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

1987 ◽  
Vol 248 (3) ◽  
pp. 889-896 ◽  
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.

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

1988 ◽  
Vol 254 (2) ◽  
pp. 547-551 ◽  
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.

A COMPARISON OF DERMATAN SULPHATE AND HEPARIN AS ANTITHROMBOTIC DRUGS

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.

Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-540

Zygote ◽  
2007 ◽  
Vol 15 (3) ◽  
pp. 225-232 ◽  
Author(s):  
A.-S. Bergqvist ◽  
J. Ballester ◽  
A. Johannisson ◽  
N. Lundeheim ◽  
H. Rodríguez-Martínez
Keyword(s):  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Negative Control ◽  
Dermatan Sulphate ◽  
Flow Cytometric ◽  
Merocyanine 540 ◽  
Incubation Duration ◽  
Flow Cytometric Study ◽  
Oviductal Fluid

SummaryGlycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p < 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p < 0.0001) higher than the control at 0–30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p < 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.

scholarly journals Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels

1983 ◽  
Vol 215 (1) ◽  
pp. 107-116 ◽  
Author(s):  
J T Gallagher ◽  
N Gasiunas ◽  
S L Schor
Keyword(s):  
Human Skin ◽  
Heparan Sulphate ◽  
Surface Membrane ◽  
Skin Fibroblasts ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Cellular Functions ◽  
Iduronic Acid ◽  
Sulphated Glycosaminoglycans

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.

scholarly journals Sulphated glycosaminoglycans in regenerating rat liver

1980 ◽  
Vol 188 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M Edward ◽  
W F Long ◽  
H H Watson ◽  
F B Williamson
Keyword(s):  
Molecular Mechanisms ◽  
Temporal Trend ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Fold Increase ◽  
Sham Operation ◽  
Dermatan Sulphate ◽  
Weight Percentage ◽  
Biphasic Pattern ◽  
Sulphated Glycosaminoglycans

The total weight percentage glycosaminoglycan content of rat liber was found to increase by 50% in the first 30 h after partial hepatectomy. The content returned to near normal by the third day, but then increased again to a second maximum at 5-6 days, only to gradually decline to normal by the ninth day, when regeneration was nearly complete. This biphasic pattern was most marked in the chondroitin sulphate A/C component, with a 6-fold increase by the sixth day. Dermatan sulphate showed the same temporal trend, whereas heparan sulphate remained relatively unaltered. No such changes were detected in the livers of rats subjected to sham operation. The possible molecular mechanisms underlying the apparent link between cellular glycosaminoglycan content and proliferative tendency are discussed.

A Low Molecular Weight Heparin Alters the Fetal Coagulation System in the Pregnant Sheep

1986 ◽  
Vol 55 (03) ◽  
pp. 342-346 ◽  
Author(s):  
M Andrew ◽  
F Ofosu ◽  
F Fernandez ◽  
A Jefferies ◽  
J Hirsh ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation System ◽  
Dermatan Sulphate ◽  
Protamine Sulphate ◽  
Pregnant Sheep ◽  
Pregnant Ewe ◽  
Inhibition Of Thrombin

SummaryStandard heparin and a LMWH, CY222 do not cross the placenta nor alter fetal coagulation when injected into the pregnant ewe. We found that another LMWH, Pharmuka-10169 (PK-10169) alters fetal coagulation without crossing the placenta in the pregnant sheep. To characterize this anticoagulant we measured the in vitro and in vivo effects of 125I-PK-10169 in maternal and fetal plasmas following administration of PK-10169 to the mother or fetus. The fetal anticoagulant activity was not neutralizable by protamine sulphate and was attributable to the inhibition of thrombin but not factor Xa. In vitro, the fetal anticoagulant activity had properties similar to dermatan sulphate : both catalyzed the inhibition of thrombin but not factor Xa by sheep plasma; and neither was neutralizable by protamine sulphate. These effects were due to the enhanced neutralization of thrombin by heparin cofactor II. We conclude that PK-10169 does not cross the placenta, but does induce the release of an endogenous dermatan sulphate-like substance which alters fetal coagulation.

scholarly journals Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

1980 ◽  
Vol 190 (2) ◽  
pp. 243-254 ◽  
Author(s):  
J T Gallagher ◽  
N Gasiunas ◽  
S L Schor
Keyword(s):  
Hyaluronic Acid ◽  
Salt Concentration ◽  
Heparan Sulphate ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Sulphated Glycosaminoglycans ◽  
Sulphated Glycosaminoglycan ◽  
Spent Media

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.

scholarly journals Proteoglycan metabolism in the connective tissue of pregnant and non-pregnant human cervix. An in vitro study

1991 ◽  
Vol 275 (2) ◽  
pp. 515-520 ◽  
Author(s):  
M Norman ◽  
G Ekman ◽  
U Ulmsten ◽  
K Barchan ◽  
A Malmström
Keyword(s):  
Connective Tissue ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
In Vitro Study ◽  
Gel Chromatography ◽  
Cervical Tissue ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycans

