Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from Swim Bladder: Isolation, Structural Analysis, and Anticoagulant Activity.

Glycosaminoglycans (GAGs) with unique structures from marine animals show intriguing pharmacological activities and negligible biological risks, providing more options for us to explore safer agents. The swim bladder is a tonic food and folk medicine, and its GAGs show good anticoagulant activity. In this study, two GAGs, CMG-1.0 and GMG-1.0, were extracted and isolated from the swim bladder of Cynoscion microlepidotus and Gadus morhua. The physicochemical properties, precise structural characteristics, and anticoagulant activities of these GAGs were determined for the first time. The analysis results of the CMG-1.0 and GMG-1.0 showed that they were chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains with molecular weights of 109.3 kDa and 123.1 kDa, respectively. They were mainly composed of the repeating disaccharide unit of -{IdoA-α1,3-GalNAc4S-ß1,4-}- (DS-A). The DS-B disaccharide unit of -{IdoA2S-α1,3-GalNAc4S-ß1,4-}- also existed in both CMG-1.0 and GMG-1.0. CMG-1.0 had a higher proportion of CS-O disaccharide unit -{-GlcA-ß1,3-GalNAc-ß1,4-}- but a lower proportion of CS-E disaccharide unit -{-GlcA-ß1,3-GalNAc4S6S-ß1,4-}- than GMG-1.0. The disaccharide compositions of the GAGs varied in a species-specific manner. Anticoagulant activity assay revealed that both CMG-1.0 and GMG-1.0 had potent anticoagulant activity, which can significantly prolong activated partial thromboplastin time. GMG-1.0 also can prolong the thrombin time. CMG-1.0 showed no intrinsic tenase inhibition activity, while GMG-1.0 can obviously inhibit intrinsic tenase with EC50 of 58 nM. Their significantly different anticoagulant activities may be due to their different disaccharide structural units and proportions. These findings suggested that swim bladder by-products of fish processing of these two marine organisms may be used as a source of anticoagulants.

Biglycan and chondroitin sulfate play pivotal roles in bone toughness via retaining bound water in bone mineral matrix.

Recent in vitro evidence shows that glycosaminoglycans (GAGs) and proteoglycans (PGs) in bone matrix may functionally be involved in the tissue-level toughness of bone. In this study, we showed the effect of biglycan (Bgn), a small leucine-rich proteoglycan enriched in extracellular matrix of bone and the associated GAG subtype, chondroitin sulfate (CS), on the toughness of bone in vivo, using wild-type (WT) and Bgn deficient mice. The amount of total GAGs and CS in the mineralized compartment of Bgn KO mouse bone matrix decreased significantly, associated with the reduction of the toughness of bone, in comparison with those of WT mice. However, such differences between WT and Bgn KO mice diminished once the bound water was removed from bone matrix. In addition, CS was identified as the major subtype in bone matrix. We then supplemented CS to both WT and Bgn KO mice to test whether supplemental GAGs could improve the tissue-level toughness of bone. After intradermal administration of CS, the toughness of WT bone was greatly improved, with the GAGs and bound water amount in the bone matrix increased, while such improvement was not observed in Bgn KO mice or with supplementation of dermatan sulfate (DS). Moreover, CS supplemented WT mice exhibited higher bone mineral density and reduced osteoclastogenesis. Interestingly, Bgn KO bone did not show such differences irrespective of the intradermal administration of CS. In summary, the results of this study suggest that Bgn and CS in bone matrix play a pivotal role in imparting the toughness to bone most likely via retaining bound water in bone matrix. Moreover, supplementation of CS improves the toughness of bone in mouse models.

Chondroitin/dermatan sulfate purified from corb (Sciaena umbra) skin and bone: In vivo assessment of anticoagulant activity.

