaluronic acid, the complex polysaccharides of connective tissue. This study is the first to demonstrate production of such a tissue-degrading enzyme by this fish pathogen. Partial characterization of the enzyme has shown it to be of the chondroitin AC lyase type that has been described for some other bacteria. No relations were found between the host origin, geographic distribution, and amount of enzyme produced by the different isolates. The enzyme is constitutive, is released into the medium as it is produced, has a molecular weight estimated at 42,600, and has an optimum pH of 6.0. Kinetic studies indicate enzyme specificities in the order chondroitin sulfate A > chondroitin sulfate C > hyaluronic acid. The enzyme showed little or no reaction with chondroitin sulfate B and none with heparin, pectic acid, arabinogalactan, gum guar, or gum acacia. The enzyme was produced by all 16 C. columnaris ...

The copolymeric structure of dermatan sulfate chains synthesized by skin fibroblasts has been examined. Chains initiated onto exogeneous p-nitrophenyl-beta-D-xylopyranoside or attached to protein in a large proteoglycan, PG-L, and two small proteoglycans, PG-S1 and PG-S2, have been compared by using high resolution electrophoresis and gel chromatography of oligosaccharides generated by specific enzymatic or chemical degradations. The results confirm that chains attached to PG-L are glucuronate-rich, whereas novel findings indicate that chains attached to either of the two PG-S variants yield closely similar oligosaccharide maps, have approximately equal glucuronate and iduronate content and contain over 90% 4-sulfated disaccharide repeating units. Dermatan sulfate chains built onto xyloside at concentrations of 50 microM and below have a copolymeric structure similar to that of chains from the two PG-S variants. These findings indicate that the polymer-modifying machinery can generate chains with extended iduronate-containing repeats also when the xylose primer is not linked to core protein.

We report studies performed on the ATPase activity of washed platelets in the presence of heparins having different molecular weights, native and desulfated dermatans and heparans

We have been interested in examining the putative biological role(s) of the major proteoglycan of adult skeletal muscle. The small proteoglycans of adult rabbit skeletal muscle and tendon were extracted and purified by sequential density-gradient ultracentrifugation, ion-exchange chromatography and gel filtration. They appeared to be homogeneous by the criterion of gel electrophoresis in SDS and to yield one major product, the core protein, after digestion with chondroitin ABC lyase, also observed after gel electrophoresis. Two major products were obtained when the intact proteoglycans were cleaved by CNBr, and those peptides were separated by SDS/PAGE and by ion-exchange chromatography. Sequencing of the N-terminal amino acids of either the intact proteoglycans or the CNBr-cleaved products allowed for comparison of the muscle and tendon proteoglycan with derived amino acid sequences previously reported for bovine bone proteoglycan. The bone and tendon proteoglycan sequences were remarkably similar, whereas those of the muscle proteoglycan differed from the other two molecules. The major site of glycosaminoglycan substitution was on a peptide fragment distant from the N-terminus, and a presumptive serine residue at position 4 from the N-terminus also appeared to be substituted, perhaps with a small glycosaminoglycan chain. These results provide some insight into the diversity of small proteoglycans of the PG-II class and provide a basis for exploring their mode of genetic expression.

By a series of experiments it could be shown that an activity test of heparin cofactor II (HC II) is only specific after a complete depletion of antithrombin III. Also when dermatan sulfate which does not enhance the action of AT III is used for the activation of HC II there is a considerable influence on the remaining thrombin activity which alters the test results. Furthermore, in a system which contains plasma as well as thrombin the formation of a clot is likely to occur which by its opacity influences photometric results. A chromogenic substrate assay is described which excludes the influence of these variables. This assay was used to examine the activity of HC II in healthy persons as well as in patients. Three members of a family were found who had a heterozygous deficiency of HC II without any history of thrombosis. On the other hand, a total of 16 patients with heterozygous deficiency of HC II suffered from recurrent thromboembolic episodes. For this reason it is assumed that a deficiency of HC II has a certain importance in the occurrence of thrombophilia though it is apparently less thrombogenic than a deficiency of other inhibitors.

Internalization of autoantibodies against double-stranded DNA (anti-dsDNA) is crucial to the pathogenesis of systemic lupus erythematosus. Anti-dsDNA may bind to cell-surface targets in order to facilitate the subsequent cell penetration of the anti-dsDNA. In this study, we observed that the 9D7 monoclonal anti-dsDNA autoantibody (9D7 mAb) penetrates into Jurkat cells via a novel alternative pathway. Endocytosis inhibitors or a lipid-raft inhibitor did not significantly change the penetration of 9D7 mAb into the Jurkat cells. However, heparin sulfate, chondroitin sulfate B, decaarginine and chondroitinase ABC significantly suppressed the internalization and the 9D7 mAb inhibited the internalization of Tat-GFP. Moreover, the penetration of the 9D7 mAb was significantly reduced in proteoglycan-deficient cells (pgs A-745). Positively charged amino acids including arginine are commonly found in the CDR of the 9D7 mAb. Point mutations to the arginine residues in the CDR of the H chain of the recombinant 9D7 mAb significantly attenuated its DNA-binding and cell-penetration abilities. These findings indicate that cell penetration of anti-dsDNA is due to the electrostatic interactions of arginine residues in the CDR with the negatively charged sulfated polysaccharides on the cell surface.

