scholarly journals Asymmetric distribution of sites with high affinity for antithrombin III in rat skin heparin proteoglycans.

1982 ◽  
Vol 257 (15) ◽  
pp. 8749-8754
Author(s):  
A A Horner ◽  
E Young
Keyword(s):  
Antithrombin Iii ◽  
Asymmetric Distribution ◽  
Rat Skin ◽  
High Affinity

scholarly journals Heterogeneity of rat skin heparin chains with high affinity for antithrombin

1987 ◽  
Vol 244 (3) ◽  
pp. 693-698 ◽  
Author(s):  
A A Horner
Keyword(s):  
Binding Sites ◽  
Anticoagulant Activity ◽  
Gradient Elution ◽  
Asymmetric Distribution ◽  
Rat Skin ◽  
High Affinity ◽  
Stepwise Increase ◽  
Beta Elimination

Subfractions of 35S-labelled rat skin heparin proteoglycans with various degrees of high affinity for antithrombin were obtained by gradient elution from a column of antithrombin-agarose. Heparin chains released from the proteoglycan preparations by beta-elimination with alkali were re-fractionated on the same column. Proportions of chains with high affinity for antithrombin (HA-chains) ranged from 17% to 76%. These separations also revealed three overlapping subfractions of HA-chains. Their proportions varied in a manner consistent with a stepwise increase in the degree of affinity of HA-chains for antithrombin, this presumably being due to the biosynthesis of increasing numbers of antithrombin-binding sites per chain. The anticoagulant activity, with respect to thrombin neutralization, ranged from 32 units/mg to 287 units/mg. It is suggested that HA-chains may have from one to five or six antithrombin-binding sites. Thus the asymmetric distribution of these sites in rat skin heparin proteoglycans is much more marked than was realized from the earlier work of Horner & Young [(1982) J. Biol. Chem. 257, 8749-8754].

Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

1987 ◽  
Vol 58 (04) ◽  
pp. 1064-1067 ◽  
Author(s):  
K Kodama ◽  
B Pasche ◽  
P Olsson ◽  
J Swedenborg ◽  
L Adolfsson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Surface Coating ◽  
Antithrombin Iii ◽  
Lower Class ◽  
Biologically Active ◽  
High Affinity ◽  
Heparin Coating ◽  
Continuous Surface ◽  
Approximate Molecular Weight

SummaryThe mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm2 The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

1983 ◽  
Vol 49 (02) ◽  
pp. 109-115 ◽  
Author(s):  
M Hoylaerts ◽  
E Holmer ◽  
M de Mol ◽  
D Collen
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Life Time ◽  
Low Molecular Weight ◽  
High Affinity ◽  
Plasma Half Life ◽  
Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

Immunochemical Studies on Antithrombin III

1979 ◽  
Author(s):  
E.J. McKay
Keyword(s):  
Antithrombin Iii ◽  
The Other ◽  
Antigenic Determinants ◽  
Immunochemical Analysis ◽  
Paradoxical Effect ◽  
Test Protocol ◽  
Agar Gel ◽  
High Affinity ◽  
Time Required ◽  
Antigen Antibody

Depressed Antithrombin III (AT) levels Increase thrombic tendency in man, therefore value in assaying this protein has been established. Immunochemical analysis of AT in clinical disease has however proved controversial, consequently systematic studies were undertaken to rationalize the requirements necessary to optimise these methods in particular electro-Immunoassay. The known binding affinity of AT for heparin has been exploited to differentiate high affinity AT from its inhibitor - protease complexes and has resulted in reports stating that heparin added to the agar gel prior to electrophoresis significantly reduces the time required for completion of antigen/antibody complexes. Our studies however have demonstrated that the antibody required for quantitative analysis must be capable of not only reacting with “native” antigenic determinants of AT but also with “neo” antigens that are exposed when inhibitor-protease complexes are formed. Heparin should not be used in the test protocol, for it has a paradoxical effect on Immunopreclpltation in gels, masking some antigenic determinants of unbound - high affinity AT on one hand, and appear to disrupt the Immunoprecipitin “rocket” formed with the inhibitor-protease complexes during electrophoresis on the other.

scholarly journals The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

1981 ◽  
Vol 197 (3) ◽  
pp. 599-609 ◽  
Author(s):  
B Casu ◽  
P Oreste ◽  
G Torri ◽  
G Zoppetti ◽  
J Choay ◽  
...  
Keyword(s):  
Uronic Acid ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Amino Sugar ◽  
Active Species ◽  
Model Compounds ◽  
High Affinity ◽  
The Common ◽  
Binding Sequence ◽  
Heparin Oligosaccharides

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].

scholarly journals Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 927-934
Author(s):  
MJ Rabiet ◽  
M Jandrot-Perrus ◽  
JP Boissel ◽  
J Elion ◽  
F Josso
Keyword(s):  
High Performance ◽  
Fluorescence Enhancement ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amidolytic Activity ◽  
Direct Inhibition ◽  
High Affinity ◽  
Factor Va

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

IS PENTASACCHARIDE THE SMALLEST HEPARIN OLIGOSACCHARIDE WITH BINDING AFFINITY TO ANTITHROMBIN III ? AN OVERVIEW

1987 ◽  
Author(s):  
J Mardiguian
Keyword(s):  
Aqueous Medium ◽  
Uronic Acid ◽  
Antithrombin Iii ◽  
Specific Binding ◽  
Factor Xa ◽  
Benzyl Ester ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Convincing Data

The binding of heparin to antithrombin III is ascribed to the presence in the heparin molecule of a specific binding site which contains a typical 3-0- sulfate group located on a glucosamine residue. It has been postulated that the smallest heparin oligosaccharide capable of high affinity binding to antithrombin III and eliciting anti-factor Xa activity is a pentasaccharide containing three glucosamine units and two uronic acid residues. Such a pentasaccharide has been recently isolated after chemical depolymerization of pig mucosal heparin and its structure found to be very close to that of a synthetic pentasaccharide prepared by other investigators. However no convincing data have, so far, excluded the possibility that an oligosaccharide composed of less than five sugar units could not be able to bind to antithrombinlll and to elicit anti-factor Xa activity. We report now the isolation of new oligosaccharides obtained by beta-eliminative chemical depolymerization of heparin using three different procedures : depolymerization of heparin benzyl ester (1) in aqueous medium and (2) in non-aqueous medium: (3) alcaline depolymerization of a periodate oxydized acetyl heparin. The data reported show that the high affinity oligosaccharides isolated after affinity chromatography on immobilized antithrombin III are distinct from the previously isolated pentasaccharide and that there is some evidence that these are tetrasaccharides resulting from the cleavage of the non reducing end of the heparin molecule

The Heparin Binding Site Of Antithrombin: Identification Of A Critical Tryptophan Residue Within The Amino Acid Sequence

1981 ◽  
Author(s):  
M Blackburn
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Heparin Binding ◽  
High Affinity ◽  
Heparin Binding Site

Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety per molecule of antithrombin III. Heparin protects against tryptophan modification, particularly at low reagent concentrations. Unlike native antithrombin, which has high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti- thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native antithrombin possess progressive antithrombin activity, inactivating these proteases at identical rates. These results indicate that the integrity of a specific tryptophan residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic cleavage techniques have been used to isolate peptides containing this tryptophan from both HNB-labeled and native antithrombin and to identify this critical tryptophan residue within the amino acid sequence of the antithrombin molecule.

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