The present work deals with the extraction and purification of chondroitin sulfate/dermatan sulfate from skin (CSG) and bone (CBG) of corb (Sciaena umbra). Electrophoresis of these polymers in barium acetate buffer on cellulose acetate revealed two fractions similar to dermatan sulfate and chondroitin sulfate. The in vivo anticoagulant activity of both chondroitin sulfate/dermatan sulfate (CS/DS) were evaluated, at 25 and 75 mg kg-1 of body weight (b.w), using activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests. Results showed that aPTT of CSG and CBG at 75 mg kg-1 of b.w were prolonged by 1.59 and 1.48-fold respectively, compared with the control. Further, toxicity studies on liver performed by the catalytic activity of transaminases in plasma, oxidative stress markers and hepatic morphological changes demonstrated that CSG and CBG at both doses are not toxics. In summary, the higher activity and lower toxicity of both CS/DS, especially at 25 mg kg-1 of b.w, recommended these compounds as a better drug candidate.

Synergistic Effects of a Mixture of Glycosaminoglycans to Inhibit Adipogenesis and Enhance Chondrocyte Features in Multipotent Cells.

BACKGROUND/AIMS: Multipotent mesenchymal stem cells affect homeostasis of adipose and joint tissues. Factors influencing their differentiation fate are of interest for both obesity and joint problems. We studied the impact of a mixture of glycosaminoglycans (GAGs) (hyaluronic acid: dermatan sulfate 1:0.25, w/w) used in an oral supplement for joint discomfort (Oralvisc™) on the differentiation fate of multipotent cells. METHODS: Primary mouse embryo fibroblasts (MEFs) were used as a model system. Post-confluent monolayer MEF cultures non-stimulated or hormonally stimulated to adipogenesis were chronically exposed to the GAGs mixture, its individual components or vehicle. The appearance of lipid laden cells, lipid accumulation and expression of selected genes at the mRNA and protein level was assessed. RESULTS: Exposure to the GAGs mixture synergistically suppressed spontaneous adipogenesis and induced the expression of cartilage extracellular matrix proteins, aggrecan core protein, decorin and cartilage oligomeric matrix protein. Hormonally-induced adipogenesis in the presence of the GAGs mixture resulted in decreased adipogenic differentiation, down-regulation of adipogenic/lipogenic factors and genes for insulin resistance-related adipokines (resistin and retinol binding protein 4), and up-regulation of oxidative metabolism-related genes. Adipogenesis in the presence of dermatan sulfate, the minor component of the mixture, was not impaired but resulted in smaller lipid droplets and the induction of a more complete brown adipocyte-related transcriptional program in the cells in the adipose state. CONCLUSIONS: The Oralvisc™ GAGs mixture can tip the adipogenic/chondrogenic fate balance of multipotent cells away from adipogenesis while favoring chondrocyte related gene expression. The mixture and its dermatan sulfate component also have modulatory effects of interest on hormonally-induced adipogenesis and on metabolic and secretory capabilities of adipose cells.

Thrombin generation assays are superior to traditional tests in assessing anticoagulation reversal in vitro.

Even though new anticoagulants are being devised with the notion that they do not require regular monitoring, when bleeding occurs, it is important to have an antidote and a reliable test to confirm whether the anticoagulant effects are persisting. We examined the effects of five heparinoids, unfractionated heparin (UFH), tinzaparin, enoxaparin, danaparoid and fondaparinux on the traditional APTT and anti-Xa assays as well as on the calibrated automated thrombogram (CAT). We also studied the ability of protamine sulphate (PS), NovoSeven, FEIBA and FFP to reverse maximum anticoagulation induced by the different heparinoids. The CAT was the only test to detect the coagulopathy of all the anticoagulants. PS produced complete reversal of UFH, and this could be monitored with all three tests. Tinzaparin can also be completely neutralised in vitro with high doses of PS, but the maximum enoxaparin reversal achieved with PS is only approximately 60%. Fondaparinux does not significantly affect the APTT and PS has no significant effect on its reversal. Only NovoSeven was able to correct the fondaparinux induced CAT abnormalities whilst having no effect on the anti-Xa level. None of the reversal agents was very effective in danaparoid spiked plasma but NovoSeven, at high dose, increased the ETP by 40% and reduced the anti-Xa level from 0.93 to 0.78 IU/ml. We conclude that the CAT is superior to the traditional coagulation tests in that it not only detects the coagulopathy of all the heparinoids but can be also be used to monitor their reversal.

