Oversulfated dermatan sulfate and heparinoid in the starfish Lysastrosoma anthosticta: Structures and anticoagulant activity.

Crude anionic polysaccharides extracted from the Pacific starfish Lysastrosoma anthosticta were separated by anion-exchange chromatography into fractions LA-F1 and LA-F2. The main fraction LA-F1 was solvolytically desulfated giving rise to preparation LA-F1-DS with a structure of dermatan core [→3)-ß-d-GalNAc-(1→4)-α-l-IdoA-(1→]n. Reduction of LA-F1 afforded preparation LA-F1-RED composed mainly of the repeating disaccharide units →3)-ß-d-GalNAc4R-(1→4)-α-l-Ido2S3S-(1→, where R was SO3- or H. Analysis of the NMR spectra of the parent fraction LA-F1 led to determine the main component as the oversulfated dermatan sulfate LA-Derm bearing sulfate groups at O-2 and O-3 of α-l-iduronic acid, as well as at O-4 of some N-acetyl-d-galactosamine residues. The minor fraction LA-F2 contained a mixture of LA-Derm and heparinoid LA-Hep, the latter being composed of the fragments →4)-α-d-GlcNS3S6S-(1→4)-α-l-IdoA2S3S-(1→ and →4)-α-d-GlcNS3S-(1→4)-α-l-IdoA2S3S-(1→. The presence of 2,3-di-O-sulfated iduronic acid residues is very unusual both for natural dermatan sulfate and heparinoid. Preparations LA-F1, LA-F2 and LA-F1-RED demonstrated significant anticoagulant effect in vitro.

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.

Dermatan sulfate obtained from the Phallusia nigra marine organism is responsible for antioxidant activity and neuroprotection in the neuroblastoma-2A cell lineage.

Neurodegenerative diseases are characterized by progressive loss of neurons in the central nervous system (CNS). Several molecules play a role in mammalian CNS regeneration, including glycosaminoglycans (GAGs). GAGs are found in abundance in many marine invertebrates, such as ascidians that belong to the phylum Chordata, which show a high CNS regeneration capacity even in adulthood. Here, we investigated the roles of dermatan sulfate, a type of GAG that was obtained from the ascidian Phallusia nigra. We investigated the neuroprotective and antioxidant properties of Phallusia nigra dermatan sulfate (PnDS) after neurotoxic damage induced by the pesticide rotenone using the Neuro-2A cell lineage. Neuroprotection was observed through a mitochondrial activity analysis. A morphometric analysis revealed long unbranched neurites after incubation with PnDS and co-incubation with PnDS and rotenone. Furthermore, PnDS showed antioxidant activity that reduced reactive oxygen species (ROS) even in co-incubation with rotenone. The reduced ROS probably occurred because PnDS increased the activity of the antioxidant enzymes superoxide dismutase and catalase and improved total antioxidant capacity, which protected cells from damage, as observed through decreased levels of lipid peroxidation. These data suggest a neuroprotective and antioxidant role of PnDS even under neurodegenerative conditions caused by rotenone.

Purification, compositional analysis, and anticoagulant capacity of chondroitin sulfate/dermatan sulfate from bone of corb (Sciaena umbra).

Chondroitin sulfate/dermatan sulfate (CS/DS) were isolated and purified for the first time from the bone of corb (Sciaena umbra) (CBG) and their chemical composition and anticoagulant activity were assessed. Infrared spectrum and agarose-gel electrophoresis for extracted CS/DS were also investigated. The results showed that the purified CS/DS obtained at a yield of 10% contains about 31.28% sulfate and an average molecular mass of 23.35 kDa. Disaccharide analysis indicated that CBG was composed of monosulfated disaccharides in positions 6 and 4 of the N-acetylgalactosamine (8.6% and 40.0%, respectively) and disulfated disaccharides in different percentages. The charge density was 1.4 and the ratio of 4:6 sulfated residues was equal to 4.64. Chondroitinase AC showed that the purified CS/DS contained mainly 74% CS and 26% DS. Moreover, the new CS/DS extracted from bone of corb showed a strong anticoagulant effect through activated partial thrombosis time (aPTT), thrombin time (TT) and prothrombin time (PT). In fact, CBG prolonged significantly (p < 0.05), aPTT and PT about 2.62 and 1.26 fold, respectively, greater than that of the negative control at a concentration of 1000 µg/mL. However, TT assay of CBG was prolonged 3.53 fold compared with the control at 100 µg/mL. The purified CS/DS displayed a promising anticoagulant potential, which may be used as a novel and soothing drug.

Chondroitin sulfate/dermatan sulfate from corb (Sciaena umbra) skin: Purification, structural analysis and anticoagulant effect.