Profound changes occur in the cervix during pregnancy. In particular, the connective tissue is remodelled. To elucidate the mechanisms behind this process, the metabolism of cervical connective tissue was studied using tissue cultures. Cervical biopsies from non-pregnant and pregnant women were incubated with [35S]sulphate. The proteoglycans of the tissue specimens were purified by ion-exchange and gel chromatography and characterized by SDS/PAGE and by enzymic degradation. In the non-pregnant cervix, the incorporation of [35S]sulphate into the proteoglycans was linear for 48 h. During the first 6 h of incubation the accumulation of chiefly one small labelled proteoglycan (apparent Mr 110,000) substituted with dermatan sulphate was recorded. This is in accordance with the known proteoglycan composition of non-pregnant cervical tissue. In addition, small amounts of two larger radioactive dermatan/chondroitin sulphate proteoglycans (apparent Mr values 220,000 and greater than 500,000) were recorded. After longer periods of incubation the proportion of heparan sulphate proteoglycans increased considerably. The pregnant tissue showed a clearly different composition of labelled proteoglycans. An increased accumulation of the two larger dermatan/chondroitin sulphate proteoglycans was seen in addition to the dominant small dermatan sulphate proteoglycan of the non-pregnant cervix. The rate of accumulation of these two proteoglycans was about 3 times higher in the pregnant tissue, whereas that of the small dermatan sulphate proteoglycan was only increased 2-fold. The fact that the concentration of proteoglycans in the pregnant cervix is approximately one-half of that in the non-pregnant cervix indicates that the turnover of proteoglycans in pregnant cervical tissue is significantly increased. The major effect of this profound change of metabolism was a 50% decrease in proteoglycan content and a 2-fold increased proportion of a dermatan sulphate proteoglycan with an apparent Mr of 220,000.

scholarly journals Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells

1978 ◽  
Vol 172 (3) ◽  
pp. 443-456 ◽  
Author(s):  
B Glimelius ◽  
B Norling ◽  
B Westermark ◽  
Å Wasteson
Keyword(s):  
Hyaluronic Acid ◽  
Malignant Glioma ◽  
Membrane Fraction ◽  
Heparan Sulphate ◽  
Glioma Cells ◽  
Gel Chromatography ◽  
Total Production ◽  
Dermatan Sulphate ◽  
Sulphated Glycosaminoglycans ◽  
Malignant Glioma Cells

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.

scholarly journals Interaction of lipoprotein lipase with native and modified heparin-like polysaccharides

1980 ◽  
Vol 189 (3) ◽  
pp. 625-633 ◽  
Author(s):  
Gunilla Bengtsson ◽  
Thomas Olivecrona ◽  
Magnus Höök ◽  
Johan Riesenfeld ◽  
Ulf Lindahl
Keyword(s):  
Lipoprotein Lipase ◽  
Chondroitin Sulphate ◽  
Hepatic Lipase ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Binding Affinities ◽  
Dermatan Sulphate ◽  
Heparin Plasma ◽  
Heparin Fractions

1. Lipoprotein lipase (EC 3.1.1.34), which was previously shown to bind to immobilized heparin, was now found to bind also to heparan sulphate and dermatan sulphate and to some extent to chondroitin sulphate. 2. The relative binding affinities were compared by determining (a) the concentration of NaCl required to release the enzyme from polysaccharide-substituted Sepharose; (b) the concentration of free polysaccharides required to displace the enzyme from immobilized polysaccharides; and (c) the total amounts of enzyme bound after saturation of immobilized polysaccharides. By each of these criteria heparin bound the enzyme most efficiently, followed by heparan sulphate and dermatan sulphate, which were more efficient than chondroitin sulphate. 3. Heparin fractions with high and low affinity for antithrombin, respectively, did not differ with regard to affinity for lipoprotein lipase. 4. Partially N-desulphated heparin (40–50% of N-unsubstituted glucosamine residues) was unable to displace lipoprotein lipase from immobilized heparin. This ability was restored by re-N-sulphation or by N-acetylation; the N-acetylated product was essentially devoid of anticoagulant activity. 5. Partial depolymerization of heparin led to a decrease in ability to displace lipoprotein lipase from heparin–Sepharose; however, even fragments of less than decasaccharide size showed definite enzyme-releasing activity. 6. Studies with hepatic lipase (purified from rat post-heparin plasma) gave results similar to those obtained with milk lipoprotein lipase. However, the interaction between the hepatic lipase and the glycosaminoglycans was weaker and was abolished at lower concentrations of NaCl. 7. The ability of the polysaccharides to release lipoprotein lipase to the circulating blood after intravenous injection into rats essentially conformed to their affinity for the enzyme as evaluated by the experiments in vitro.

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