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PubMed2002

Analysis of heparan sulfate oligosaccharides with ion pair-reverse phase capillary high performance liquid chromatography-microelectrospray ionization time-of-flight mass spectrometry.

Heparan sulfate, a cell surface bound glycosaminoglycan polysaccharide, has been implicated in numerous biological functions. Heparan sulfate molecules are highly complex and diverse, yet deceivingly look simple and similar, rendering structure--function correlation tedious. Current chromatographic and mass spectrometric techniques have limitations for analyzing glycosaminoglycan samples that are in low abundance and that are large in size, due to their highly acidic nature arising from a large number of sulfate and of carboxylate groups. A new methodology was developed using capillary ion-paired reverse-phase C18 HPLC directly coupled to ESI-TOF-MS to address the above issues. On the basis of HS disaccharide analysis, dibutylamine was found to be the best suited for HS analysis among many ion-pairing agents investigated. Next, analysis of oligosaccharides derived from heparosan, the precursor for heparan sulfate, was undertaken to demonstrate its greater applicability in a more complex structural analysis. The established chromatographic conditions enabled the characterization of heparosan oligosaccharides of sizes up to tetracontasaccharide with high resolution in a single run and were amenable to negative ion electrospray MS in which sodium adduction and fragmentation were avoided. To date, these are the largest nonsulfated HS precursor oligosaccharides to be characterized by LC/MS. Finally, the current methodology was applied to the characterization of the biologically important ATIII binding pentasaccharide and its precursors, which differ from each other by sulfation pattern and/or degree of sulfation. All of these pentasaccharides were well-resolved and characterized by the LC/MS system with (34)SO(4) as a mass spectral probe. This newly developed methodology facilitates the purification and rapid characterization of biologically significant HS oligosaccharides, and will thus expedite their synthesis. These findings should undoubtedly pave the way in deciphering multiple functional arrangements, ascribed to many biological activities, which are predictably embedded in a single large chaotic, yet well-organized HS polysaccharide chain. Development of newer techniques for HS oligosaccharide analysis is greatly needed in the postgenome era as attention shifts to the functional implications of proteins and carbohydrates in general and HS in particular.

Antithrombin III↗
PubMed2006

Enzymatic degradation of oligosaccharides in pinto bean flour.

The use of dry edible beans is limited due to the presence of flatulence factors, the raffinose oligosaccharides. Our objective was to investigate the process for the removal of oligosaccharides from pinto bean using enzymatic treatment and to compare it to removal by soaking and cooking methods. Crude enzyme preparation was produced by six fungal species on wheat bran- and okara-based substrates with soy tofu whey. The loss of raffinose oligosaccharides after soaking pinto beans for 16 h at the room temperature was 10%, after cooking for 90 min was 52%, and after autoclaving for 30 min was 58%. On the other hand, the treatment using crude alpha-galactosidase (60 U mL(-1)) produced by Aspergillus awamori NRRL 4869 from wheat bran-based substrate with soy tofu whey on pinto bean flour for 2 h completely hydrolyzed raffinose oligosaccharides. These results supported that the enzymatic treatment was the most effective among various processing methods tested for removing the raffinose oligosaccharides, and hence, crude alpha-galactosidases from fungi have potential use in the food industry.

Aspergillus↗
PubMed2006

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria.

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.

Bacteria↗
PubMed1998

Oligosaccharide excretion in adult Gaucher disease.

Gaucher disease is a lysosomal storage disease characterized by storage of glucocerebroside due to lysosomal glucocerebrosidase deficiency. Increased urinary excretion of sialyloligosaccharides and mannosylglycoasparagines has been described for two patients with the infantile form of the disease, probably as a consequence of obstruction of lysosomal functioning due to the glycolipid accumulation in lysosomes. By thin-layer chromatography, we found increased urinary oligosaccharide excretion in a series of adult non-neuronopathic patients. Oligosaccharide patterns were comparable between patients and also with the pattern observed in infantile Gaucher disease. Composition was analysed by methanolysis and gas chromatography. Mannose and N-acetylglucosamine are the main carbohydrates in all oligosaccharide bands. A statistically significant correlation was found between oligosaccharide excretion and the severity of the disease expressed as severity score index. Patients treated with enzyme replacement therapy showed a reduction up to 65% of the original oligosaccharide excretion after 1 year of treatment, comparable with the reduction in spleen volume.