In this study, chondroitin sulfate/dermatan sulfate was isolated and purified from the skin of corb (Sciaena umbra) (CSG) with a yield of 6.2%. Chemical and structural analysis showed that CSG consisted of high sulfate content 28.74% and an average molecular weight of 15.46 KDa. The separation of CSG by agarose-gel electrophoresis revealed the presence of DS and CS. Structural analysis of the purified CS/DS by means of SAX-HPLC after treatment with specific chondroitinases showed that this polymer was composed of nonsulfated disaccharide, monosulfated disaccharides and disulfated disaccharides in various percentages. The results also suggest that the percentage of CS and DS recovred in CSG were 24% and 76%, respectively. Anticoagulant activity in vitro was measured in plasma using classical anticoagulation tests: activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. The findings thus indicated that the purified CS/DS exhibits a remarkably high anticoagulant effect.

Sequencing the Dermatan Sulfate Chain of Decorin.

Glycomics represents one of the last frontiers and most challenging in omic analysis. Glycosylation occurs in the endoplasmic reticulum and the Golgi organelle and its control is neither well-understood nor predictable based on proteomic or genomic analysis. One of the most structurally complex classes of glycoconjugates is the proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains. Previously, our laboratory solved the structure of the chondroitin sulfate chain of the bikunin PG. The current study examines the much more complex structure of the dermatan sulfate GAG chain of decorin PG. By utilizing sophisticated separation methods followed by compositional analysis, domain mapping, and tandem mass spectrometry coupled with analysis by a modified genetic algorithm approach, the structural motif for the decorin dermatan sulfate chain was determined. This represents the second example of a GAG with a prominent structural motif, suggesting that the structural variability of this class of glycoconjugates is somewhat simpler than had been expected.

Glycosaminoglycans and glycolipids as potential biomarkers in lung cancer.

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.

Quantification and comparison of acidic polysaccharides in edible fish intestines and livers using HPLC-MS/MS.

Fish intestines and livers are usually considered as delicious and nutritious food in China. Acidic polysaccharides are important nutrients in these food of animal origin, but there is currently little information regarding their quantitative distributions. The present study demonstrated a method to quantify acidic polysaccharides simultaneously by analyzing their disaccharides produced from the acid hydrolysis using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry. The recoveries for these acidic polysaccharides were all 97%-115% with relative standard deviation of 3.0%-9.0%. All of the acidic polysaccharides had good linearities. Then this method was applied to determine the composition of acidic polysaccharides in 5 edible fish livers and intestines. Besides well-known glycosaminoglycans (GAGs) including hyaluronic acid (HA), Chondroitin sulfate (CS), dermatan sulfate (DS) and heparin (HP), 4 novel acidic polysaccharides including 2 GAGs and 2 non-GAGs comprised of hexose-hexuronic acid repeating units were also found. CS and HP were the major acidic polysaccharides components in fish intestines and livers, respectively. The absolute amounts of acidic polysaccharides differed greatly in these fish tissues, but their proportions showed similarity in the same type of tissues. The present study demonstrated an effective method for acidic polysaccharides quantification, and revealed acidic polysaccharides compositions of edible fish livers and intestines.

Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin.

Because of the complexity and global nature of the heparin supply chain, the control of heparin quality during manufacturing steps is essential to ensure the safety of the final active pharmaceutical ingredient (API). For this reason, there is a need to develop consistent analytical methods able to assess the quality of heparin early in production (i.e., as the crude heparin before it is purified to API under cGMP conditions). Although a number of analytical techniques have been applied to characterize heparin APIs, few of them have been applied for crude heparin structure and composition analyses. Here, to address this issue, NMR spectroscopy and chemometrics were applied to characterize 88 crude heparin samples. The samples were also analyzed by strong anion exchange HPLC (SAX-HPLC) as an orthogonal check of the purity levels of the crudes analyzed by NMR. The HPLC data showed that the chemometric analysis of the NMR data differentiated the samples based on their purity. These orthogonal approaches differentiated samples according their glycosaminoglycan (GAG) composition and their mono and disaccharide composition and structure for each GAG family (e.g., heparin/heparan, dermatan sulfate, and chondroitin sulfate A). Moreover, quantitative HSQC and multivariate analysis (PCA) were used to distinguish between crude heparin of different animal and tissue sources.

Purification and sequence characterization of chondroitin sulfate and dermatan sulfate from fishes.

