A COMPARISON OF THE BINDING OF ANTITHROMBIN III AND HEPARIN COFACTOR II TO HEPARINS, NATURALLY OCCURRING GLYCOSAMINOGLYCANS AND OTHER SULPHATED POLYMERS

1987 ◽  
Author(s):  
J Dawes ◽  
D S Pepper
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
Antithrombin Iii ◽  
Molar Ratio ◽  
Heparin Binding ◽  
Dextran Sulphate ◽  
Heparin Cofactor Ii ◽  
Wide Range ◽  
Pentosan Polysulphate ◽  
Binding Sequence

Antithrombin III (ATIII) and heparin cofactor II (HCII) are currently thought to be the most important protein mediators of the anticoagulant and antithrombotic activities of glycosamino-glycans. A simple, quantitative method for assessing the affinity of a protein for a sulphated polymer in the liquid phase, based on competition with immobilised heparin, has been developed. Using this technique, the binding of ATIII and HCII to a wide range of glycosaminoglycans and other sulphated polymers have been compared, and the contributions to binding of size, degree of sulphation and backbone structure of the polymers analysed.In the presence of the high protein concentrations found in plasma, unfractionated heparin inhibited the binding of ATIII to immobilised heparin with a Ki of 1 x 10-6. Binding was destroyed by N-desulphation. 1 Results with a range of low molecular weight (LMW) heparins and heparan sulphates are consistent with the view that they all contain the ATIII-binding sequence, but at a lower molar ratio than heparin. Highly sulphated synthetic polymers such as dextran sulphate bound ATIII by a different mechanism, which was molecular weight-dependent.The affinity of HCII for heparins increased markedly with heparin chain length. Binding was largely, but not entirely, mediated by sulphate residues. HCII bound to heparan and dermatan sulphates with lower affinities than to heparin, and to synthetic sulphated polymers with similar or higher affinities. Pentosan polysulphate (SP54) bound HCII as effectively as did heparin. Binding of HCII to dextran sulphate was highly dependent on molecular weight. The affinity of HCII for a sulphated polymer appears to depend both on its chain length and density of sulphation.Thus the profiles of binding of ATIII and HCII to glycosaminoglycans and other sulphated polymers are quite different. This technique is useful both for investigating the interactions of existing therapeutic anticoagulants and assessing new products.

The Additive Effect of Low Molecular Weight Heparins on Thrombin Inhibition by Dermatan Sulfate

1993 ◽  
Vol 70 (03) ◽  
pp. 443-447 ◽  
Author(s):  
Benilde Cosmi ◽  
Giancarlo Agnelli ◽  
Edward Young ◽  
Jack Hirsh ◽  
Jeffrey Weitz
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Additive Effect ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
Sds Page

SummaryThe aim of this study was to investigate the mechanism by which the anticoagulant activity of dermatan sulfate (DS) is increased by low molecular weight heparin (LMWH). In platelet poor plasma, LMWH enhances the effect of DS on thrombin (IIa) inhibition as determined by thrombin clotting times and with a chromogenic substrate assay. Analysis of the results of the chromogenic assays using either the algebraic fractional or the graphic isobole method suggests that LMWH has an additive effect on the anti-IIa activity of DS. This additive effect was lost when the experiments were repeated in plasma immunodepleted of antithrombin III (ATIII), indicating that the anti-IIa activity of LMWH is ATIII-dependent. To further explore the mechanism of the interaction between LMWH and DS, 125I-labeled IIa was added to plasma in the presence or absence of DS and/or LMWH and the formation of IIa-inhibitor complexes was assessed using SDS-PAGE followed by autoradiography. DS addition selectively increases the formation of heparin cofactor II (HCII)-IIa complexes, whereas LMWH enhances ATIII-IIa complex generation. Compared to plasma containing DS alone, the formation of ATIII-IIa complexes also is increased when the combination of DS and LMWH is added. These findings suggest that the additive effect of LMWH on the anti-IIa activity of DS reflects their different modes of IIa inhibition; DS potentiates IIa inhibition by HCII, while LMWH catalyses ATIII-dependent IIa inactivation. The potential clinical significance of these findings requires further investigation.

scholarly journals Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3380-3385 ◽  
Author(s):  
MA Delorme ◽  
N Saeed ◽  
A Sevcik ◽  
L Mitchell ◽  
L Berry ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Psoas Muscle ◽  
Chronic Hemodialysis ◽  
Gel Chromatography ◽  
Thrombin Time ◽  
Heparin Cofactor Ii ◽  
Dermatan Sulfate Proteoglycan

Abstract A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.

