Isolation of Human Antithrombin-III by Affinity Chromatography on Heparin-Agarose

Methods were developed for the isolation of gm. quantities of human antithrombin-III (AT-III) from Cohn Fraction IV-1 of human plasma using heparin covalently attached to agarose. Attachment of heparin carboxyl groups to alkylamino-agarose yielded a support with no affinity for AT-III. Linkage via the heparin hydroxyl groups yielded a support with approximately 1 mg of heparin/ml agarose and with a low capacity for binding AT-III. Linkage of heparin to agarose thru its amino groups yielded a heparin-agarose with the highest capacity for AT-III. Reaction of heparin containing a free α-amino group with cyanogen bromide activated agarose resulted in agarose substituted with 5 mg of heparin/ml. The conditions of buffer, pH, ionic strength and temperature which maximized AT-III binding were 0.05M sodium phosphate, 0.02M sodium citrate, 0.15M NaCl, pH 8.3 and 4°. The heparin-agarose bound 0.1-0.2 mg of AT-IIl/ml. The AT-III isolated by affinity chromatography was further purified by gel permeation and yielded a homogeneous product as judged by Polyacrylamide disc gel electrophoresis of native and reduced protein and by sedimentation velocity (S20, w = 4.1). This material had an activity of 1700 units/A280 as measured by inhibition of human thrombin. The AT-III is stable to heating at 60° for 10 hours in a buffer of 0.5M sodium citrate at pH 7-8. Injection of bovine thrombin (3000 units/Kg) into heparinized dogs (150 units/Kg) decreased circulating AT-III levels to 50%. (Supported by NHLI, Contract NOl-HB-4-2946).

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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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Pasteurization Of Antithrombin III: Stabilization By Heparin And Lyotropic Anions

10.1055/s-0038-1653120 ◽

1981 ◽

Author(s):

Thomas F Busby ◽

Donald H Atha ◽

Kenneth C Ingham

Keyword(s):

Sodium Citrate ◽

Antithrombin Iii ◽

Antithrombin Activity ◽

At Iii ◽

Exclusion Chromatography ◽

Direct Binding ◽

Chaotropic Anions ◽

Increasing Temperature ◽

Denaturation Curve ◽

Lyotropic Anions

Pasteurization of Antithrombin III (AT III) for 10 hours at 60°C is necessary to reduce the risk of transfusion hepatitis. Addition of appropriate stabilizers can largely prevent the loss of antithrombin activity which otherwise occurs during pasteurization. Studies of the mechanism of denaturation and stabilization have been facilitated by the use of 1,8-anilinonaphthalene sulfonate (ANS) which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature as the protein unfolds. The extent of the increase in ANS fluorescence correlated roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure exclusion chromatography. The midpoint, Td, of the thermal denaturation curve increased by 13 and 19°C in the presence of 0.5 M and 1.0 M sodium citrate respectively. Phosphate, sulfate, and EDTA were also strong stabilizers while the chaotropic anions, iodidé and thiocyanate were potent destabilizers. Heparin, at 10 mg/ml, increased Td by 7°, presumbly through a direct binding mechanism. Reducing agents increased ANS fluorescence by an amount similar to that seen with thermally denatured samples, an effect which was inhibited by heparin but not by citrate. Furthermore, incorporation of 14C-iodoacetamide into AT III during thermal titration was coincident with the increase in ANS fluorescence suggesting that disulfide cleavage is the event which triggers the unfolding of the protein. Samples pasteurized for 10 hours at 60°C in the presence of 0.5 M and 1.0 M citrate retained full antithrombin activity but exhibited evidence of minor alterations in the ability to bind heparin.