Adult↗
PubMed1998

Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease.

Many studies have reported changes in the carbohydrate structure of serum glycoproteins in disease, but this information is often of limited value for understanding disease mechanisms because it is obtained with simple and/or indirect methodologies that determine only one structural feature. On the other hand, more detailed carbohydrate methodologies are time-consuming and require a lot of purified material. Using haptoglobin (Hp) as a model protein, a new procedure was devised that determined the oligosaccharide composition of very small amounts of Hp in a relatively short time. The Hp was purified by batch affinity-chromatography, oligosaccharides were removed with PNGase F, and the oligosaccharide composition of charged species was determined using HPAEC/PAD (Dionex carbohydrate analyser). The method was applied to the analysis of Hp from eight healthy individuals and 37 patients with different inflammatory diseases or cancers. Twenty-seven oligosaccharides were consistently detected, but the majority could not be identified. However, by calculating retention times relative to the sialylated biantennary peak (Neu5Ac(alpha)2-3/6Gal(beta)1-4GlcNAc(beta)1-2Man(alpha)1-6(Neu 5Ac(alpha)2-3/6Gal(beta)1-4GlcNAc(beta)1-2Man(alpha)1-3)Man(beta)1-4G lcNAc(beta)1-4GlcNAc) it was possible to compare profiles quantitatively. Although no peak was identified as disease-specific, characteristic and reproducible profiles were obtained. Particularly striking were reductions in the major peaks in Crohn's disease, rheumatoid arthritis, stomach cancer, accompanied by increases in unidentified peaks. Previous studies suggested that many of the unknown peaks were due to increased sialylation and fucosylation. Only small changes in patterns were observed for breast and ovarian cancer. The new procedure will be very useful in the characterization of oligosaccharide composition of glycoproteins in clinical specimens.

Adult↗
PubMed1999

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl.

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal beta1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac alpha2,3Gal beta1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.

Acetylgalactosamine↗
PubMed1999

Novel multi-dimensional heteronuclear NMR techniques for the study of 13C-O-acetylated oligosaccharides: expanding the dimensions for carbohydrate structures.

Complex carbohydrates have critical roles in a wide variety of biological processes. An understanding of the molecular mechanisms that underlie these processes is essential in the development of novel oligosaccharide-based therapeutic strategies. Unfortunately, obtaining detailed structural information for larger oligosaccharides (> 10 residues) can be exceedingly difficult, especially where the amount of sample available is limited. Here we demonstrate the application of 13C O-acetylation in combination with novel NMR experiments to obtain much of the information required to characterize the primary structure of oligosaccharides. (H)CMe COH-HEHAHA and H(CMe)COH-HEHAHA experiments are presented that use heteronuclear Hartmann-Hahn transfer to correlate the acetyl groups with sugar ring protons in peracetylated oligosaccharides. The in-phase, pure absorption nature of the correlation peaks in these experiments allows measurement of both chemical shifts and, importantly, 1H-1H coupling constants that are used to define the stereochemistry of the sugar ring. The (HCMe)COH and (HCMe)COH-RELAY experiments provide additional methods for obtaining chemical shift assignments for larger oligosaccharides to define the sites of glycosidic linkages from the patterns of acetylation.

Acetylation↗
PubMed1997

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin beta-subunit by disruption of disulfide bonds.

The human chorionic gonadotropin beta-subunit (hCGbeta) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGbeta produced in Chinese hamster ovary (CHO) cells: wild-type hCGbeta (betaWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of betaWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled betaWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGbeta molecule.

Acetylglucosamine↗
PubMed1997

Heparin oligosaccharides: inhibitors of the biological activity of bFGF on Caco-2 cells.