Interaction Between Low Molecular Weight Dextran Sulphate and Bentonite adsorbed Plasma

1979 ◽  
Author(s):  
T. Matsuda ◽  
M. Ogawara ◽  
T. Seki ◽  
R. Miura ◽  
M. Yokouchi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Low Molecular Weight ◽  
Thrombin Time ◽  
Dextran Sulphate ◽  
Bothrops Atrox ◽  
Plasmin Inhibitor ◽  
Mean Molecular Weight

Effects of low molecular weight dextran sulphate (D.S.; mean molecular weight = 6,0006,500; S content = 17-19%) and/or bentonite (Wako Pure Chemical Industries, Osaka, Japan) adsorbed plasma (supernatant from plasma mixed with 300 mg/ml of bentonite for 10 minutes at 37°C), which contains no fibrinogen nor antithrombin III but α2 macro-globulin, α1-antitrypsin and α2-plasmin inhibitor, on thrombin time or batroxobin (purified fraction of venom of Bothrops atrox) time of fibrinogen solution were investigated. Addition of D.S. or bentonite adsorbed plasma to fibrinogen solution resulted in prolongations of thrombin time and batroxobin time. These results indicate that both D.S. and bentonite adsorbed plasma inhibit conversion of fibrinogen to fibrin by thrombin or batroxobin. However, when appropriately diluted bentonite adsorbed plasma and D.S. were added to fibrinogen solution simultaneously, thrombin times and batroxobin times were shorter compared to those when either bentonite adsorbed plasma or D.S. but not both was added. From these results, it is concluded that D.S. and bentonite adsorbed plasma interact to inhibit each other.

Chromatography of heparin on Sepharose–lysine: molecular size fractionation and its relevance to thrombin and antithrombin III binding

1979 ◽  
Vol 57 (10) ◽  
pp. 1183-1190 ◽  
Author(s):  
M. W. C. Hatton ◽  
L. R. Berry ◽  
H. Kaur ◽  
A. Koj ◽  
E. Regoeczi
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Binding Capacity ◽  
Anticoagulant Activity ◽  
Average Molecular Weight ◽  
Molecular Size ◽  
Lysine Content ◽  
Heparin Binding ◽  
Significant Difference ◽  
Lysine Residues

Batches of Sepharose–lysine, which varied in lysine content from 35 to 430 μmol/g of dry gel, were prepared by varying the quantity of CNBr in the activation reaction. The batches were tested for heparin binding by using a controlled chromatographic procedure. Sepharose–lysine, containing < 150 μmol of lysine/g, did not significantly bind heparin whereas conjugates with > 400 μmol/g retained the entire heparin load. For intermediate batches of Sepharose–lysine (150–400 μmol/g) the quantity of heparin bound largely paralleled the lysine content. Thus, Sepharose–lysine of an intermediate lysine content separated heparin into an unretained fraction and a bound fraction which was recovered from the column by eluting with 1 M NaCl. On testing for anticoagulant activity by factor Xa inhibition assay, no significant difference in specific anticoagulant activity was observed between these heparin fractions and the heparin load. However, from gel filtration studies, a substantial difference in molecular size was noted. An unretained heparin fraction from Sepharose–lysine was of a lower average molecular weight than the parent heparin. In contrast, a retained heparin peak was of a higher average molecular weight compared with the parent heparin. These observations were confirmed by studying the chromatographic properties of low (10 000) and high (23 000) molecular weight heparin samples on various Sepharose–lysine batches. A model is proposed to explain this discriminating property of Sepharose–lysine. For a conjugate containing 400 μmol/g, the mean lysine spacing is calculated at 47 Å (1 Å = 0.1 nm), which is approximately equivalent to five to six disaccharide units in heparin.The property of Sepharose–lysine to bind heparin was compared with the affinities of the mucopolysaccharide for both thrombin and antithrombin III. Evidence has been proposed for the involvement of lysine residues of antithrombin III in this process. Our investigations suggest that lysine, in addition to arginine, groups of thrombin are also involved in heparin binding. By specifically modifying two to four lysine residues using nitrous acid, the heparin-binding capacity of the enzyme and its plasma clotting activity were largely destroyed, although the esterase activity was retained.