Chondroitin sulfate (CS) and dermatan sulfate (DS) were extracted and purified from skins or bones of salmon (Salmo salar), snakehead (Channa argus), monkfish (Lophius litulon) and skipjack tuna (Katsuwonus pelamis). Size, structural sequences and sulfate groups of oligosaccharides in the purified CS and DS could be characterized and identified using high performance liquid chromatography (HPLC) combined with Orbitrap mass spectrometry. CS and DS chain structure varies depending on origin, but motif structure appears consistent. Structures of CS and DS oligosaccharides with different size and sulfate groups were compared between fishes and other animals, and results showed that some minor differences of special structures could be identified by hydrophilic interaction chromatography-liquid chromatography-fourier transform-mass/mass spectrometry (HILIC-LC-FT-MS/MS). For example, data showed that salmon and skipjack CS had a higher percentage content of high-level sulfated oligosaccharides than that porcine CS. In addition, structural information of different origins of CS and DS was analyzed by principal component analysis (PCA) and results showed that CS and DS samples could be differentiated according to their molecular conformation and oligosaccharide fragments information. Understanding CS and DS structure derived from different origins may lead to the production of CS or DS with unique disaccharides or oligosaccharides sequence composition and biological functions.

Biological function of unique sulfated glycosaminoglycans in primitive chordates.

Glycosaminoglycans with unique sulfation patterns have been identified in different species of ascidians (sea squirts), a group of marine invertebrates of the Phylum Chordata, sub-phylum Tunicata (or Urochordata). Oversulfated dermatan sulfate composed of [4-α-L-IdoA-(2-O-SO3)-1 â†’ 3-ß-D-GalNAc(4-OSO3)-1]n repeating disaccharide units is found in the extracellular matrix of several organs, where it seems to interact with collagen fibers. This dermatan sulfate co-localizes with a decorin-like protein, as indicated by immunohistochemical analysis. Low sulfated heparin/heparan sulfate-like glycans composed mainly of [4-α-L-IdoA-(2-OSO3)-1 â†’ 4-α-D-GlcN(SO3)-1 (6-O-SO3)-1]n and [4-α-L-IdoA-(2-O-SO3)-1 â†’ 4-α-D-GlcN(SO3)-1]n have also been described in ascidians. These heparin-like glycans occur in intracellular granules of oocyte assessory cells, named test cells, in circulating basophil-like cells in the hemolymph, and at the basement membrane of different ascidian organs. In this review, we present an overview of the structure, distribution, extracellular and intracellular localization of the sulfated glycosaminoglycans in different species and tissues of ascidians. Considering the phylogenetic position of the subphylum Tunicata in the phylum Chordata, a careful analysis of these data can reveal important information about how these glycans evolved from invertebrate to vertebrate animals.

Implementation of infrared and Raman modalities for glycosaminoglycan characterization in complex systems.

Glycosaminoglycans (GAGs) are natural, linear and negatively charged heteropolysaccharides which are incident in every mammalian tissue. They consist of repeating disaccharide units, which are composed of either sulfated or non-sulfated monosaccharides. Depending on tissue types, GAGs exhibit structural heterogeneity such as the position and degree of sulfation or within their disaccharide units composition being heparin, heparan sulfate, chondroitine sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. They are covalently linked to a core protein (proteoglycans) or as free chains (hyaluronan). GAGs affect cell properties and functions either by direct interaction with cell receptors or by sequestration of growth factors. These evidences of divert biological roles of GAGs make their characterization at cell and tissue levels of importance. Thus, non-invasive techniques are interesting to investigate, to qualitatively and quantitatively characterize GAGs in vitro in order to use them as diagnostic biomarkers and/or as therapeutic targets in several human diseases including cancer. Infrared and Raman microspectroscopies and imaging are sensitive enough to differentiate and classify GAG types and subtypes in spite of their close molecular structures. Spectroscopic markers characteristic of reference GAG molecules were identified. Beyond these investigations of the standard GAG spectral signature, infrared and Raman spectral signatures of GAG were searched in complex biological systems like cells. The aim of the present review is to describe the implementation of these complementary vibrational spectroscopy techniques, and to discuss their potentials, advantages and disadvantages for GAG analysis. In addition, this review presents new data as we show for the first time GAG infrared and Raman spectral signatures from conditioned media and live cells, respectively.

Structural characterization and anti-inflammatory activity of two novel polysaccharides from the sea squirt, Ascidiella aspersa.