A number of growth factors, including members of the fibroblast growth factor (FGF) family - hepatocyte growth factor, vascular endothelial growth factor and heparin-binding epidermal growth factor - are dependent on heparan sulphate (HS) for biological activity mediated through their high-affinity signal-transducing receptors. This obligate requirement for HS prompted the search for antagonists of HS function that could be used as anti-growth factor drugs for the treatment of cancer. Basic FGF (bFGF) was the focus of this study. Caco-2, a human colon carcinoma cell line, was adapted to growth in serum-free medium so that investigation of its growth factor requirements for growth and migration could be performed in defined conditions (Jayson GC, Evans GS, Pemberton PW, Lobley RW, Allen T 1994, Cancer Res, 54, 5718-5723). This cell line multiplied and moved in a dose-dependent manner in response to bFGF. Here, we show that the mitogenic response to bFGF is dependent on the presence of heparan sulphate. A library of heparin oligosaccharides with uniform composition but variable length was generated [general formula [IdoA(2S)-GlcNS(6S)n], and oligosaccharides of defined lengths were tested for their ability to inhibit the biological activity of bFGF. While intact heparin and heparin-derived fragments of 12 monosaccharide units did not affect bFGF-induced cell division or bFGF-induced cell migration, octasaccharides and decasaccharides potently inhibited the bFGF-induced growth and migration responses. In particular, octasaccharides completely inhibited these biological activities at 10 microg ml-, a clinically achievable and tolerable concentration. This study shows that the length of an oligosaccharide determines its ability to block the biological activity of bFGF. The observation that the biological activity of cell-surface heparan sulphate can be antagonized in this way in a human carcinoma cell line suggests that oligosaccharides should be investigated further as anti-growth factor agents for the treatment of cancer. In addition, the results suggest that the clinical evaluation of low-molecular weight heparin (LMWH) as an anti-cancer agent might benefit from subfractionation of the LMWH, to remove oligosaccharides of 12 or more residues.

Animals↗
PubMed2002

Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions.

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.

Animals↗
PubMed1999

Effect of nondigestible oligosaccharides on large-bowel functions, blood lipid concentrations and glucose absorption in young healthy male subjects.

OBJECTIVE: To study the effect of the intake of 15 g nondigestible oligosaccharides per day on various parameters of large-bowel function, as well as on blood lipid concentrations and glucose absorption in man. DESIGN: Latin square, randomized, double-blind, diet-controlled. SETTING: Metabolic research unit. SUBJECTS: Twelve apparently healthy men (mean age 23 years), recruited from the Institute's pool of volunteers, no drop-outs. INTERVENTIONS: Four treatment periods of 3 weeks: inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and control; analyses of stool weight, intestinal transit, faecal pH, short-chain fatty acids, bile acids, faecal enzymes, blood lipids and glucose absorption. RESULTS: As compared to the control treatment: higher concentration of faecal acetate (inulin and GOS, P < 0.05) and valerate (inulin, P < 0.05), significantly lower concentration of faecal deoxycholic acid (inulin and FOS, P < 0.05 and P< 0.02, respectively) and beta-glucuronidase activity (inulin and GOS, P < 0.05 and P < 0.02 respectively). Other changes of faecal parameters and those of blood lipids and glucose absorption were statistically not significant. CONCLUSIONS: RESULTS indicate that nondigestible oligosaccharides are (partly) fermented in the human colon, but in healthy young men the effects are limited. Also the consumption of 15 g nondigestible oligosaccharides does not seem to alter blood lipid concentrations and glucose absorption in our young healthy adults.

Adult↗
PubMed2006

Glycosynthase-based synthesis of xylo-oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi.