Inhibition of thrombin generation in recalcified plasma.

Eight inhibitors of thrombin generation were compared in recalcified unfrozen plasma. Individual or pooled normal citrated plasma was supplemented on polystyrol flat-bottom wells (23 degrees C) with increasing concentrations of low-molecular-weight heparin, heparin, danaparoid, fondaparinux, hirudin, argatroban, corn trypsin inhibitor, or aprotinin. Thrombin was generated by addition of 5 microl fresh 250 mmol/l CaCl2 to 50 microl plasma in polystyrol flat-bottom wells and incubation for 20 min at 37 degrees C (recalcified coagulation activity assay). Arginine stopped hemostasis activation and then the generated thrombin activity was specifically quantified. The approximate 50% inhibitory concentrations of plasmatic anticoagulants for individual or pooled normal plasma are, respectively, 0.6 or 3.7 mIU/ml low-molecular-weight heparin, 0.3 or 1.6 mIU/ml heparin, 0.7 or 6.1 mU/ml danaparoid, 0.023 or 0.18 microg/ml fondaparinux, 75 or 230 pg/ml hirudin, 0.026 or 0.24 microg/ml argatroban, 1 or 2 U/ml corn trypsin inhibitor, and 2 or 4 KIU/ml aprotinin. The 50% inhibitory concentration values for corn trypsin inhibitor or aprotinin at plasmatic concentrations above 4-100 U/ml might increase pathologically the thrombin generation. The recalcified coagulation activity assay is a sensitive method to measure prothrombotic tendencies of blood or subtle concentrations of any plasmatic anticoagulant. It is suggested to analyze the individual patient's sensibility to certain plasmatic anticoagulants.

Inhibition of cultured bovine aortic endothelial cell proliferation by sodium spirulan, a new sulfated polysaccharide isolated from Spirulina platensis.

Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, which consists of two types of disaccharide repeating units, O-hexuronosyl-rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methylrhamnose (acofriose) with sulfate groups, other minor saccharides and sodium ion. Vascular endothelial cells are present on the inner surface of blood vessels in a monolayer and have anticoagulant properties. To address the question whether Na-SP influences the maintenance of endothelial cell monolayers, we investigated the proliferation of cultured bovine aortic endothelial cells treated with Na-SP. It was found that Na-SP has an inhibitory activity on endothelial cell proliferation accompanied with suppression of whole protein synthesis but without non-specific cell damage. The inhibitory activity of Na-SP was the strongest when compared to that of heparan sulfate, heparin, dextran sulfate, dermatan sulfate, chondroitin sulfate A/C and hyaluronan. Furthermore, it was shown that the inhibitory activity of Na-SP disappeared by either desulfation or depolymerization. The present data suggest that Na-SP is a unique sulfated polysaccharide that strongly inhibits vascular endothelial cell proliferation, and the inhibitory activity requires polymerization of sulfated O-rhamnosyl-acofriose repeating units.

Selective and sustained inhibition of surface-bound thrombin activity by intimatan/heparin cofactor II and its relevance to assessing systemic anticoagulation in vivo, ex vivo and in vitro.

We compare the relative activities of surface-bound and fluid-phase thrombin and their inhibition by heparin and Intimatan, a novel heparin cofactor II (HCII) agonist. In vitro, we compared the observed amidolytic activities of fluid-phase and surface-bound thrombin with the expected activities based upon 125I-specific activity. In vivo, we compared the inhibitory effects of heparin and Intimatan on thrombin activity bound to injured vessel walls. In vitro, the correlations between observed and expected activities of fluid-phase and surface-bound thrombin, were: r = 0.9974, p < 0.001; and r = 0.9678, p < 0.001; respectively. In vivo, injured vessel wall surface-bound thrombin activity persisted for > 24 h. This activity was not inhibited by heparin, but was inhibited by Intimatan, p < 0.001. We conclude that surface-bound thrombin is as active as fluid-phase thrombin and remains protected from inhibition by heparin, thereby contributing to vessel wall thrombogenicity following injury. In contrast, surface-bound thrombin is inhibited by Intimatan, thereby effectively decreasing vessel wall thrombogenicity following injury in vivo.

