Structure and contribution to the heparin cofactor II-mediated inhibition of thrombin of naturally oversulphated sequences of dermatan sulphate

Dermatan sulphate (DS) obtained from bovine and pig mucosa and pig skin, and charge-enriched fractions of a selected DS preparation, were characterized in terms of charge density, M(r) and disaccharide composition of chondroitin ABC lyase digests, and by 13C-n.m.r. spectroscopy. Besides the major IdoA-GalNAc4SO3 sequences, all DS preparations contain about 10% disulphated disaccharide sequences (mostly IdoA2SO3-GalNAc4SO3, with minor amounts of IdoA-GalNAc4,6SO3). DS fragments (prepared by radical-catalysed depolymerization of DS and retaining the internal structure of the parent polysaccharide) as well as Smith degraded fragments [SD-DS, obtained by controlled degradation of periodate-oxidized and borohydride-reduced DS (RO-DS)] with the general structure GalNAc4SO3(IdoA2SO3-GalNAc4SO3)n-R (where R is the remnant of a glycol-split uronic acid, and n = 2-3 and 3-4) were characterized by one- and two-dimensional 1H-n.m.r., 13C-n.m.r. and disaccharide composition analysis. In accordance with previous findings [Maimone and Tollefsen (1990) J. Biol. Chem. 265, 18263-18271], only fragments with n > or = 3 significantly enhance the heparin cofactor II-mediated inhibition of thrombin. In natural DS preparations and their fractions, this activity (as well as the antithrombotic activity in an animal model) appears to require IdoA2SO3-containing sequences. The heparin cofactor II activity of DS, RO-DS and SD-DS fragments decreases with decreasing M(r). However, RO-DS fragments are more active than DS fragments of similar M(r), probably because of the extra flexibility endowed by glycol-split IdoA residues.

Download Full-text

Increased sulphation improves the anticoagulant activities of heparan sulphate and dermatan sulphate

Biochemical Journal ◽

10.1042/bj2480889 ◽

1987 ◽

Vol 248 (3) ◽

pp. 889-896 ◽

Cited By ~ 52

Author(s):

F A Ofosu ◽

G J Modi ◽

M A Blajchman ◽

M R Buchanan ◽

E A Johnson

Keyword(s):

Functional Property ◽

Heparan Sulphate ◽

Dermatan Sulphate ◽

Heparin Cofactor Ii ◽

Comparable Amount ◽

Important Functional Property ◽

Sulphated Glycosaminoglycans ◽

Catalytic Effects ◽

Inhibition Of Thrombin

Heparan sulphate and dermatan sulphate have both antithrombotic and anticoagulant properties. These are, however, significantly weaker than those of a comparable amount of standard pig mucosal heparin. Antithrombotic and anticoagulant effects of glycosaminoglycans depend on their ability to catalyse the inhibition of thrombin and/or to inhibit the activation of prothrombin. Since heparan sulphate and dermatan sulphate are less sulphated than unfractionated heparin, we investigated whether the decreased sulphation contributes to the lower antithrombotic and anticoagulant activities compared with standard heparin. To do this, we compared the anticoagulant activities of heparan sulphate and dermatan sulphate with those of their derivatives resulphated in vitro. The ratio of sulphate to carboxylate in these resulphated heparan sulphate and dermatan sulphate derivatives was approximately twice that of the parent compounds and similar to that of standard heparin. Anticoagulant effects were assessed by determining (a) the catalytic effects of each glycosaminoglycan on the inhibition of thrombin added to plasma, and (b) the ability of each glycosaminoglycan to inhibit the activation of 125I-prothrombin in plasma. The least sulphated glycosaminoglycans were least able to catalyse the inhibition of thrombin added to plasma and to inhibit the activation of prothrombin. Furthermore, increasing the degree of sulphation improved the catalytic effects of glycosaminoglycans on the inhibition of thrombin by heparin cofactor II in plasma. The degree of sulphation therefore appears to be an important functional property that contributes significantly to the anticoagulant effects of the two glycosaminoglycans.