It is now recognized that certain polysaccharides can exhibit anti-inflammatory activity, including the glycosaminoglycan (GAG) heparin that is widely used as an anti-coagulant drug. However, it would be desirable to identify molecules that retain the anti-inflammatory actions of heparin, but that are devoid of significant anti-coagulant activity. In the present study we have identified a number of novel GAG and GAG-like polysaccharides (VRP327) from marine organisms, most of which were resistant to digestion by heparinase II and chondroitinase ABC. Fourier transform infra-red spectrum (FTIR) revealed species with variable degrees of sulphation and monosaccharide analysis revealed a range of sugar compounds, which in some cases included sugars not present in mammalian GAGs. (1)H NMR spectra of these species are consistent with the structures of complex polysaccharides. From an initial screening cascade to remove compounds having significant anti-coagulant activity and no overt cytotoxicity, we identified a high molecular weight oversulphated dermatan sulphate (VRP327) isolated from the tunicate Ascidiella aspersa which was fully characterised by NMR spectroscopy. This material was depolymerised to produce well characterized low molecular weight fractions which were demonstrated to be non-toxic, with low levels of anti-coagulant activity, and to have demonstrable anti-inflammatory activity assessed in several in vitro and in vivo models. The identification of low molecular weight polysaccharides having significant anti-inflammatory activity without significant anti-coagulant activity may provide novel templates for the development of a novel class of anti-inflammatory drugs.

A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses.

INTRODUCTION: Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III). METHODS: Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs. RESULTS: The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them. CONCLUSION: The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay.

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: a brand-new attempt at chondroitin sulfate isomers separation.

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of -10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 µm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.

Sea, carbohydrates and clotting: a triad on the road of drug discovery.

Cardiovascular diseases (CDs) are the principal cause of death in the world. Anticoagulation is the commonest therapeutic strategy for treatments of CDs in clinical settings. Although possessed of numerous downsides, heparin is the main clinical anticoagulant/antithrombotic agent used so far. Novel sulfated polysaccharides like the marine dermatan sulfate, sulfated fucans and galactans are also able to block clot and thrombus formation. These relatively new marine glycans call special attention mostly due to their unique structures and distinct mechanisms of action. This structural uniqueness is seen by the peculiar aspect of these polysaccharides being made of clear and regular sulfation patterns. The structures have been reported only in polysaccharides from marine invertebrates like sea urchins and cucumbers. This report intends to prove the promising combination of the triad sea-carbohydrates-clotting in drug discovery of the cardiovascular field.

Characterisation of hyaluronic acid and chondroitin/dermatan sulfate from the lumpsucker fish, C. lumpus.

The lumpsucker, Cyclopterus lumpus, a cottoid teleost fish found in the cold waters of the North Atlantic, and North Pacific, was identified as a possible source of GAGs. The GAGs present in the C. lumpus dorsal hump and body wall tissue were isolated and purified. Two fractions were analysed by NMR and their GAG structures determined as hyaluronic acid and CS/DS chains. The latter fraction contained GlcA (65% of the total uronic acids) and IdoA (the remaining 35%). All uronic acid residues were unsulfated, whilst 86% of the GalNAc was 4-sulfated and 14% was 6-sulfated. The presence of GlcA-GalNAc4S, IdoA-GalNAc4S and GlcA-GalNAc6S disaccharide fragments was confirmed. The isolated GAGs obtained from each tissue were biochemically characterised. The lumpsucker offers a high yield source of GAGs, which compares favourably with other sources such as shark cartilage.

Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling.

Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates.

Brain chondroitin/dermatan sulfate, from cerebral tissue to fine structure: extraction, preparation, and fully automated chip-electrospray mass spectrometric analysis.

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs) are covalently linked to proteins, building up a wide range of proteoglycans, with a prevalent expression in the extracellular matrix (ECM). In mammalian tissues, these GAG species are often found as hybrid CS/DS chains. Their structural diversity during chain elongation is produced by variability of sulfation in the repeating disaccharide units. In central nervous system, a large proportion of the ECM is composed of proteoglycans; therefore, CS/DS play a significant role in the functional diversity of neurons, brain development, and some brain diseases. A requirement for collecting consistent data on brain proteoglycan glycosylation is the development of adequate protocols for CS/DS extraction and detailed compositional and structure analysis. This chapter will present a strategy, which combines biochemical tools for brain CS/DS extraction, purification, and fractionation, with a modern analytical platform based on chip-nanoelectrospray multistage mass spectrometry (MS) able to provide information on the essential structural elements such as epimerization, chain length, sulfate content, and sulfation sites.

Ascidian dermatan sulfates attenuate metastasis, inflammation and thrombosis by inhibition of P-selectin.

BACKGROUND: Cancer-associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti-inflammatory activities that are linked to inhibition of P-selectin and/or L-selectin. P-selectin-mediated platelet-tumor cell and tumor cell-endothelium interactions facilitate the initial steps of metastasis. OBJECTIVES AND METHODS: The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P-selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. RESULTS: Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2-GalNAc)(n) , but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P-selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC-38 colon carcinoma and B16-BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl(3) -induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P-selectin and thereby the binding of activated platelets during thrombus formation. CONCLUSIONS: Collectively, these findings provide evidence that specific inhibition of P-selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.