Use of quantitative affinity chromatography for characterizing high-affinity interactions: Binding of heparin to antithrombin III

SummaryAntithrombin III was purified from normal plasma by DEAE- Sephadex chromatography and heparin affinity chromatography; the protein was subsequently radiolabelled with 125I. 125I-anti- thrombin III alone and 125I-antithrombin III in the presence of high affinity 35S-heparin fractions were injected into normal humans. 125I-radiolabel and protein bound 35S-radioactivity were followed separately. In semilogarithmic plots 125I-antithrombin III disappeared according to a double exponential curve with a half-life in the second phase of 56.8 hr in the absence of heparin and of 33.7 hr in the presence of heparin. Protein bound 35S- radioactivity disappeared much faster than the 125I-radiolabel. These data support the concept that heparin disappears as free heparin from the equilibrium heparin – antithrombin III ⇄ heparin + antithrombin III. Immuno-reactive antithrombin III decreased from 100% to 85-90% immediately after injection of 125I-antithrombin III in the presence of heparin and returned to normal values within 30 min. This suggests that antithrombin III is transiently sequestered, possibly in trimolecular complexes consisting of antithrombin III, heparin and either lipases or other vascular bound proteins.

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Differential role of fractionated heparin in antithrombin-III proteolysis

Blood ◽

10.1182/blood.v59.3.576.576 ◽

1982 ◽

Vol 59 (3) ◽

pp. 576-581 ◽

Cited By ~ 5

Author(s):

E Marciniak

Keyword(s):

Affinity Chromatography ◽

Antithrombin Iii ◽

Enzyme Inhibitor ◽

Anticoagulant Activity ◽

Lower Affinity ◽

Inhibitor Complex ◽

High Affinity ◽

Highly Active ◽

At Iii

Abstract Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.

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Differential role of fractionated heparin in antithrombin-III proteolysis

Blood ◽

10.1182/blood.v59.3.576.bloodjournal593576 ◽

1982 ◽

Vol 59 (3) ◽

pp. 576-581

Author(s):

E Marciniak

Keyword(s):

Affinity Chromatography ◽

Antithrombin Iii ◽

Enzyme Inhibitor ◽

Anticoagulant Activity ◽

Lower Affinity ◽

Inhibitor Complex ◽

High Affinity ◽

Highly Active ◽

At Iii

Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.

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Comparison of the behaviour in vivo of two molecular forms of antithrombin III

Biochemical Journal ◽

10.1042/bj2250557 ◽

1985 ◽

Vol 225 (3) ◽

pp. 557-564 ◽

Cited By ~ 23

Author(s):

T H Carlson ◽

A C Atencio ◽

T L Simon

Keyword(s):

Affinity Chromatography ◽

Antithrombin Iii ◽

Molecular Forms ◽

Rabbit Plasma ◽

High Affinity ◽

Free Iodine ◽

Exponential Equations ◽

Total Body ◽

Heparin Affinity

The distribution and rates of catabolism were determined for rabbit antithrombin III (AT) isoforms differing in affinity for heparin-agarose. After isolation from rabbit plasma by heparin-affinity chromatography, the very-high-affinity form, ATvh, was labelled with 131I, and its high-affinity congener, ATh, with 125I. The two forms were separated from free iodine by heparin-affinity chromatography, and then injected simultaneously into young recipient rabbits. The disappearance-of-plasma-AT-radioactivity data were fitted to three exponential equations, and the resulting constants were used to calculate fractional catabolic rates and the various pool sizes for ATh and ATvh. The fractions of plasma ATh and ATvh catabolized daily (j3) were 0.763 +/- 0.023 and 1.88 +/- 0.057 day-1 respectively. Average values of jT, the daily fractional catabolic rates for the total-body ATh and ATvh, were 0.2633 +/- 0.0113 and 0.3832 +/- 0.0211 day-1. The ratios, ATh/ATvh, of the sizes of the plasma, non-circulating vascular-associated and extra-vascular compartments were 0.593, 0.990 and 1.30. By using a previous estimate of a 9:1 ATh/ATvh ratio in rabbit plasma and the calculated compartment sizes, the calculated relative ratio of ATh to ATvh was 5.4:1 in the non-circulating vascular-associated compartment. The data fit a model of antithrombin III distribution and catabolism in which a pool of endothelial-cell heparin-like receptors is seen to mediate the transfer of the inhibitor from plasma to interstitium with catabolism serving as an alternative to this transport.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646057 ◽

1987 ◽

Vol 58 (04) ◽

pp. 1064-1067 ◽

Cited By ~ 41

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.

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The Different Forms of Antithrombin III in Serum

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651855 ◽

1977 ◽

Vol 38 (02) ◽

pp. 0494-0503 ◽

Cited By ~ 18

Author(s):

D. S Pepper ◽

D Banhegyi ◽

J. D Cash

Keyword(s):

Molecular Weight ◽

Affinity Chromatography ◽

Human Serum ◽

Degradation Product ◽

Antithrombin Iii ◽

Gel Filtration ◽

Free Form ◽

Polymeric Form ◽

At Iii ◽

Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657178 ◽

1982 ◽

Vol 47 (03) ◽

pp. 244-248 ◽

Cited By ~ 47

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.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657333 ◽

1983 ◽

Vol 49 (02) ◽

pp. 109-115 ◽

Cited By ~ 4

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).

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Faculty Opinions recommendation of Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727713903.793534341 ◽

2017 ◽

Author(s):

Mario Lebendiker

Keyword(s):

Affinity Chromatography ◽

High Affinity ◽

One Step

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Immunochemical Studies on Antithrombin III

10.1055/s-0039-1684699 ◽

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.

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