Download Full-text

Effect of oversulphated chondroitin and dermatan sulphate upon thrombin and factor Xa inactivation by antithrombin III or heparin cofactor II

Biochemical Journal ◽

10.1042/bj2540547 ◽

1988 ◽

Vol 254 (2) ◽

pp. 547-551 ◽

Cited By ~ 32

Author(s):

M F Scully ◽

V Ellis ◽

N Seno ◽

V V Kakkar

Keyword(s):

Antithrombin Iii ◽

Chondroitin Sulphate ◽

Factor Xa ◽

Dermatan Sulphate ◽

Physiological Control ◽

Heparin Cofactor Ii ◽

High Affinity ◽

Human Thrombin ◽

Sulphated Glycosaminoglycans ◽

Inhibition Of Thrombin

The kinetics of inhibition of human thrombin and Factor Xa by antithrombin III or heparin cofactor II were examined under pseudo-first-order conditions as a function of the concentration of naturally occurring oversulphated chondroitin and dermatan sulphates. The sulphated glycosaminoglycans (GAGs) studied were chondroitin sulphate D (CSD) (GlcA-2-SO4-GalNAc-6-SO4), chondroitin sulphate K (CSK) (GlcA-3-SO4-GalNAc-4-SO4), chondroitin sulphate H (CSH) (IdA-GalNAc-4,6-diSO4) and polysulphated dermatan sulphate (DPS) (IdA-2-SO4 or -3-SO4-GalNAc-4,6-diSO4). The data for the antithrombin III inhibition of thrombin showed a low degree of maximal potentiation of this interaction (congruent to 10-fold), which would appear to be characteristic of GAGs devoid of the high-affinity antithrombin III binding site. In contrast there was a greater potentiation of the inhibition of thrombin by heparin cofactor II with DPS showing an activity comparable to heparin in this interaction at a concentration two orders of magnitude lower than dermatan sulphate. DPS potentiated antithrombin III-Factor Xa interaction by 1200-fold, similar to that shown by high-affinity heparin of 6 kDa. The antithrombin III-Factor Xa interaction was potentiated by all other GAGs studied to a degree similar to that of heparin pentasaccharide with high affinity for antithrombin III. The findings suggest more stringent structural requirements for GAG stimulation of antithrombin-thrombin interaction than for antithrombin-Factor Xa or heparin cofactor-thrombin interaction, which may also be of significance in physiological control of haemostasis.

Download Full-text

A COMPARISON OF DERMATAN SULPHATE AND HEPARIN AS ANTITHROMBOTIC DRUGS

10.1055/s-0038-1642933 ◽

1987 ◽

Author(s):

R E Merton ◽

T W Barrowcliffe ◽

D P Thomas

Keyword(s):