The effects of danaparoid, dalteparin and heparin on tissue factor-induced experimental disseminated intravascular coagulation and bleeding time in the rat.

Danaparoid and heparin, on the basis of anti-activated factor X (anti-FXa) activity, were equipotent in accelerating the rate of interaction of FXa and antithrombin III. In rat tissue factor-induced disseminated intravascular coagulation (DIC) models, an intravenous administration of danaparoid inhibited the decrease in plasma fibrinogen and platelet counts and the increase in serum fibrinogen degradation products. Expressed on the basis of anti-FXa activity, these effects were comparable with those of dalteparin and heparin. In rat mesenteric small artery and vein, less bleeding was observed after intravenous administration of danaparoid than after dalteparin or heparin. Danaparoid did not affect adenosine diphosphate- or collagen-induced platelet aggregation, and showed weaker inhibitory effects on aggregation induced by thrombin, or collagen + thrombin, than did dalteparin or heparin. These findings suggest that danaparoid may be useful for the prevention of DIC and has less tendency to cause bleeding than dalteparin or heparin, probably as a result of its weaker ability to inhibit platelet aggregation.

Pharmacological actions of sulodexide.

This report summarizes the results of some of the studies that have evaluated the pharmacokinetic, pharmacodynamic, anticoagulant, and antithrombotic properties of Sulodexide, which consists of a mixture of electrophoretically fast moving heparin (80% of the mass) and dermatan sulfate (the balance), with an average product (Mr) <8000. The low molecular weight (Mr) of the constituents of Sulodexide would predict that the product has the high bioavailability associated with low-Mr heparin and low-Mr dermatan sulfate. Given orally, subcutaneously, or by intravenous injection, Sulodexide exhibits antithrombotic and profibrinolytic properties in several animal models of venous and arterial thrombosis and has relatively high affinity for endothelial (and possibly other) cells. Additionally, in a large multicenter clinical trial involving 3986 patients who had recovered from acute myocardial infarction, oral Sulodexide was associated with a 32% reduction in death and a significant reduction of left ventricular thrombus formation. Compared with heparin, low-Mr heparin, and unfractionated and low-Mr dermatan sulfates, the doses of Sulodexide required for antithrombotic efficacy suggest that the combination of heparin and dermatan sulfate in Sulodexide provides a more effective antithrombotic mechanism than heparin/low-Mr heparins (which catalyze the antiprotease actions of antithrombin III) or dermatan sulfate/low-Mr dermatan sulfate (which catalyze thrombin inhibition by heparin cofactor II).

The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation.

IP-10 is a member of the chemokine family of cytokines and is induced in a variety of cells in response to interferon gamma and lipopolysaccharide. The self-aggregation common to many chemokines, including IP-10, has hindered the identification of a specific IP-10 receptor. Using an IP-10 alkaline phosphatase fusion protein that fortuitously blocks this self-aggregation, we have identified an IP-10 binding site on a variety of cells including endothelial, epithelial, and hematopoietic cells. This binding site has a Kd of 25 nM, is inhibited by recombinant murine or human IP-10, and is dependent on the presence of cell surface heparan sulfate proteoglycans (HSPG). This conclusion is based on the findings that IP-10 binding to cells is: (a) inhibited by heparin and heparan sulfate; (b) sensitive to a 1 M NaCl wash; (c) eliminated by treatment with heparinase and trypsin; and (d) absent on mutant CHO cells that do not express cell surface HSPG. Platelet factor 4 (PF4), but not IL-8, monocyte chemoattractant protein-1, RANTES, monocyte inflammatory protein (MIP)-1 alpha, or MIP-1 beta, can compete effectively with IP-10 for binding to the cell surface. Furthermore, IP-10 shares with PF4 the ability to inhibit endothelial cell proliferation (IC50 = 150 nM). These studies demonstrate specificity in the interaction of chemokines and HSPG, and they define IP-10 and PF4 as a distinct subset of chemokines sharing an HSPG-binding site and angiostatic properties.

