A Highly Purified Antithrombin III Concentrate Prepared From Human Plasma Fraction IV-1 by Affinity Chromatography

Vox Sanguinis ◽  
1994 ◽  
Vol 67 (2) ◽  
pp. 117-124 ◽  
Author(s):  
Wytold R. Lebing ◽  
David J. Hammond ◽  
James E. Wydick ◽  
George A. Baumbach
Keyword(s):  
Affinity Chromatography ◽  
Human Plasma ◽  
Antithrombin Iii ◽  
Plasma Fraction

A Highly Purified Antithrombin III Concentrate Prepared From Human Plasma Fraction IV-1 by Affinity Chromatography

Vox Sanguinis ◽  
1994 ◽  
Vol 67 (2) ◽  
pp. 117-124
Author(s):  
Wytold R. Lebing ◽  
David J. Hammond ◽  
James E. Wydick lll ◽  
George A. Baumbach
Keyword(s):  
Affinity Chromatography ◽  
Human Plasma ◽  
Antithrombin Iii ◽  
Plasma Fraction

Affinity Chromatography Used To Identify Asa-Binding Proteins From Human Plasma

1981 ◽  
Author(s):  
C M Kirchmaier ◽  
G Fink ◽  
N Bender ◽  
K Breddin
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Analytical Methods ◽  
Gel Electrophoresis ◽  
Covalent Binding ◽  
Plasma Fraction ◽  
Acetyl Group ◽  
Sepharose 4B ◽  
Poly Acrylamide

By the formerly used analytical methods it was not possible to show the binding of Acetylsalicylicacid (ASA) to proteins, if the acetyl-group of the ASA was not transferred to the protein. Using affinity chromatography it was possible to demonstrate the simultaneous non covalent binding of several proteins of a human plasma fraction to the immobilized ligand ASA.To this purpose we synthesized a new photoreactive ASA-analogue N3-ASA, which was coupled to SEPHAROSE 4B, using 3,3’- /mino-bis-propylamine as a spacer.The isolated proteins were identified by SDS-poly- acrylamide gel-electrophoresis and by immunoelectrophoresis. After eluation of the column albumin and two proteins with a molecular weight of about 200 000 Daltons bound to ASA were found.Affinity chromatography using the N3-ASA analogue may be a new useful tool to investigate the non covalent binding of ASA to proteins and to reveal unknown ASA- effects not mediated by the transfer of the acetyl- group

scholarly journals Heparin cofactor activities in a family with hereditary antithrombin III deficiency: evidence for a second heparin cofactor in human plasma

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 111-118 ◽  
Author(s):  
MJ Griffith ◽  
T Carraway ◽  
GC White ◽  
FA Dombrose
Keyword(s):  
Venous Thrombosis ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Functional Properties ◽  
Plasma Levels ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Normal Plasma ◽  
Antithrombin Iii Deficiency ◽  
Inhibit Factor

Abstract Plasma levels of antithrombin-heparin cofactor, determined by heparin- dependent antithrombin assay, and antithrombin III antigen were measured in 22 members of a large kindred predisposed to venous thrombosis. While 11 members had reduced plasma levels of both antithrombin-heparin cofactor and antithrombin III antigen, the levels of antithrombin-heparin cofactor were always greater than the levels of antithrombin III antigen: 66% (+/- 7%) and 49% (+/- 5%) of normal plasma, respectively. Pooled normal plasma and plasma from one of the affected family members (60% antithrombin-heparin cofactor and 47% antithrombin III antigen) were fractionated by heparin-agarose affinity chromatography. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 0.4 M NaCl and did not cross- react with antibody specific for antithrombin III and did not inhibit factor Xa at an appreciable rate in the presence of heparin, was designated heparin cofactor A. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 2.0 M NaCl, was functionally and antigenically identified as antithrombin III. The concentrations of heparin cofactor A in normal and patient plasma were similar (4.5 x 10(-7) M), while the concentration of antithrombin III in patient plasma (8.0 x 10(-7) M) was only 50% of normal (1.6 x 10(-6) M). The functional properties of both heparin cofactor A and antithrombin III obtained from patient plasma were normal. From the results of the present study it would appear that the antithrombin- heparin cofactor concentrating measured in patient plasma reflects the combined concentrations of heparin cofactor A and antithrombin III. Since heparin cofactor A does not cross-react with antibody to antithrombin III, the concentration of antithrombin III antigen in patient plasma is thus lower than the concentration of antithrombin- heparin cofactor.

