Further Investigations on Antithrombin III in the Plasmas of Patients with the Abnormality of ‘Antithrombin III Budapest’

1975 ◽  
Vol 33 (03) ◽  
pp. 564-572 ◽  
Author(s):  
Géza Sas ◽  
Duncan S Pepper ◽  
John D Cash
Keyword(s):  
Electrophoretic Mobility ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Molecular Size ◽  
High Concentration ◽  
Abnormal Protein ◽  
Crossed Immunoelectrophoresis ◽  
Normal Plasma ◽  
At Iii ◽  
Cofactor Activity

SummaryAntithrombin III (AT-III) was studied in a thrombophilic family with an abnormal AT-III molecule (antithrombin III Budapest) using a modified crossed Immunoelectrophoresis technique, gel filtration, ‘rocket’ Immunoelectrophoresis and a heparin cofactor assay.When plain agarose was applied in the first phase of the crossed Immunoelectrophoresis, the normal and the pathological AT-III revealed identical electrophoretic mobility. However, when heparin was mixed with agarose in the first phase of electrophoresis, the propositus’ plasma displayed a different AT-III pattern from normal plasma. His plasma contained the first component of the normal plasma (Immune Antithrombin III1, IAT-III1) in a concentration of only 5% of normal, and a protein in high concentration which although immunoreactive to AT-III antisera, had an electrophoretic mobility similar (but not identical) to that of IAT-III2. This ab-normal protein had no heparin cofactor activity and a molecular size greater than normal plasma AT-III. Unlike normal AT-III, the addition of heparin did not change the molecular size of the pathologic AT-III molecule significantly.The abnormal protein was present in lower concentrations in the patient’s children and at the time of study they had no clinical or laboratory evidence of intravascular coagulation.

A New Variant of Antithrombin III. Study of three Related Cases.

1979 ◽  
Author(s):  
M. Wolf ◽  
C. Boyer ◽  
J.M. Lavergne ◽  
M.J. Larrieu
Keyword(s):  
Electrophoretic Mobility ◽  
Antithrombin Iii ◽  
Chromogenic Substrates ◽  
Double Peak ◽  
Enzymatic Assays ◽  
Crossed Immunoelectrophoresis ◽  
New Variant ◽  
At Iii ◽  
Cofactor Activity ◽  
Two Peaks

A qualitative abnormality of antithrombin III (AT III) was demonstrated in three member from the same family. The mother (58 y.) had recurrent episodes of venous thrombosis an pulmonary embolism whereas the affected daughters (20 and 29 y.) are asymptomatic. AT III was investigated in presence and absence Of heparin hy enzymatic assays using chromogenic substrates (S-2238 and S-2160) and by rocket - and crossed - immunoelectro phoresis using two different monospecific anti-AT III antisera. In the three patients the qualitative abnormality of AT III was suggested by the discrepancy between a norml level of AT III antigen and decreased heparin-cofactor activity (49, 51, 54%). By crossed immunoe1ectrophoresis, patients’ AT III migrated as a single peak in absence of heparin, with the same electrophoretic mobility as that of the control. In presence of heparin (25 u/ml), crossed immunoelectrophoresis demonstrated the presence of two peaks, one with a normal, and the other with a decreased electrophoretic mobility. A double peak was also observed by rocket immunoelectrophoresis in presence of heparin. In this family with a variant of AT III, the three heterozygous affected cases demonstrate two popu1ations of AT III, with a different affinity for heparin.

Antithrombin III Alger: A New Homozygous AT III Variant

1986 ◽  
Vol 55 (02) ◽  
pp. 218-221 ◽  
Author(s):  
A M Fischer ◽  
P Cornu ◽  
C Sternberg ◽  
F Mériane ◽  
M D Dautzenberg ◽  
...  
Keyword(s):  
Family History ◽  
Antithrombin Iii ◽  
Antigen Concentration ◽  
Crossed Immunoelectrophoresis ◽  
The Family ◽  
At Iii ◽  
Molecular Abnormality ◽  
Slow Moving ◽  
Cofactor Activity ◽  
Plasma Heparin

SummaryA qualitative abnormality of antithrombin III (AT III) was found in the plasma of a 41-year old patient. The plasmatic AT III antigen concentration was 130% and the progressive anti-F IIa and anti-F Xa activities were normal (105% and 137%). The plasma heparin cofactor activity was less than 10%, when measured by F Ila or F Xa inhibition. Crossed immunoelectrophoresis of AT III in the presence of heparin revealed in the plasma an abnormal slow-moving peak. When tested by affinity chromatography on heparin Sepharose, this abnormal AT III did not bind to heparin. Among the investigated relatives, 5 subjects had normal AT III levels, whatever the test used, the nine others having reduced levels of antithrombin heparin cofactor activity (45-61%) but normal levels of immunoreactive AT III (97-122%). Consanguinity was found in the family history. We therefore considered our patient as homozygous for an AT III molecular abnormality affecting the binding site for heparin.