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Heparin-Affinity of Antithrombin III in a Family With Congenital Antithrombin III Deficiency

10.1055/s-0039-1684713 ◽

1979 ◽

Author(s):

G. Sas ◽

D. Bánhegyi ◽

I. Petö

Keyword(s):

Affinity Chromatography ◽

Antithrombin Iii ◽

Normal Person ◽

Agarose Gel ◽

Functional Methods ◽

At Iii ◽

Antithrombin Iii Deficiency ◽

Heparin Affinity

We found a new thrombophilic family with antithrombin III/ AT-III / deficiency. In the members of this family both immunologic and functional methods revealed similarly decreased levels of AT-III. Gelfiltration displayed identical size of AT-III molecule of patient and normal alike. On the basis of tnese findings we assumed that in this family normal AT-III is produced Dut only in diminished quantity. Experiments on the heparin-affinity of AT-III did not support this assumption. The AT-III of the proposita migrated slower than that of normal person in the heparinized agarose gel. In the course of the heparin-affinity chromatography the AT-III of the proposita could be eluted at lower salt concentrations than normal AT-III.Thus we conclude that even in the case of “true” AT-III deficiency the molecule might have some qualitative deviation from normal.

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Isolation and Partial Characterization of Equine Antithrombin III.

10.1055/s-0039-1684573 ◽

1979 ◽

Cited By ~ 1

Author(s):

D.W. Estry ◽

T.G. Bell ◽

G.H. Tishkoff ◽

J.C. Mattson ◽

S.C. Estry

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Stable Complex ◽

Sds Page ◽

At Iii ◽

Human Thrombin ◽

Horse Plasma ◽

Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

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Immunchemical Investigation on Antithrombin III/AT-III/in Presence of Heparin

10.1055/s-0039-1684679 ◽

1979 ◽

Author(s):

D. Bánuergyi ◽

G. Sas

Keyword(s):

Affinity Chromatography ◽

Antithrombin Iii ◽

Original Method ◽

Normal Plasma ◽

At Iii ◽

Different Sources

Various components of AT-III car, be differentiated by crossed ioununo-eleetrophoresis in the presence of heparin as we described previously / Sas et al. 1975 /. The migration of heparin is discontinuous in this original method, i.e. at the end of the electrophoresis the plate does not contain heparin at all. This fact rises the possibility pf the artefactual nature of these AT-III components. To exclude this effect we introduced a modification using double plates incorporated with heparin. The second plate was used as a heparin “reservoir”. By this modification we obtained constant heparin flow in the course of electrophoresis. We found four different AT-III components in normal plasma comparing with three AT-III eomportints of the original method.Investigating heparins originated from different sources we found a higher mobility of AT-III in the pnsenee of mucous heparin than that of lung heparin. This observation corresponds well with the results obtamed by heparin-agarose affinity chromatography.

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Studies On The Effects Of Protamine On The Antithrombin III Heparin Complex

10.1055/s-0038-1653154 ◽

1981 ◽

Author(s):

Y Okajima ◽

M Nakagawa ◽

T Kitani ◽

S Urano ◽

R Hino ◽

...

Keyword(s):

Affinity Chromatography ◽

Electrophoretic Mobility ◽

Antithrombin Iii ◽

Free Fraction ◽

The Third ◽

Antithrombin Activity ◽

Crossed Immunoelectrophoresis ◽

At Iii ◽

Neutralizing Agent ◽

Phoretic Mobility

Protamine has been employed as principal neutralizing agent for heparin but there are few reports on the effect of protamine to antithrombin III (AT 3III)-heparin complex, and this interaction was analyzed by means of column chromatography, crossed immunoelectrophoresis, and heparin sepharose affinity chromatography. Sephadex G-200 chromatography of heat defibrinated plasma treated with 3H- heparin revealed radioactivity in all protein fractions and protein free fraction. Immediate antithrombin activity was detected in AT III fractions. When the plasma treated with 3H labeled heparin was fractioned on sephadex G-200 after neutralization by protamine, most of radioactivity was eluted in the void volume and the radioactivity of other fractions was markedly decreased, and the immediate antithrombin activity was lost in any fractions and progressive antithrombin activity was detected in the third protein peak. Electrophoretic mobility of heparin treated AT III was increased depending to the heparin concentrations but when the heparinized plasma was neutralized with protamine, electrophoretic mobility of AT III was recovered. Meanwhile protamine treatment did not affect on the electro phoretic mobility of not heparinized control AT III. When AT III bound heparin sepharose was eluted with protamine solution and eluted fractions were chromatographed on sephadex G-75, AT III was separated from protamine. These results indicate that protamine dissociates AT III from AT III-heparin complex with binding to heparin and that the separated AT III recovers original progressive antithrombin activity.