Thrombin Generation ◽

Ex Vivo ◽

Heparan Sulphate ◽

Fold Increase ◽

Plasma Samples ◽

Dermatan Sulphate ◽

Heparin Cofactor Ii ◽

Antithrombotic Activity ◽

The Mean

Dermatan sulphate (DS) has been shown to accelerate thrcmbin inhibition by its action on heparin cofactor II (HCII) but has no effect on anti thrombin III (Tollefson et al., 1983). In this study, we have examined the in vitro anticoagulant effect of a purified preparation of DS (free from heparin and heparan sulphate), m comparison with that of unfractionated heparin (UEH). We have also studied the effect of DS and UEH in preventing experimental venous thrombosis in rabbits and in inhibiting thrombin generation, both in vitro and in ex vivo plasma samples.Dermatan had low activity in vitro by APTT and anti-Xa assays (< 5 iu/ mg). When thrombin generation was measured in vitro, 1 μg/ml UEH was sufficient to inhibit thrombin formation. Although 1 μg/ml DS reduced thrombin generation to 40% of control values, there was no further reduction when the concentration of DS was increased to 8 μg/ml.When DS was injected into rabbits (n = 10), a dose of 150 μg/kg inpaired thrombogenesis m a Wessler stasis model. The mean thrombus score was reduced to 25% of the control values, although thrombosis could not be completely prevented, even after an eight-fold increase m dose (1250 μg/kg). When the duration of stasis was extended from 10 to 20 minutes, there was no impairment of thrombosis (mean thrombus score 100%) following 1250 μg/kg of DS. Thrombin generation measured in ex vivo plasma after 150 μg/kg of DS was 72% (s.e.m. 63-81) of that measured in pre-injection plasma. In contrast, 150 μg/kg of heparin prevented thrombosis after both 10 and 20 minutes stasis (mean score 0%) and thrombin generation was reduced to 17% (s.e.m. 12-23) of control values m ex vivo plasma samples.Unlike heparin, DS does not completely abolish thrcmbin generation in vitro and is not as potent as UEH in inhibiting thrombin generation m ex vivo plasma. While DS has demonstrable antithrombotic activity under defined conditions, it is less effective than heparin and increasing doses of DS do not improve antithrombotic effectiveness in this model.

Download Full-text

The Venous Antithrombotic Profile of Naroparcil in the Rabbit

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648977 ◽

1994 ◽

Vol 72 (06) ◽

pp. 874-879 ◽

Cited By ~ 11

Author(s):

Jean Millet ◽

Jocelyne Theveniaux ◽

Neil L Brown

Keyword(s):

Oral Administration ◽

Bleeding Time ◽

Dermatan Sulfate ◽

Factor Xa ◽

Bleeding Risk ◽

Thrombin Time ◽

Heparin Cofactor Ii ◽

Antithrombotic Activity ◽

Thrombin Inhibition ◽

Maximal Reduction

SummaryThe venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.

Download Full-text

Structural studies on heparan sulphate from human lung fibroblasts. Characterization of oligosaccharides obtained by selective periodate oxidation of d-glucuronic acid residues followed by scission in alkali

Biochemical Journal ◽

10.1042/bj1910103 ◽

1980 ◽

Vol 191 (1) ◽

pp. 103-110 ◽

Cited By ~ 15

Author(s):

Ingrid Sjöberg ◽

Lars-Ȧke Fransson

Keyword(s):

Uronic Acid ◽

Glucuronic Acid ◽

Human Lung ◽

General Structure ◽

Heparan Sulphate ◽

Periodate Oxidation ◽

Exchange Chromatography ◽

Lung Fibroblasts ◽

Human Lung Fibroblasts ◽

Iduronic Acid

1. 3H- and 35S-labelled heparan sulphate was isolated from monolayers of human lung fibroblasts and subjected to degradations by (a) deaminative cleavage and (b) periodate oxidation/alkaline elimination. Fragments were resolved by gel- and ion-exchange-chromatography. 2. Deaminative cleavage of the radioactive glycan afforded mainly disaccharides with a low content of ester-sulphate and free sulphate, indicating that a large part (approx. 80%) of the repeating units consisted of uronosyl-glucosamine-N-sulphate. Blocks of non-sulphated [glucuronosyl-N-acetyl glucosamine] repeats (3–4 consecutive units) accounted for the remainder of the chains. 3. By selective oxidation of glucuronic acid residues associated with N-acetylglucosamine, followed by scission in alkali, the radioactive glycan was degraded into a series of fragments. The glucuronosyl-N-acetylglucosamine-containing block regions yielded a compound N-acetylglucosamine–R, where R is the remnant of an oxidized and degraded glucuronic acid. Periodate-insensitive uronic acid residues were recovered in saccharides of the general structure glucosamine–(uronic acid–glucosamine)n–R. 4. Further degradations of these saccharides via deaminative cleavage and re-oxidations with periodate revealed that iduronic acid may be located in sequences such as glucosamine-N-sulphate→iduronic acid→N-acetylglucosamine. Occasionally the iduronic acid was sulphated. Blocks of iduronic acid-containing repeats may contain up to five consecutive units. Alternating arrangements of iduronic acid- and glucuronic acid-containing repeats were also observed. 5. 3H- and 35S-labelled heparan sulphates from sequential extracts of fibroblasts (medium, EDTA, trypsin digest, dithiothreitol extract, cell-soluble and cell-insoluble material) afforded similar profiles after both periodate oxidation/alkaline elimination and deaminative cleavage.