Fate of Thrombin and Thrombin-Antithrombin-III Complex Adsorbed to a Heparinized Biomaterial: Analysis of the Enzyme-Inhibitor Complexes Displaced by Plasma

1983 ◽  
Vol 50 (04) ◽  
pp. 873-877 ◽  
Author(s):  
Mark W C Hatton ◽  
Gordon Rollason ◽  
Michael V Sefton
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Enzyme Inhibitor ◽  
Gel Filtration ◽  
Similar Rate ◽  
Subsequent Treatment ◽  
Enzyme Inactivation ◽  
Plasma Fraction ◽  
Thrombin Antithrombin Iii Complex ◽  
Free Plasma

SummaryHeparin covalently-linked to polyvinyl alcohol (PVA) is a biomaterial which is of potential value as a non-thrombogenic coating. 125I-labelled thrombin adsorbed to heparin-PVA beads was not dislodged by phosphate-buffered saline, pH 7.4, although radioactivity was progressively displaced from the adsorbent by fibrinogen-free human plasma. Analysis by gel filtration and affinity chromatography showed that the released radioactivity was distributed between (thrombin-antithrombin-III) complex (approx. 70%) and, probably, (thrombin-α-2-macroglobulin) complex (approx. 30%). Less efficient thrombin displacement was obtained by either bovine serum albumin (5% w/v) or antithrombin-III-free human plasma: in the latter case, the dislodged enzyme was presumably associated with α-2-macroglobulin. Purified α-2-macroglobulin did not displace thrombin from heparin-PVA. The quantity of thrombin displaced by an α- 2-macroglobulin-free plasma fraction compared well with fibrinogen-free plasma: The eluted enzyme was largely associated with antithrombin-III. Purified antithrombin-III did not displace thrombin from heparin-PVA despite causing >70% inactivation of the bound enzyme. Subsequent treatment with fibrinogen-free plasma dislodged (thrombin-antithrombin-III) at a similar rate to that of bound thrombin. We conclude that plasma contains a component(s) which displaces (thrombin-antithrombin-III) complex from immobilised heparin: presumably this leaves the heparin sites free for further use in enzyme inactivation.

scholarly journals Heparin cofactor activities in a family with hereditary antithrombin III deficiency: evidence for a second heparin cofactor in human plasma

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 111-118
Author(s):  
MJ Griffith ◽  
T Carraway ◽  
GC White ◽  
FA Dombrose
Keyword(s):  
Venous Thrombosis ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Functional Properties ◽  
Plasma Levels ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Normal Plasma ◽  
Antithrombin Iii Deficiency ◽  
Inhibit Factor

Plasma levels of antithrombin-heparin cofactor, determined by heparin- dependent antithrombin assay, and antithrombin III antigen were measured in 22 members of a large kindred predisposed to venous thrombosis. While 11 members had reduced plasma levels of both antithrombin-heparin cofactor and antithrombin III antigen, the levels of antithrombin-heparin cofactor were always greater than the levels of antithrombin III antigen: 66% (+/- 7%) and 49% (+/- 5%) of normal plasma, respectively. Pooled normal plasma and plasma from one of the affected family members (60% antithrombin-heparin cofactor and 47% antithrombin III antigen) were fractionated by heparin-agarose affinity chromatography. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 0.4 M NaCl and did not cross- react with antibody specific for antithrombin III and did not inhibit factor Xa at an appreciable rate in the presence of heparin, was designated heparin cofactor A. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 2.0 M NaCl, was functionally and antigenically identified as antithrombin III. The concentrations of heparin cofactor A in normal and patient plasma were similar (4.5 x 10(-7) M), while the concentration of antithrombin III in patient plasma (8.0 x 10(-7) M) was only 50% of normal (1.6 x 10(-6) M). The functional properties of both heparin cofactor A and antithrombin III obtained from patient plasma were normal. From the results of the present study it would appear that the antithrombin- heparin cofactor concentrating measured in patient plasma reflects the combined concentrations of heparin cofactor A and antithrombin III. Since heparin cofactor A does not cross-react with antibody to antithrombin III, the concentration of antithrombin III antigen in patient plasma is thus lower than the concentration of antithrombin- heparin cofactor.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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.

A Hitherto Undescribed Naturally Occurring Plasma Antagonist of Activated Factor X Inhibitor (Antithrombin III)

1979 ◽  
Author(s):  
E. T. Yin ◽  
W. J. Salsgiver ◽  
O. Tangen
Keyword(s):  
Equilibrium State ◽  
Human Plasma ◽  
Antithrombin Iii ◽  
Circumstantial Evidence ◽  
Incubation Mixture ◽  
Factor X ◽  
Normal Human Plasma ◽  
Plasma Component ◽  
Naturally Occurring ◽  
Normal Human

Circumstantial evidence suggested that normal human plasma contained a substance regulating the neutralization of F.Xa by F.Xa inhibitor(XaI), (Yin et.al.,Adv.Exper. Med. & Biol., 52 : 239, 1975, Plenum Press, N.Y.).This plasma component has now been isolated and partially purified in our laboratory, and tentatively designated as “Anti-XaI”.In experiments employing purified components, when Anti-XaI was incubated at 37°C with F.Xa, Xal and heparin for two minutes at pH7.5, the amount of F.Xa inhibited was inversely proportional to the Anti-XaI concentration. But, when the F.Xa was replaced by thrombin in the incubation mixture, the neutralization of thrombin clotting activity was undisturbed.Anti-XaI was found to be neither PF3 nor PF4.These and other data strongly suggest that the “Antithrombin III pathway” is more complex than currently believed to be. In circulating blood an equilibrium state must exist between Anti-XaI and XaI.Under certain conditions when the Anti-XaI activity is predominant the rate of F.Xa neutralization bv XaI then becomes slower than the activation of prothrombin to thrombin by F.Xa.

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