A chromatographic method was developed for the detection and quantification of the contaminant oversulfated chondroitin sulfate (OSCS) and the impurity dermatan sulfate in heparin active pharmaceutical ingredient (API). The HPLC analysis of heparin is carried out using a polymer-based strong anion exchange (SAX) column with gradient elution from 0.125 M sodium chloride to 2.5 M sodium chloride buffered mobile phase. The limit of detection (LOD) and limit of quantitation (LOQ) for the contaminant OSCS in heparin were determined to be 0.03% and 0.1%, respectively. The LOD and LOQ for dermatan sulfate, an impurity in heparin sulfate, were determined to be 0.1% and 0.8%, respectively. This manuscript is not a policy document and is not intended to replace either of the methods (capillary electrophoresis and NMR) currently required by the FDA.

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Abeta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Abeta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Abeta(1-42) species on Ca(2+) homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca(2+) that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca(2+) increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

In the present study, inhibitory effects of several sulfated and nonsulfated glycoconjugates were evaluated on the in vitro asexual growth of Babesia bovis. Among the selected sulfated glycoconjugates, dextran sulfate, heparin, heparan sulfate, fucoidan, and chondroitin sulfate B strongly inhibited the parasitic growth, and all but chondroitin sulfate B induced a significant accumulation of extracellular merozoites in culture. In contrast, chondroitin sulfate A, keratan sulfate, and protamine sulfate, as well as nonsulfated dextran and hyaluronic acid, did not influence the growth. These findings indicate that the asexual growth of B. bovis merozoites is inhibited by specific sulfated glycoconjugates, possibly providing us with an important insight into the molecular interaction(or interactions) during the process of the erythrocyte invasion by B. bovis merozoites.

Hunter disease (mucopolysaccharidosis type II, MPS II) is an X-linked lysosomal storage disease caused by deficiency of iduronate-2-sulfatase. Accumulation of chondroitin sulfate B and heparan sulfate in various tissues is the biochemical consequence of MPS II. Children with Hunter disease are normal at birth, and symptoms occur between 2 and 10 years of age. Typical symptoms include coarse facies with enlarged tongue and prominent forehead as well as a short, stocky built stature with short neck. The cardiovascular, respiratory and gastrointestinal systems may be affected, and oral, dermatological and psychiatric as well as neurological complications are described. Life expectancy is markedly reduced and may be limited to 12 years for severely affected patients. The most common causes of death are airway obstruction and cardiac failure. The most severe symptoms may result from neurological symptoms or complications including hydrocephalus, spinal cord compression, cervical myelopathy, optic nerve compression, and hearing impairment. Patients may also develop carpal tunnel syndrome, sleep apnoea, seizures or mental retardation. This review describes characteristic neurological manifestations in MPS II and its underlying pathophysiology. In addition, an appraisal is given whether or not enzyme replacement therapy may be able to improve in particular the neurological symptoms of Hunter disease.

Summary Chondroitin 4-sulfate proteoglycan (C4S-PG) was localized both in rat and human central nervous system (CNS) by monoclonal and polyclonal antisera recognizing the 4-sulfate disaccharide (C4S). In the rat the whole CNS was studied in serial coronal sections. A positive extracellular meshwork was observed both in white and grey matters. In the white matter (WM) C4S-PG formed a network around myelinated axons, sparing myelin sheaths and axoplasms. The neuropil of the grey matter (GM) showed a positive meshwork constituted by delicate intermingling filaments. The cytoplasms of neuronal, glial and endothelial cells were negative. Stronger straining than in the neuropil was observed around the soma and the proximal part of the cell processes of some neurons located in the cortex, in the deep cerebellar nuclei and in some other CNS nuclei. A similar pattern was also observed in human CNS, the only difference being a smaller amount of cortical neurons surrounded by a rim of C4S-PG. This study shows that a PG bearing C4S disaccharide is located extracellularly in the rodent and human CNS and that C4S disaccarides can be present in different types of CNS proteoglycans (PGs).

Two recently identified structural elements important for glycosaminoglycan-mediated activation of human leuserpin-2 (hLS2) were investigated in detail by functional analysis of variants secreted by transiently transfected COS cells. Highly specific requirements with respect to the nature of the involved amino acids as well as to their spatial arrangements were found to be crucial for efficient activation of hLS2 by dermatan sulfate. In contrast, binding and activation of hLS2 by heparin seem to be determined mainly by the positive charge density of the involved inhibitor segment. A dimeric repeat enriched in acidic amino acids turned out to exert a dual role with respect to structure and function of hLS2. First, in the absence of functional activators the negatively charged dimer interacts intramolecularly with the glycosaminoglycan-binding site. Second, the acidic dimer is instrumental in glycosaminoglycan-mediated activation of hLS2. The monomers constituting the acidic dimer are functionally not equivalent.

The small proteoglycans (PGs) of cartilage matrix represent a small fraction of the total mass of PGs, but with a small size they can be present in equivalent moles to the large PGs. Three types of PGs with a wide skeletal and extraskeletal distribution, biglycan (PGI), decorin (PGII) and fibromodulin have distinct but homologous core proteins containing leucin-rich sequences. Carbohydrate substituants (one or two chondroitin sulfate/dermatan sulfate chains for decorin and biglycan respectively, chains of keratan sulfate for fibromodulin and oligosaccharides) present variations from tissue to tissue and with age and other factors. Decorin and fibromodulin appear to interact with collagen and to participate in the regulation of collagen matrices. In vitro experiments indicate a role for small PGs in adhesion, multiplication, differentiation, and migration of cells. Recent data on molecular biology of the small PGs contribute to a better understanding of their functions and make the evaluation of their role in hereditary diseases.