Download Full-text

S PROTEIN/VITRONECTIN NEUTRALIZES THE ANTICOAGULANT ACTIVITY OF GLYCOSAMINOGLYCANS IN THE INHIBITION OF THROMBIN BY HEPARIN COFACTOR II

10.1055/s-0038-1643633 ◽

1987 ◽

Author(s):

K T Preissner ◽

P Sie

Keyword(s):

Dermatan Sulfate ◽

Enzyme Inhibitor ◽

Anticoagulant Activity ◽

Coagulation System ◽

Binary Complex ◽

Heparin Cofactor Ii ◽

S Protein ◽

Adhesive Protein ◽

Inhibition Of Thrombin

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin against fast inactivation by antithrombin III. The interference of S protein with glycos-aminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in a purified system. In the presence of 0.3 μg/ml heparin, or 0.5 μg/ml pentosan polyphosphate (SP 54), or 2 μg/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo-first order rate constants by about 17-fold (heparin), or about 7-fold (SP 54), but only by about 2-fold for dermatan sulfate at a physiological ratio of S protein to heparin cofactor II. Likewise, S protein significantly counteracted the anticoagulant activity of heparin and SP 54 bot not of dermatan sulfate when tested in an inhibition assay using various concentrations of glycosaminoglycans. For heparin, the activity of S protein at the point of 50% inhibition of thrombin was expressed in the range 0.06-0.6 μg/ml (0.01-0.1 U/ml) and for SP 54 in the range 0.3-2 pg/ml. Exposure of the glycos-aminoglycan-binding region of S protein by reduction and carb-oxymethylation of the protein even increased the neutralizing activity of S protein towards heparin and SP 54. S protein not only was found together with thrombin in a binary complex. S protein also became incorporated into a ternary complex with thrombin and heparin cofactor II as judged by crossed immunoelectrophoresis, regardless whether complex formation was initiated by heparin or dermatan sulfate. These findings underline the role of S protein as potent glycosaminoglycan-neutral-izing protein in plasma and as scavenger protein which may bind to enzyme-inhibitor complexes of the coagulation system.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj1430379 ◽

1974 ◽

Vol 143 (2) ◽

pp. 379-389 ◽

Cited By ~ 25

Author(s):

Lars-Åke Fransson ◽

Lars Cöster ◽

Birgitta Havsmark ◽

Anders Malmström ◽

Ingrid Sjöberg

Keyword(s):

Periodate Oxidation ◽

Exchange Chromatography ◽

Dermatan Sulphate ◽

Chondroitinase Abc ◽

Pig Skin ◽

Isolation And Characterization ◽

Smith Degradation ◽

Iduronic Acid ◽

Acidic Conditions

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-SO4 (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-SO4 residues are unsulphated to a large extent.

Download Full-text

Dermatan sulphate induces plasminogen activator release in the perfused rat hindquarters

Blood ◽

10.1182/blood.v70.6.1858.bloodjournal7061858 ◽

1987 ◽

Vol 70 (6) ◽

pp. 1858-1860 ◽

Cited By ~ 1

Author(s):

M Abbadini ◽

GJ Zhu ◽

A Maggi ◽

J Pangrazzi ◽

MB Donati ◽

...

