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The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked ␤-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked ␣-fucose and one or two nonsulfated, 4-linked ␤-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.

The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastinactivated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.Heparan sulfate proteoglycans are complex macromolecules that consist of a protein backbone to which heparan sulfate chains are covalently linked (1). They are ubiquitous compounds found in a wide variety of vertebrate and invertebrate tissues (2) and are actively synthesized by cells in culture (3). These proteoglycans have been found to be present on the plasma membrane and in the extracellular matrix (4, 5) and exhibit a peculiar structural variability according to the tissue and species of origin (2,6). Despite their wide occurrence, little is known of their biological function. They have been implicated in several biological processes such as cell-cell recognition (7), tissue differentiation (8), organization of extracellular matrices (9), and cell-matrix and cell-substrate adhesion (10).The availability of two heparitinases (11, 12) and a heparinase (EC 4.2.2.7) from Flavobacterium heparinum (12, 13), which can be used in conjunction to elucidate the distribution and grouping in the polymeric chain of disaccharides with various degrees of sulfation and with different hexuronic acid moieties, has enabled us to undertake the structural study of a proteoglycan isolated from the conditioned medium of endothelial cell cultures that appears to be highly characteristic of this cell type. Using these enzymes, we have determined that the glycosaminoglycan chain of this proteoglycan contains heparin segments and have developed a strategy for the elucidation of the sequence of the disaccharide repeating units that may be applicable to the study of other structurally related compounds. MATERIALS AND METHODSSubstrates, Enzymes, and Materials. Heparin from bovine intestinal mucosa and heparan sulfate from bovine pancreas were gifts from P. Bianchini (Opocri...

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The correlation between structure, anticlotting, antithrombotic and hemorrhagic activities of heparin, heparan sulfate, low molecular weight heparins and heparin-like compounds from various sources that are in used in clinical practice or under development is briefly reviewed. Heparin-like molecules composed exclusively of iduronic acid 2-O-sulfate residues have weak anticlotting activities, whereas molecules that contain both iduronic acid 2-O sulfate, iduronic acid and small amounts of glucuronic acid, such as heparin, or mixed amounts of glucuronic and iduronic acids (mollusk heparins) possess high anticlotting and anti-Xa activities. These results also suggest that a proper combination of these elements might produce a strong antithrombotic agent. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate derived from bovine pancreas and a sulfated fucan from brown algae have a potent antithrombotic activity in arterial and venous thrombosis model "in vivo" with a negligible activity upon the serine-proteases of the coagulation cascade "in vitro". These and other results led to the hypothesis that antithrombotic activity of heparin and other antithrombotic agents is due at least in part by their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate. All the antithrombotic agents derived from heparin and other heparinoids have hemorrhagic activity. Exceptions to this are a heparan sulfate from bovine pancreas and a sulfated fucan derived from brown algae, which have no hemorrhagic activity but have high antithrombotic activities "in vivo". Once the structure of these compounds are totally defined it will be possible to design an ideal antithrombotic.

The lumen of the Golgi apparatus is the subcellular site where galactose is transferred, from UDP-galactose, to the oligosaccharide chains of glycoproteins, glycolipids, and proteoglycans. The nucleotide sugar, which is synthesized in the cytosol, must first be transported into the Golgi lumen by a specific UDP-galactose transporter. Previously, a mutant polarized epithelial cell (MDCKII-RCA r ) with a 2% residual rate of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., RodriguezBoulan, E., and Simons, K. (1988) J. Biol. Chem. 263, 16283-16290). The mutant has an enrichment in glucosyl ceramide and cell surface glycoconjugates bearing terminal N-acetylglucosamine, as well as a 75% reduction in sialylation of cell surface glycoproteins and glycosphingolipids.We have now studied the biosynthesis of galactose containing proteoglycans in this mutant and the corresponding parental cell line. Wild-type Madin-Darby canine kidney cells synthesize significant amounts of chondroitin sulfate, heparan sulfate, and keratan sulfate, while the above mutant synthesizes chondroitin sulfate and heparan sulfate but not keratan sulfate, the only proteoglycan containing galactose in its glycosaminoglycan polymer. The mutant also synthesizes chondroitin 6-sulfate rather than only chondroitin 4-sulfate as wild-type cells. Together, the above results demonstrate that the Golgi membrane UDP-galactose transporter is rate-limiting in the supply of UDP-galactose into the Golgi lumen; this in turn results in selective galactosylation of macromolecules. Apparently, the K m for galactosyltransferases involved in the synthesis of linkage regions of heparan sulfate and chondroitin sulfate are significantly lower than those participating in the synthesis of keratan sulfate polymer, glycoproteins, and glycolipids. The results also suggest that the 6-Osulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate.Proteoglycans are complex macromolecules consisting of a protein core to which glycosaminoglycans are covalently linked. Their strategic localization in the plasma membrane and extracellular matrix makes them important intermediates between cells and their environment (1, 2). They have been implicated to play a role in cell-cell (3) and cell-matrix interactions (4), organization of basement membranes (5), control of macromolecules' diffusion (6), and also interactions with a variety of ligands such as growth factors, hormones, and neurotransmitters (7).In most GAGs, 1 the repeating disaccharide units are composed of one amino sugar and one uronic acid, the only exception being keratan sulfate in which galactose replaces the sugar acid. Most GAGs are attached to serine of the core protein by a tetrasaccharide of xylose-galactose-galactose-glucuronic acid (8, 9). Keratan sulfate is an exception; keratan sulfate I, from cornea, is N-linked to proteins and keratan sulfate II, from skeletal tis...