Glycosynthases are synthetic enzymes derived from retaining glycosidases in which the catalytic nucleophile has been replaced. The mutation allows irreversible glycosylation of sugar acceptors using glycosyl fluoride donors to afford oligosaccharides without any enzymatic hydrolysis. Glycosynthase technology has proven fruitful for the facile synthesis of useful oligosaccharides, therefore the expansion of the glycosynthase repertoire is of the utmost importance. Herein, we describe for the first time a glycosynthase, derived from a retaining xylanase, that synthesizes a range of xylo-oligosaccharides. The catalytic domain of the retaining endo-1,4-beta-xylanase from Cellulomonas fimi (CFXcd) was successfully converted to the corresponding glycosynthase by mutation of the catalytic nucleophile to a glycine residue. The mutant enzyme (CFXcd-E235G) was found to catalyze the transfer of a xylobiosyl moiety from alpha-xylobiosyl fluoride to either p-nitrophenyl beta-xylobioside or benzylthio beta-xylobioside to afford oligosaccharides ranging in length from tetra- to dodecasaccharides. These products were purified by high performance liquid chromatography in greater than 60% combined yield. 1H and 13C NMR spectroscopic analyses of the isolated p-nitrophenyl xylotetraoside and p-nitrophenyl xylohexaoside revealed that CFXcd-E235G catalyzes both the regio- and stereo-selective synthesis of xylo-oligosaccharides containing, exclusively, beta-(1 --> 4) linkages.

Carbohydrate Conformation↗
PubMed1965

The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group U (o 43) lipopolysaccharides.

1. A series of oligosaccharides was isolated from Salmonella milwaukee lipopolysaccharide by partial acid hydrolysis. 2. Structural studies on these oligosaccharides indicated that the O-specific side chain of this lipopolysaccharide has a repeating pentasaccharide unit that is probably alpha-d-galactosyl-(1-->3)-beta-d-galactosyl- (1-->3)-N-acetylgalactosaminyl-(1-->3)-N-acetyl- d-glucosaminyl-(1-->4)-l-fucose. 3. Another oligosaccharide, which is not structurally related to the repeating pentasaccharide unit, has also been isolated and it is indistinguishable from an oligosaccharide obtained from Salmonella ;rough' (R) lipopolysaccharides. The isolation of this and similar core oligosaccharides from all chemotype VI lipopolysaccharides supports the view that Salmonella S-lipopolysaccharides have a common core that is probably identical with RII lipopolysaccharide.

Antigens↗
PubMed1974

The copolymeric structure of pig skin dermatan sulphate. Isolation and characterization of L-idurono-sulphate-containing oligosaccharides from copolymeric chains.

Dermatan sulphate was degraded by testicular hyaluronidase and an oversulphated fraction was isolated by ion-exchange chromatography. This preparation, which contained fairly long segments derived from the non-reducing terminal portion of the molecule, was subjected to periodate oxidation under acidic conditions. The oxidized iduronic acid residues were cleaved by reduction-hydrolysis (Smith-degradation) (Fransson & Carlstedt, 1974) or by alkaline elimination. The oligosaccharides so obtained contained both GlcUA (glucuronic acid) and IdUA-SO(4) (sulphated iduronic acid) residues. Copolymeric oligosaccharides obtained after alkaline elimination were cleaved by chondroitinase-AC into disaccharide and higher oligosaccharides. Since the corresponding oligosaccharides obtained by Smith-degradation were unaffected by this enzyme, it was concluded that the carbohydrate sequences were GalNAc-(IdUA-GalNAc)(n)-GlcUA-GalNAc. The iduronic acid-containing sequences were resistant to digestion with chondroitinase-ABC. It was demonstrated that the presence of unsulphated N-acetylgalactosamine residues in these sequences could be responsible for the observed effect. This information was obtained in an indirect way. Chemically desulphated dermatan sulphate was found to be a poor substrate for the chondroitinase-ABC enzyme. Moreover, digestion with chondroitinase-ABC of chondroitinase-AC-degraded dermatan sulphate released periodate-resistant iduronic acid-containing oligosaccharides. It is concluded that copolymeric sequences of the following structure are present in pig skin dermatan sulphate: [Formula: see text] N-acetylgalactosamine moieties surrounding IdUA-SO(4) residues are unsulphated to a large extent.

Animals↗
PubMed2005

Mode of action of xylogalacturonan hydrolase towards xylogalacturonan and xylogalacturonan oligosaccharides.