AT III Barcelona: A Familial Quantitative-Qualitative AT III Deficiency

1988 ◽  
Vol 59 (01) ◽  
pp. 013-017 ◽  
Author(s):  
E Grau ◽  
J Fontcuberta ◽  
J Félez ◽  
I de Diego ◽  
R Soto ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Normal Range ◽  
Affinity Adsorption ◽  
Crossed Immunoelectrophoresis ◽  
At Iii ◽  
Spanish Family ◽  
Slow Moving ◽  
Cofactor Activity ◽  
Ph Range ◽  
Thrombotic Tendency

SummaryA quantitative and qualitative deficiency of antithrombin III (AT III) was found in four members of a Spanish family with thrombotic tendency. In all affected members, levels of AT III antigen and activity (heparin cofactor activity) were reduced to 50% of the normal range. When crossed immunoelectrophoresis (CIE) was performed in the presence of heparin, an abnormal slow-moving peak was found. Crossed immunoelectrofocusing (CIEF) from normal and affected individuals showed that normal AT III migrated between pH 4.9–5.3 while the AT III under study was asymetrically distributed between two pH ranges: 4.9–5.3 and 4.6–4.8. Affinity adsorption of affected members’ plasma to heparin-sepharose beads revealed one population of AT III in the supernatant corresponding to the abnormal AT III, devoid of heparin cofactor activity and showing a peak between pH range: 4.6–4.8 in CIEF.Our data supports the view that a quantitative-qualitative deficiency was present in the heterozygous state in all the affected family members. Both normal and abnormal ATIII were present in plasma of the affected individuals. This abnormal ATIII was characterized by a lack of affinity for heparin. This familial ATIII deficiency was named ATIII Barcelona.

High Molecular Weight Forms of Antithrombin III Complexes in Blood

1983 ◽  
Vol 49 (01) ◽  
pp. 032-036 ◽  
Author(s):  
E Marciniak ◽  
G Gora-Maslak
Keyword(s):  
Antithrombin Iii ◽  
Serum Proteins ◽  
Gel Filtration ◽  
Factor Xa ◽  
Molecular Size ◽  
Molecular Forms ◽  
Factor X ◽  
Monomeric Complex ◽  
At Iii ◽  
Antibody Competition

SummaryA double antibody competition radioimmunoassay was developed that allowed to detect specifically as little as 15 ng antithrombin III (AT III) per ml of the assayed material. In normal plasma examined by this assay, AT III concentration averaged 199 ± 21 μg/ml. Complexes of AT III with thrombin or factor X a crossreacted with free AT III in 87% and 95%, respectively. Molecular forms of AT III produced in plasma treated with coagulation enzymes, or in serum, were assessed by measuring immunoreactive AT III in fractions obtained by gel filtration chromatography on Sephadex G-200. AT III bound by thrombin in fibrinogen free-plasma ranged in molecular size from 160,000 to above 250,000. Similar aggregation occurred when monomeric complex of purified AT III and thrombin, of 90,000 Mr, was added to plasma. Presence of heparin intensified the degree of aggregation. In factor Xa-treated plasma AT III was converted into components with 160,000 Mr, or less. No complexes below 200,000 Mr were present in serum. They decreased in size to 160,000 Mr after affinity chromatography on heparin-Sepharose. These results indicated that blood represents a unique milieu conducive to aggregation of bound AT III. It appears, however, that AT III complexes present in blood may not only aggregate, but also associate with other serum proteins through unstable binding most likely caused by the enzyme component of the complex.

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

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.

Antithrombin III Geneva: A Hereditary Abnormal AT III with Defective Heparin Cofactor Activity

1987 ◽  
Vol 57 (02) ◽  
pp. 154-157 ◽  
Author(s):  
P A de Moerloose ◽  
G Reber ◽  
Ph Vernet ◽  
Ph Minazio ◽  
C A Bouvier
Keyword(s):  
Antithrombin Iii ◽  
Normal Pattern ◽  
Crossed Immunoelectrophoresis ◽  
At Iii ◽  
Normal Mean ◽  
Cofactor Activity ◽  
Normal Inhibition ◽  
Heparin Affinity ◽  
Two Populations ◽  
Inhibition Of Thrombin

SummaryA 43-year-old man presented a pulmonary embolism. The unusual circumstances of apparition, the age and the increased heparin requirements suggested an antithrombin III (AT III) deficiency. AT III activity was low in the propositus and seven other members of his family (mean 55%), but immunologic levels were normal (mean 110%). Crossed immunoelectrophoresis in absence of heparin showed a normal pattern, but in presence of heparin showed an abnormal peak as compared with controls. Kinetics experiments showed a normal inhibition of thrombin and Xa in absence of heparin, but abnormal in presence of heparin. Affinity chromatography on heparin-Sepharose revealed two populations of AT III, one of which was devoid of heparin cofactor activity. The toponym AT III Geneva is proposed for this new familial abnormal AT III with defective heparin cofactor activity.