Differential effects of glycosaminoglycans on neurite outgrowth from hippocampal and thalamic neurones.

Chondroitin sulphate proteoglycans are expressed in a temporally restricted pattern from embryonic day 17 to postnatal day 0 in both the thalamus and the cortical subplate, to which thalamic neurones transiently project. To study whether chondroitin sulphate proteoglycans could be specifically involved in the modulation of thalamic axon outgrowth, we compared neurite outgrowth from cultured rat embryonic hippocampal and thalamic neurones, in the presence of chondroitin sulphate type C (isolated from shark cartilage) and chondroitin sulphate type B (dermatan sulphate; isolated from bovine mucosa). When added to the culture medium, both types of glycosaminoglycan lowered the adhesion to laminin and polylysine of both hippocampal and thalamic neurones. However, only chondroitin sulphate specifically modified the pattern of thalamic but not hippocampal neurone outgrowth, promoting axon growth. The morphological changes induced by chondroitin sulphate were concentration dependent and correlated with the selective binding of chondroitin sulphate to the neuronal plasma membrane and its subsequent internalisation. Chondroitin sulphate loosely bound to the surface of hippocampal neurones, but was not internalised. These results indicate that proteoglycans, and in particular the glycosaminoglycan component of these molecules, can differentially modulate neurite outgrowth, depending on their biochemical composition and on the type of neurones they bind to; this would be a possible mechanism of controlling axon guidance in vivo.

Influence of glycosaminoglycans on neurite morphology and outgrowth patterns in vitro.

The neuritic growth patterns obtained on substrates made of several glycosaminoglycans (GAGs) bound to type I collagen were analysed and compared in primary cultures of chick embryo dorsal root ganglion grown in serum-free supplemented medium. In 2-day cultures grown on type I collagen or heparan sulphate (HS)-collagen surfaces, ganglionic explants exhibit a dense, symmetrical network of long, parallel neuritic processes and very few flat migrating non-neuronal cells. In contrast, on either dermatan sulphate (DS), chondroitin-6-sulphate (C6S) or hyaluronic acid (HA)-bound collagen substrates, neurons form irregular nerve fibre patterns; indeed, neurites follow convoluted paths and often, after abrupt turns, totally reverse their direction of extension. Experiments were carried out in which a choice was given to growing neural processes between collagen or GAG-collagen substrates. While growth cones elongating over type I collagen easily cross the border with HS-bound collagen surface and indiscriminately extend on this substrate, in contrast, neurites generally avoid surfaces coated with DS, C6S or HA and change their direction of growth in order to stay on collagen. The binding of DS, C6S or HA, but not HS, to type I collagen thus decreases its ability to promote neurite elongation. The interaction of neuronal cells with these extracellular matrix components by restricting neurites in their paths of extension may, therefore, play a role in the patterning of the nervous circuitry.

Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor.

Proliferation of smooth muscle cells from the pulmonary arteries and aortas of fetal calves is inhibited by heparin in vitro. This effect is reversible and dose dependent. Comparisons with effects of other polysaccharides indicate that only extensively sulfated polysaccharides inhibit proliferation of smooth muscle cells but that specific structural features of heparin are required to achieve maximum effect. Heparin-Sepharose chromatography of medium containing fetal calf serum reduces the ability of that medium to promote growth of smooth muscle cells from fetal pulmonary arteries, suggesting that heparin may remove soluble growth factors in serum. However, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is identical in media supplemented either with serum prepared from fetal calf plasma, in which platelet-derived growth factor (PDGF) is not detectable, or with fetal calf serum, which contains relatively abundant PDGF (114 pg/ml). Thus, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is not mediated solely by decreased availability or activity of exogenous PDGF. These studies suggest that morphogenesis of the smooth muscle investment of the pulmonary arteries could be regulated by local production of heparin-like inhibitors of smooth muscle cell growth.