scholarly journals The Mode of Action of CY216 and CY222 in Plasma

1992 ◽  
Vol 67 (01) ◽  
pp. 033-041 ◽  
Author(s):  
Suzette Béguin ◽  
Simone Wielders ◽  
J C Lormeau ◽  
H Coenraad Hemker
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Time Course ◽  
Antithrombin Iii ◽  
Platelet Rich Plasma ◽  
Gel Permeation Chromatography ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Material ◽  
At Iii

SummaryThree fractions of the low molecular weight heparin CY216 (fraxiparin, mean molecular weight [MMW] 5,090), with MMWs of respectively, 3,090, 4,400 and 7,910 were prepared by gel permeation chromatography. From CY222 (MMW 3,770) as well as from CY216 and its three fractions the material with high affinity to antithrombin III (AT III) was obtained by chromatography on immobilised AT III. The molecular weight distribution of each of the ten preparations thus obtained was determined by high performance liquid chromatography, while the content of AT III binding material was determined by stoichiometric titration of AT III, monitored by intrinsic fluorescence enhancement.We measured the effect of all heparins on the decay of endogenous thrombin in plasma and on the overall generation of thrombin in plasma, triggered via the extrinsic or via the intrinsic pathway. From these data we calculated the time course of prothrombin conversion, i. e. the course of factor Xa activity as expressed by prothrombinase activity.It was found that in platelet-poor plasma the anticoagulant properties of the heparins are largely dependent on their antithrombin action, which is determined by their content of high affinity material with a MW of 5,400 or higher. The specific antithrombin activity of all heparins, when expressed in terms of material with high affinity to antithrombin III (HAM) with a MW >5,400 is 13.0 min-1/(μg/ml) (range 10.5-15.9). The anticoagulant potency is not influenced by the presence of low-affinity material and hardly by material with MW <5,400.In platelet-rich plasma, however, the presence of non-AT III binding material enhances the inhibition, presumably by neutralising heparin binding material originating from activated platelets. The ultra low MW fractions (<3,400) show a similar activity in PPP and in PRP.

scholarly journals Homologous Over-Expression of Chain Length Determination Protein EpsC Increases the Molecular Weight of Exopolysaccharide in Streptococcus thermophilus 05-34

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhengyuan Zhai ◽  
Shuxin Xie ◽  
Hongxing Zhang ◽  
Huaxi Yi ◽  
Yanling Hao
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
High Performance ◽  
Gel Permeation Chromatography ◽  
Streptococcus Thermophilus ◽  
Monosaccharide Composition ◽  
Molar Ratio ◽  
Gas Chromatography Mass Spectrometry ◽  
Over Expression ◽  
Length Determination

In Streptococcus thermophilus, EpsC is a polysaccharide co-polymerase which is involved in determining the chain length of EPS synthesized by the Wzx/Wzy-dependent pathway. Our previous study found that there was a positive correlation between transcription level of epsC and molecular weight of EPS in S. thermophilus 05-34. To further investigate the effects of EpsC on EPS biosynthesis, this gene was over-expressed in S. thermophilus 05-34 in this study. Reverse transcription qPCR and Western blotting confirmed the successful transcription and translation of epsC in 05-34, respectively. The yield of EPS was not affected by the over-expression of EpsC. Gas chromatography-mass spectrometry (GC-MS) showed that the monosaccharide composition was still composed of galactose and glucose in a molar ratio of 1.0:0.8, whereas high performance gel permeation chromatography (HPGPC) indicated that the molecular weight of EPS was increased from 4.62 × 105 Da to 9.17 × 105 Da by the over-expression of EpsC. In addition, S. thermophilus 05epsC which could produce higher molecular weight EPS improved the viscoelasticity and water-holding capacity of yogurt, but significantly reduced the level of syneresis in yogurt. In summary, these results indicated that homologous over-expression of EpsC in S. thermophilus could increase the molecular weight of EPS and improve the microrheological or physical properties of yogurt.