Interaction Of Atherogenic (VLdL; LdL) And Non Atherogenic Lipoproteins (HDL) With Heparin Fractions Of Various Molecular Size

1981 ◽  
Author(s):  
P Duthilleul ◽  
P Fievet ◽  
L Bouillet ◽  
J C Fruchart
Keyword(s):  
Gel Filtration ◽  
Molecular Size ◽  
Two Dimensional ◽  
Fluorimetric Method ◽  
Molecular Weights ◽  
Crossed Immunoelectrophoresis ◽  
At Iii ◽  
Heparin Fractions ◽  
Inhibition Of Thrombin

Some anticoagulant properties of various heparin fractions (with molecular weights from 5 000 to 20 300 daltons) have been investigated in purified and plasmatic systems with and without Lipoproteins isolated by ultracentrifugation from hyperlipemic subjects (type IIa). Amidolytic (Tos-Gly-Pro-Arg-pNA) and coagulometric (Yin and Wessler) anti Xa activity of a HMW fraction (231 USP/mg. MW: 20 300) was decreased from 15 %m both systems supplied with LDL or VLDL since HDL have no effect. No influence was detected on anti Ila activity (Phe-pro-arg-AIE fluorimetric method). LMW (20 USP/mg ; MW 6 000 - 8 000) and ULMW (20 USP.MW 4 000-5 000) anti Xa and anti Ila activities was modified neither by VLDL/LDL neither by HDL. These findings point out to differences in the mechanisms of inhibition of thrombin and Xa by various molecular weigh heparin fractions.Two dimensional crossed Immunoelectrophoresis in agarose 1 % with mixing various quantities of each heparin fraction (0 - 100 μg/ml) in the first phase of electrophoresis of LDL/VLDL, LDL/VLDL and AT III, LDL/VLDL and FXa reveal the presence of LDL or VLDL/heparin fractions complexes and LDL or VLDL/AT III complexes, which are isolated by Gel filtration on Ultrogel A 6. We conclude that FXa can escape from neutralization by inactive complexes.

Respective Roles of Antithrombin III and Alpha 2 Macroglobulin in Thrombin Inactivation

1981 ◽  
Vol 45 (01) ◽  
pp. 051-054 ◽  
Author(s):  
A M Fischer ◽  
J Tapon-Bretaudiere ◽  
A Bros ◽  
F Josso
Keyword(s):  
Antithrombin Iii ◽  
Practical Interest ◽  
High Plasma ◽  
At Iii ◽  
Human Material ◽  
Cofactor Activity

SummaryIn order to investigate the mechanism of thrombin inactivation in the presence of both antithrombin III (AT III) and α 2-macroglobulin (α 2 M), thrombin and the inhibitors have been purified from human material and thrombin inactivation studied using purified reagents either alone or added to defibrinated plasma. Comparison of clotting and amidolytic activities of residual thrombin allowed to measure the amount of thrombin bound to α 2 M. In a purified reagent system as well as in plasma, part of exogenous thrombin is bound to α 2 M. The amount of bound thrombin is related to α 2 M concentration. Conversely, previous plasma α 2 M depletion by immunoabsorption increases the consumption of heparin-cofactor activity by exogenous thrombin. Thus AT III and α 2 M compete for thrombin inactivation. This finding could be of practical interest in clinical situations associating high plasma α 2 M levels and a decrease of AT III concentration.

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.

Antithrombin III, Heparin Cofactor and Antifactor Xa in Relation to Age, sex And Pathological Conditions

1979 ◽  
Author(s):  
F. Panicucci ◽  
A. Sacripanti ◽  
E. Pinori ◽  
M. Vispi ◽  
B. Conte ◽  
...  
Keyword(s):  
Oral Contraceptive ◽  
Antithrombin Iii ◽  
Normal Subjects ◽  
Cerebral Thrombosis ◽  
Positive Correlation ◽  
Pathological Conditions ◽  
At Iii ◽  
The Mean ◽  
Antifactor Xa ◽  
Cofactor Activity

Determinations of AT-III activity, heparin cofactor activity, antifactor Xa activity and AT-III protein were carried out in 200 healthy adults, evenly distributed within age and sex groups, in 60 patients with cerebral thrombosis and in 20 oral contraceptive users. There was a positive correlation between AT-III protein and its activitiesin normal subjects and in patients with cerebral thrombosis. In oral contraceptive users the positive correlation was between AT-III protein and its activities, antifactor Xa activity excepted. The mean AT-III protein and heparin cofactor activity values decreased in males with age and were significantly lower in the groups between 50 and 70 years. The mean AT-III protein and heparin cofactor activity values decreased slightly in women in fertile age and were lower in the 40 to 50 age-group. The mean AT-III protein and its activities values did not show any variation in the patients with cerebral thrombosis. The mean antifactor Xa activity value in the women, taking the pill for 3 months, decreased, whereas the other AT-III activities and AT-III protein were unchanged.

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