Keyword(s):

Plasminogen Activator ◽

Local Level ◽

Tissue Type ◽

Dermatan Sulphate ◽

Antithrombotic Activity ◽

Sds Page ◽

Different Doses

Heparin or heparin-like substances have been described to induce the release of plasminogen activator (PA) activity in different animal perfusion models. In this paper we report that Dermatan Sulphate (DS) is able to induce PA activity release in the perfused rat hindquarters. Perfusion of different doses of DS (0.1 to 0.8 mg/mL) stimulates a release of PA activity that is maximum after the initial two minutes of perfusion. The amount of PA activity released rises progressively within a certain concentration range of DS (0.1 to 0.4 mg/mL) and declines thereafter (0.6 to 0.8 mg/mL). The type of PA activity increased during DS perfusion was characterized by SDS-PAGE and fibrin autography as tissue-type PA (t-PA) on the basis of its mol wt (67,000 d) and inhibition by a specific anti t-PA antiserum. This effect might be considered as potentially contributing to the antithrombotic activity of DS, at least at the local level.

Download Full-text

Dermatan sulphate induces plasminogen activator release in the perfused rat hindquarters

Blood ◽

10.1182/blood.v70.6.1858.1858 ◽

1987 ◽

Vol 70 (6) ◽

pp. 1858-1860 ◽

Cited By ~ 29

Author(s):

M Abbadini ◽

GJ Zhu ◽

A Maggi ◽

J Pangrazzi ◽

MB Donati ◽

...

Keyword(s):

Plasminogen Activator ◽

Local Level ◽

Tissue Type ◽

Dermatan Sulphate ◽

Antithrombotic Activity ◽

Sds Page ◽

Different Doses

Abstract Heparin or heparin-like substances have been described to induce the release of plasminogen activator (PA) activity in different animal perfusion models. In this paper we report that Dermatan Sulphate (DS) is able to induce PA activity release in the perfused rat hindquarters. Perfusion of different doses of DS (0.1 to 0.8 mg/mL) stimulates a release of PA activity that is maximum after the initial two minutes of perfusion. The amount of PA activity released rises progressively within a certain concentration range of DS (0.1 to 0.4 mg/mL) and declines thereafter (0.6 to 0.8 mg/mL). The type of PA activity increased during DS perfusion was characterized by SDS-PAGE and fibrin autography as tissue-type PA (t-PA) on the basis of its mol wt (67,000 d) and inhibition by a specific anti t-PA antiserum. This effect might be considered as potentially contributing to the antithrombotic activity of DS, at least at the local level.

Download Full-text

The structure of Estonian personal values: a lexical approach

European Journal of Personality ◽

10.1002/per.439 ◽

2002 ◽

Vol 16 (3) ◽

pp. 221-235 ◽

Cited By ~ 22

Author(s):

Toivo Aavik ◽

Jüri Allik

Keyword(s):

Principal Component Analysis ◽

Personal Values ◽

General Structure ◽

Principal Component ◽

Higher Order ◽

The Self ◽

The Other ◽

Two Dimensional ◽

Lexical Approach ◽

Estonian Language

The main purpose of this paper is to investigate the variety of value describing words and interrelation of value categories in the Estonian language. To accomplish this aim, a psycholexical approach was adopted, during which a set of 560 value‐related words was selected from the Estonian Orthological Dictionary and the results were compared with the Schwartz Values Survey (SVS). When principal‐component analysis was applied on the self‐ratings of a reduced list of 78 value‐related words, six factors emerged and were labelled as benevolence, self‐enhancement, broadmindedness, hedonism, conservatism, and self‐realization. However, all these themes are interrelated and load on a singular secondary dimension. The constructs measured by SVS and the value categories in Estonian were only partially interchangeable; moderate correlations imply an imperfect correspondence: each theme was related to many categories on the other questionnaire. However, a significant general structure refers to the same two‐dimensional level of higher‐order values described by Schwartz in 1992. Copyright © 2002 John Wiley & Sons, Ltd.

Download Full-text