The Effects of Human Thrombomodulin on the Inactivation of Thrombin by Its Serum Inhibitors

1987 ◽  
Vol 58 (03) ◽  
pp. 806-810 ◽  
Author(s):  
E Anne Thompson ◽  
Hatem H Salem
Keyword(s):  
Molecular Weight ◽  
Protein C ◽  
Antithrombin Iii ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Cell Protein ◽  
Heparin Cofactor Ii ◽  
Sds Page ◽  
Dependent Protein ◽  
Molecular Weight Fractions

SummaryThrombomodulin is an endothelial cell protein which accelerates thrombin-dependent protein C activation by over 1000 fold. In this study, the effect of thrombomodulin on the inactivation of thrombin by its serum inhibitors was evaluated. 125I-thrombin was incubated at 37° C with serum and the resulting complexes separated by SDS-PAGE. Antithrombin III was the major complex formed with some 125I-thrombin bound to heparin cofactor II and highermolecular weight fractions. Inclusion of thrombomodulin at increasing concentrations inhibited 125I-thrombin binding to antithrombin III and the higher molecular weight fractions but had little effect on thrombin-heparin cofactorII complex formation. Similar results were obtained using a purified antithrombin III/heparin cofactor II system.Kinetic studies, using purified antithrombin III, revealed that thrombomodulin acts as a weak competitive inhibitor towards antithrombin III (Ki = 39 nM).We postulate that in the microcirculation, where the ratio of thrombomodulin to antithrombin III is relatively high, thrombin bound to thrombomodulin may be protected from inactivation by antithrombin III and can thus promote efficient activation of protein C.

scholarly journals Antimicrobial Evaluation of N-Alkyl Betaines and N-Alkyl-N,N-Dimethylamine Oxides with Variations in Chain Length

2000 ◽  
Vol 44 (9) ◽  
pp. 2514-2517 ◽  
Author(s):  
Christine R. Birnie ◽  
Daniel Malamud ◽  
Roger L. Schnaare
Keyword(s):  
Antimicrobial Activity ◽  
Biological Activity ◽  
Chain Length ◽  
Amine Oxide ◽  
Molar Ratio ◽  
Microdilution Method ◽  
Wide Range ◽  
Broth Microdilution Method ◽  
Antimicrobial Evaluation ◽  
Chain Lengths

ABSTRACT Alkyl betaines and alkyl dimethylamine oxides have been shown to have pronounced antimicrobial activity when used individually or in combination. Although several studies have been conducted with these compounds in combinations, only equimolar concentrations of the C12/C12 and C16/C14chain lengths for the betaine and the amine oxide, respectively, have been investigated. This study investigates the antimicrobial activity of a wide range of chain lengths (C8 to C18) for both the betaine and amine oxide and attempts to correlate their micelle-forming capabilities with their biological activity. A broth microdilution method was used to determine the MICs of these compounds singly and in various molar ratio combinations. Activity against bothStaphylococcus aureus and Escherichia coli was investigated. Antimicrobial activity was found to increase with increasing chain length for both homologous series up to a point, exhibiting a cutoff effect at chain lengths of approximately 16 for betaine and 14 for amine oxide. Additionally, the C18 oleyl derivative of both compounds exhibited activity in the same range as the peak alkyl compounds. Critical micelle concentrations were correlated with MICs, inferring that micellar activity may contribute to the cutoff effect in biological activity.

scholarly journals Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3380-3385
Author(s):  
MA Delorme ◽  
N Saeed ◽  
A Sevcik ◽  
L Mitchell ◽  
L Berry ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Psoas Muscle ◽  
Chronic Hemodialysis ◽  
Gel Chromatography ◽  
Thrombin Time ◽  
Heparin Cofactor Ii ◽  
Dermatan Sulfate Proteoglycan

A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.

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