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Homozygous Variant of Antithrombin III that Lacks Affinity for Heparin, AT III Kumamoto

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646520 ◽

1989 ◽

Vol 61 (01) ◽

pp. 020-024 ◽

Cited By ~ 28

Author(s):

Kenji Okajima ◽

Hidetsugu Ueyama ◽

Youichiro Hashimoto ◽

Yasuto Sasaki ◽

Keiko Matsumoto ◽

...

Keyword(s):

Affinity Chromatography ◽

Autosomal Dominant ◽

Antithrombin Iii ◽

Factor Xa ◽

Heparin Cofactor Ii ◽

At Iii ◽

Antifactor Xa ◽

Cofactor Activity ◽

Inhibition Of Thrombin ◽

Plasma Heparin

SummaryAbnormal antithrombin III (AT III) was found in the plasma of a 31-year-old female who suffered from recurrent thrombotic episodes. Heparin cofactor activity was 28% of normal and undetectable when measured by inhibition of thrombin and factor Xa (F. Xa), while both progressive antithrombin and antifactor Xa activities were normal. The concentration of plasma AT III antigen was 37 mg/dl. Analysis by crossed-immunoelec- trophoresis (CIE) in the presence of heparin and affinity chromatography on heparin-Sepharose revealed that the propositus’ AT III did not bind to heparin. When heparin cofactor II (HC II) was removed from propositus’ plasma, heparin cofactor activity of AT III was not detected. Thus, HC II seemed to account for the plasma heparin cofactor activity found in the presence of thrombin. The patient’s parents and three of her brothers demonstrated qualitative abnormality of AT III; heparin cofactor activity was 30-50% of normal levels in the presence of both thrombin and F. Xa. These findings indicate that the propositus’ AT III lacks affinity for heparin and the mode of its inheritance seems to be autosomal dominant and, hence, the propositus would be a homozygote. For this variant, the name of AT III Kumamoto is proposed.

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Isolation and Partial Characterization of Equine Antithrombin III

10.1055/s-0038-1665845 ◽

1979 ◽

Author(s):

D. W. Estry ◽

T. G. Bell ◽

G. H. Tishkoff ◽

J. C. Mattson ◽

S. C. Estry

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Stable Complex ◽

Sds Page ◽

At Iii ◽

Human Thrombin ◽

Horse Plasma ◽

Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. in order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichiometry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

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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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Polyethylene glycol can be validly omitted from chromogenic peptide substrate assay for antithrombin III.

Clinical Chemistry ◽

10.1093/clinchem/32.8.1554 ◽

1986 ◽

Vol 32 (8) ◽

pp. 1554-1556

Author(s):

H J Kolde

Keyword(s):

Polyethylene Glycol ◽

Antithrombin Iii ◽

Tween 80 ◽

Test System ◽

Chromogenic Substrate ◽

Peptide Substrate ◽

Chromogenic Substrates ◽

Bovine Thrombin ◽

Test Kit ◽

Human Thrombin

Abstract To investigate whether the characteristics of a commercial test kit for antithrombin III (Berichrom Antithrombin III) could be influenced by surfactants such as Tween-80 or polyethylene glycol (PEG), we performed some experiments with the original kit reagents and with the reagents dissolved in surfactants. Neither the reliability of the calibration curve nor the data for precision and assay kinetics were amended by the addition of either PEG to the (human) thrombin reagent or Tween-80 to the chromogenic substrate. In the same test system, assays with some other chromogenic substrates and with bovine thrombin showed comparable behavior. Evidently, if one follows the working scheme proposed for this kit, the use of surfactants is not warranted.

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