Purification and Partial Characterization of a Hereditary Abnormal Antithrombin III Fraction of a Patient with Recurrent Thrombophlebitis

1980 ◽  
Vol 44 (02) ◽  
pp. 087-091 ◽  
Author(s):  
T H Tran ◽  
H Bounameaux ◽  
C Bondeli ◽  
H Honkanen ◽  
G A Marbet ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Agarose Gel ◽  
Normal Range ◽  
Antigen Concentration ◽  
Superficial Thrombophlebitis ◽  
Antithrombin Activity ◽  
Crossed Immunoelectrophoresis ◽  
At Iii ◽  
Heparin Affinity

SummaryA relatively low heparin cofactor activity (0.60 U/ml) was observed in a patient with recurrent superficial thrombophlebitis of the left leg. However, the antigen concentration was in the normal range (1.04 U/ml) and the progressive antithrombin activity was normal. The crossed immunoelectrophoresis in presence of heparin in agarose gel separated the patient's AT-III antigen in 2 fractions with different mobilities. The patient's AT-III was purified for further characterization. The last step of the purification procedure, a heparin-agarose chromatography, led to a separation and a purification of 2 AT-III fractions with different heparin affinities: an abnormal AT-III with reduced heparin affinity and a normal AT-III with a heparin affinity similar to that of AT-III isolated from normal plasmas. Abnormal and normal AT-III share several identical properties as molecular weight, ability to form complexes with thrombin and progressive antithrombin activity.

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.

Biological Characteristics Op Antithrombin III In An Antithrombin III Deficient Family

1981 ◽  
Author(s):  
S Kondo ◽  
T Matsuo ◽  
Y Ohoki ◽  
O Matsuo
Keyword(s):  
Antithrombin Iii ◽  
Recurrent Thrombosis ◽  
Normal Range ◽  
Japanese Family ◽  
Neutralization Activity ◽  
Normal Value ◽  
Antithrombin Activity ◽  
At Iii ◽  
Molecular Abnormality ◽  
Antifactor Xa

In the familial AT III deficiency of a Japanese family, the propositus (a-39-yr old female) and her mother had episodes of recurrent thrombosis and their AT III levels as measured immunologically and biologically were below the normal value. In the plasma of her brother, the AT III concentration as measured immunologically was half of the normal value, but his biological antithrombin activity was within the normal range. The progressive antithrombin activity and antifactor Xa activity of plasma samples in this familial AT III deficiency were within the normal range. Measurements of the rate of thrombin neutralization activity revealed that the brother’s plasma was in the normal range, but the plasma of the propositus and of her mother showed rates of thrombin neutralization activity which were somewhat below the normal value. The rate of thrombin neutralization activity per mg protein of AT III was highest in the plasma of the brother, and became slower in the mother, propositus, and pooled normal plasma in that order. In the plasma of this familial AT III deficiency, the rate of Xa neutralization activity was much slower than the normal value. It is postulated that since the antithrombin of the brother of the propositus was found to react as normal in the neutralization of thrombin, he does not have episodes of thrombosis. Such characteristic hyperfunction of antithrombin in the plasma of the brother may be due to some molecular abnormality of AT III within this hereditary deficient family.

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.

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.

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.

ANTITHROMBIN III GLASGOW: A VARIANT WITH INCREASED HEPARIN AFFINITY AND REDUCED ABILITY TO INACTIVATE THROMBIN, ASSOCIATED WITH FAMILIAL THROMBOSIS

1987 ◽  
Author(s):  
D A Lane ◽  
G DO Lowe ◽  
A Flynn ◽  
H Ireland ◽  
E Thompson ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Crossed Immunoelectrophoresis ◽  
Reducing Conditions ◽  
High Incidence ◽  
At Iii ◽  
Heparin Plasma ◽  
Active Pool ◽  
A Minor ◽  
Heparin Affinity ◽  
Substrate Hydrolysis

