The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
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.

Isolation and Partial Characterization of Equine Antithrombin III.

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 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.

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

1977 ◽  
Author(s):  
W.H. Holleman ◽  
L.J. Coen ◽  
J.O. Capobianco ◽  
G.H. Barlow
Keyword(s):  
Affinity Chromatography ◽  
Sodium Citrate ◽  
Antithrombin Iii ◽  
Sedimentation Velocity ◽  
Hydroxyl Groups ◽  
Amino Groups ◽  
Bovine Thrombin ◽  
Homogeneous Product ◽  
At Iii ◽  
Human Thrombin

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).

scholarly journals Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein

1980 ◽  
Vol 189 (2) ◽  
pp. 247-253 ◽  
Author(s):  
T M Turpeenniemi-Hujanen ◽  
U Puistola ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polymeric Form ◽  
Lysyl Hydroxylase ◽  
Enzyme Form

Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol.

Isolation and Partial Characterization of Equine Antithrombin III

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.

Characterization Of Rabbit Antithrombin III

1981 ◽  
Author(s):  
D Estry ◽  
J C Mattson ◽  
T G Bell ◽  
G H Tishkoff
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Heparin Binding ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Immunologic Assays

The rabbit is a well established model for studying the disseminated intravascular coagulation (DIC) associated with endotoxic syndromes. In order to establish the role of antithrombin III (AT III) in the modulation of DIC in the rabbit, characterization of rabbit AT III was undertaken. Rabbit antithrombin III, isolated according to modifications of the method of Thaler and Schmer, has a molecular weight comparable to that of human AT III (62,000 daltons) as measured by mobility on SDS-PAGE gels. Mixtures of rabbit and human AT III co-migrate as a single band on 7.5% SDS-PAGE gels. Rabbit AT III possesses both progressive and heparin activated (immediate) antithrombin activity in assays using human thrombin. Antisera raised against rabbit AT III demonstrates no cross reactivity with human AT III suggesting that despite physiologic and molecular weight similarities, antigenic differences are present. Incubation of rabbit antithrombin III with specific antisera, either prior to or after addition of heparin, did not alter the ability of antithrombin III to inhibit thrombin in either the immediate or progressive assays indicating that the antigenic determinants are not found in either the heparin binding or active thrombin binding sites. Crossed immunoelectrophoresis (IEP) demonstrates that antisera to rabbit AT III reacts with both free rabbit antithrombin III and AT III-thrombin complexes and can therefore be used in immunologic assays to quantitate total rabbit AT III (bound and free) and in crossed IEP to demonstrate the mobility of both free and complexed AT III.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

scholarly journals Complex Formation of Covalently Crosslinked Fibrin Oligomers with Agarose Coupled Fibrinogen and Fibrin

1977 ◽  
Author(s):  
R. von Hugo ◽  
R. Hafter ◽  
A. Stemberger ◽  
H. Graeff
Keyword(s):  
Molecular Weight ◽  
Complex Formation ◽  
Affinity Chromatography ◽  
High Molecular Weight ◽  
Calcium Chloride ◽  
Gel Filtration ◽  
Void Volume ◽  
Soluble Fibrin ◽  
Derivatives Of

Crosslinked high molecular weight derivatives of fibrin (fibrinoligomers) were observed during intravascular coagulation. It was the purpose of this study to investigate the complex formation of fibrin oligomers with fibrinogen and fibrinmonomer. Fibrinogen coupled to agarose (Fg-ag) as well as fi-brinmonomer coupled to agarose (Fm-ag) was used as substrate. Soluble oligomers of fibrin were produced by incubating fibrinogen with thrombin, calcium-chloride, cystein and F XIII. They were separated from fibrinogen by gel filtration. Γ-dimers were demonstrated in fractions from the void volume and the shoulder prior to the fibrinogen peak. These fractions were subjected to affinity chromatography. Crosslinked oligomers of fibrin were not adsorbed on Fg-ag, yet adsorption occured on Fm-ag. This indicates that fibrin oligomers have no affinity to fibrinogen, yet readily form complexes with fibrin. This could mean that in vivo they compete with fibrinogen for the fibrinmonomer part of soluble fibrin monomer complexes, and hence have a tendency to increase in size.

NEUTRALIZING EFFECT OF PLATELET FACTOR 4 OR HISTIGINE-RICH GLYCOPROTEIN AGAINST LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN

1987 ◽  
Author(s):  
K Takahashi ◽  
M Niwa ◽  
N Sakuragawa
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
Bleeding Tendency ◽  
Human Origin ◽  
Platelet Factor ◽  
At Iii ◽  
Antifactor Xa

Purpose: Low molecular weight(LMW) heparin shows stronger antifactor Xa(F-Xa) and weaker anti-thrombin(TH) activities compared with unfractionated(UF) heparin, and shows less bleeding tendency in the cases of clinical use. Platelet factor 4(Pf-4) and histidine-rich glycoprotein(HRG) neutralize heparin. We investigated on the heparin neutralizing effects of them to both kinds of heparinMaterials and methods: LMW heparin(Kabi and Pharmuka) and UF heparin(Novo) were used. Antithrombin III(AT-III), HRG(human origin ) and pf-4( bovine origin ) were purified by our methodsTH(Green-Cross) and F-Xa(Sigma) were used. Reaction mixtures for anti-TH or anti-F-Xa were as follows: 1 vol of AT-III( 0.1 U/ml)+ 1 vol of heparin( 10 ug/ml)+l vol of pf-4 or HRG(varied)→incubated for 5 min→+l vol of TH(5 U/ml) or F-Xa( 7 nKat/ml)→incubated for 5 min→ + S-2238 or S-2222→ recorded at 405 nm.Results: (1) Pf-4 showed the equivalent anti-TH effect on both kinds of heparin, and 3 ug of pf-4 neutralized 1 ug of heparinOn F-Xa neutralizing effect, 13 ug of pf-4 neutralized 1 ug of UF heparin, but could not neutralize LMW heparin. (2) HRG showed the same results on anti-TH effect of both kinds of heparin, but could not neutralize the anti-F-Xa effect of LMW heparin on the same amount of HRG which neutralized that of UF heparin. Conclusion: Anti-F-Xa effect of. LMW heparin could not be easily neutralized by pf-4 or HRG compared with that of UF heparin.

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

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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