XGH (xylogalacturonan hydrolase; GH 28) is an enzyme that is capable of degrading XGA (xylogalacturonan), which is a polymer of alpha-D-galacturonic acid, highly substituted with beta-D-xylose. XGA is present in cell walls of various plants and exudates, such as gum tragacanth. XGA oligosaccharides were derived from an XGH digestion of gum tragacanth, then fractionated, and analysed for their sugar composition and structure by matrix-assisted laser-desorption ionization-time-of-flight MS and nanospray MS. Several oligosaccharides from XGA were identified with different galacturonic acid/xylose ratios including five oligosaccharide isomers. Although XGH can act as an endo-enzyme, product-progression profiling showed that the disaccharide GalAXyl was predominantly produced from XGA by XGH, which indicated also an exolytic action. The latter was further supported by degradation studies of purified oligosaccharide GalA4Xyl3. It was shown that XGH acted from the non-reducing end towards the reducing end of this oligosaccharide, and showed the processive character of XGH. The results from this study further show that although XGH prefers to act between two xylosidated GalA units, it tolerates unsubstituted GalA units in its -1 and +1 subsites.

Carbohydrate Conformation↗
PubMed2005

Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa.

One of the mechanisms contributing to the protection by breast-feeding of the newborn against enteric diseases is related to the ability of human milk oligosaccharides to prevent the attachment of pathogenic bacteria to the duodenual epithelium. Indeed, a variety of fucosylated oligosaccharides, specific to human milk, form part of the innate immune system. In the present study, we demonstrate the specific blocking of PA-IIL, a fucose-binding lectin of the human pathogen Pseudomonas aeruginosa, by milk oligosaccharides. Two fucosylated epitopes, Lewis a and 3-fucosyl-lactose (Lewis x glucose analogue) bind to the lectin with dissociation constants of 2.2x10(-7) M and 3.6x10(-7) M respectively. Thermodynamic studies indicate that these interactions are dominated by enthalpy. The entropy contribution is slightly favourable when binding to fucose and to the highest-affinity ligand, Lewis a. The high-resolution X-ray structures of two complexes of PA-IIL with milk oligosaccharides allow the precise determination of the conformation of a trisaccharide and a pentasaccharide. The different types of interaction between the oligosaccharides and the protein involve not only hydrogen bonding, but also calcium- and water-bridged contacts, allowing a rationalization of the thermodynamic data. This study provides important structural information about compounds that could be of general application in new therapeutic strategies against bacterial infections.

Adhesins, Bacterial↗
PubMed2006

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol.

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.

Alkaloids↗
PubMed1984

Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4.

Oligosaccharides of well-defined molecular size were prepared from heparin by nitrous acid depolymerization, affinity chromatography on immobilized antithrombin III (see footnote on Nomenclature) and gel chromatography on Sephadex G-50. High affinity (for antithrombin III) octa-, deca-, dodeca-, tetradeca-, hexadeca- and octadeca-saccharides were prepared, as well as oligosaccharides of larger size than octadecasaccharide. The inhibition of Factor Xa by antithrombin III was greatly accelerated by all of these oligosaccharides, the specific anti-Factor Xa activity being invariably greater than 1300 units/mumol. The anti-Factor Xa activity of the decasaccharide was not significantly decreased in the presence of platelet factor 4, even at high platelet factor 4/oligosaccharide ratios. Measurable but incomplete neutralization of the anti-Factor Xa activities of the tetradeca- and hexadeca-saccharides was observed, and complete neutralization of octadeca- and larger oligo-saccharides was achieved with excess platelet factor 4. The octa-, deca-, dodeca-, tetradeca- and hexadeca-saccharides had negligible effect on the inhibition of thrombin by antithrombin III, whereas specific anti-thrombin activity was expressed by the octadeca-saccharide and by the larger oligosaccharides. An octadecasaccharide is therefore the smallest heparin fragment (prepared by nitrous acid depolymerization) that can accelerate thrombin inhibition by antithrombin III. The anti-thrombin activities of the octadecasaccharide and larger oligosaccharides were more readily neutralized by platelet factor 4 than were their anti-Factor Xa activities. These findings are compatible with two alternative mechanisms for the action of platelet factor 4, both involving the binding of the protein molecule adjacent to the antithrombin III-binding site. Such binding results in either steric interference with the formation of antithrombin III-proteinase complexes or in displacement of the antithrombin III molecule from the heparin chain.

Anticoagulants↗