A functional anti thrombin III CAT III) deficiency has been identified in three generations of a family with a high incidence of deep venous thrombosis. The deficiency presented as ~50% reduction in its heparin cofactor activity compared to its antigen concentration. No abnormality was detected by crossed immunoelectrophoresis of plasma in the presence or absence of heparin. Plasma from the propositus was precipitated with dextran sulphate, applied to heparin-Sepharose and the AT III stepwise eluted with NaCl . The AT III had a reduced ability to inactivate thrombin, when this was monitored by substrate hydrolysis or by SDS PA gel e1ectrophoresis. Its mobility was normal when examined by the latter technique on 10-20X gels under reducing and non-reducing conditions. Patient AT III was reapplied to heparin-Sepharose and eluted with a NaCl gradient. A minor active pool eluted in the same concentration range, 0.5-1.2M, used to purify normal AT III, while predominantly inactive AT III eluted at higher NaCl concentrations, 1.2-2.0M. Further purification of variant AT III was achieved by passage of heparin-Sepharose eluates through a thrombin-Sepharose column and structural studies are being undertaken on this material. It is concluded that AT III Glasgow has increased affinity for heparin but reduced ability to inactivate thrombin. It can be differentiated from two other AT III variants that bind heparin with high affinity, Milano and Northwick Park, but has similar features to those of AT III Chicago.

ANTITHROMBIN III NORTHWICK PARK: CHARACTERIZATION OF AN INACTIVE HIGH MW COMPLEX WITH INCREASED AFFINITY FOR HEPARIN

1987 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
A M Flynn ◽  
H Ireland ◽  
M Panico ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Exchange Chromatography ◽  
Terminal Sequence ◽  
Structural Investigations ◽  
Crossed Immunoelectrophoresis ◽  
Sds Page ◽  
Affected Family Member ◽  
At Iii ◽  
Salt Gradient

It has been shown previously that antithrombin III Northwick Park (AT III NWP) has reduced ability to inactivate thrombin and is characterised by an additional anodal component on crossed immunoelectrophoresis. We have applied plasma from an affected family member to heparin-Sepharose and eluted the AT III with a salt gradient. Evidence will be presented that the anodal component has* higher affinity for heparin than normal AT III. Furthermore, this variant component is present in plasma as a MW >120,000 inactive complex whose tryptic peptide FAB map contains numerous signals not characteristic of normal AT . 111 . This complex can be reduced with dithiothreitol to two non identical bands on SDS PAGE with MW ~60,000, only one of which reacts with anti-AT III. Using ion-exchange chromatography and HPLC these two components have been isolated and separated. The N-terminal sequence of the protein that does not react with anti-AT III is believed to be Asp-Ala-His-Ile-Ser-Glu. Structural investigations on the variant AT III are underway.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

1980 ◽  
Vol 44 (02) ◽  
pp. 092-095 ◽  
Author(s):  
T H Tran ◽  
C Bondeli ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Superficial Thrombophlebitis ◽  
Heparin Concentration ◽  
Heparin Binding Site ◽  
At Iii ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryTwo different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

Comparison of Progressive Antithrombin Activity and the Concentration of Three Thrombin Inhibitors in Nephrotic Syndrome

1981 ◽  
Vol 46 (03) ◽  
pp. 623-625 ◽  
Author(s):  
B Boneu ◽  
F Bouissou ◽  
M Abbal ◽  
P Sie ◽  
C Caranobe ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Antithrombin Iii ◽  
Heparin Therapy ◽  
Thrombin Inhibitors ◽  
Compensatory Mechanism ◽  
Dramatic Reduction ◽  
Antithrombin Activity ◽  
Α2 Macroglobulin ◽  
At Iii ◽  
A Prospective Study

SummaryIn order to compare the plasmatic progressive antithrombin activity to the concentration of three thrombin inhibitors, antithrombin III (AT III), α2 macroglobulin (α2, M), α1 anti-trypsin (α1 AT) in nephrotic syndrome, a prospective study was carried out on a group of 28 children affected with the disease. A dramatic reduction of the level of AT III and of α1 AT, two inhibitors of molecular weight close to that of albumin, was observed. The decreased level of AT III was counterbalanced by an increase in α2 M. This phenomenon accounts for the increased progressive antithrombin activity observed in all the affected children. It is suggested that the above compensatory mechanism explains the absence of thrombotic accidents in this series and that the benefit of heparin therapy is doubtful in these conditions.

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