scholarly journals Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1428-1434 ◽  
Author(s):  
MA Blajchman ◽  
F Fernandez-Rachubinski ◽  
WP Sheffield ◽  
RC Austin ◽  
S Schulman
Keyword(s):  
Serine Protease ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
White Female ◽  
Polymorphism Analysis ◽  
Heparin Binding ◽  
At Iii ◽  
Polymerase Chain ◽  
Heparin Affinity

Abstract Antithrombin-III-Stockholm is a new structural variant of antithrombin- III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT- III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha- thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.

scholarly journals Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1428-1434 ◽  
Author(s):  
MA Blajchman ◽  
F Fernandez-Rachubinski ◽  
WP Sheffield ◽  
RC Austin ◽  
S Schulman
Keyword(s):  
Serine Protease ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
White Female ◽  
Polymorphism Analysis ◽  
Heparin Binding ◽  
At Iii ◽  
Polymerase Chain ◽  
Heparin Affinity

Antithrombin-III-Stockholm is a new structural variant of antithrombin- III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT- III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha- thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.

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.

Biochemical and Functional Study of Antithrombin III in Newborn Infants

1982 ◽  
Vol 47 (01) ◽  
pp. 056-058 ◽  
Author(s):  
M M McDonald ◽  
W E Hathaway ◽  
E B Reeve ◽  
B D Leonard
Keyword(s):  
Protein Concentration ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Newborn Infants ◽  
Factor Xa ◽  
Functional Study ◽  
Functional Studies ◽  
Sds Page ◽  
At Iii ◽  
Heparin Affinity

SummaryAntithrombin III (AT-III) was isolated by heparin affinity chromatography from adult venous and newborn term and preterm umbilical cord blood. The purified proteins were compared by SDS-PAGE, rocket immuno-electrophoresis, protein concentration by microbiuret relative to optical density at 280 nm, heparin cofactor specific activity, progressive neutralization of thrombin and factor Xa at 37°C and pH related antithrombin kinetics. The structural evaluations revealed a fetal AT-III of molecular weight, charge and electrophoretic migration indistinguishable from adult AT-III. The functional studies showed that, on an equimolar basis, the rates of thrombin and Xa interactions with fetal AT-III were as rapid as those with adult AT-III. The catalytic rates of various concentrations of heparin were also equal. The newborn infant, therefore, displays a quantitative but not qualitative deficiency of AT-III.

EVIDENCE FOR A HOMOLOGY BETWEEN THE HEPARIN AND HEPARAN SULFATE BINDING REGIONS TO ANTITHROMBIN III

1987 ◽  
Author(s):  
A M Fischer ◽  
J Tapon-Bretaudière ◽  
M D Dautzenberg ◽  
C Sternberg ◽  
J Choay
Keyword(s):  
Heparan Sulfate ◽  
Antithrombin Iii ◽  
Competitive Inhibition ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Heparin Binding ◽  
At Iii ◽  
Binding Regions ◽  
Partial Homology ◽  
Type 3

As we have previously demonstrated, heparan sulfate, a glycosaminoglycan physiologically present on the endothelial wall, is, like heparin, unable to potentiate the inhibitory effect against Factors IIa and Xa of two abnormal type 3 antithrombin III (AT III) variants. We report here that a synthetic pentasaccharide which constitutes the sequence of the heparin binding site to AT III is also unable to potentiate these two AT III variants in an anti-Factor Xa assay. According to these data, we speculated the existence of a homology between the heparin and heparan sulfate binding regions to normal AT III. We thus studied the competitive inhibition by the pentasaccharide of heparin and heparan sulfate in their potentiation of AT III activity. Such a competitive inhibition can be observed in an AT III antiFactor IIa assay because the pentasaccharide which exhibits a high anti-Factor Xa activity is devoid of any anti-Factor IIa activity. In the absence of pentasaccharide, with both heparin and heparan sulfate, a plateau is reached in AT III potentiation for concentrations of respectively 2.5 ng and 17.2 μg (corresponding to the same anti-Factor IIa activity of 0.4 u/ml for both glycosaminoglycans). In the presence of 5.5 μg of pentasaccharide, the inhibition of heparin cofactor activity is 70%. With heparan sulfate, the inhibition by the same amount of pentasaccharide is less pronounced, being only 30%. These results strongly suggest the existence of a partial homology between heparin and heparan sulfate binding sites to AT III. For heparan sulfate, the exact sequence of this site remains to be identified .

Studies Of The Heterogeneity Of Purified Antithrombin III

1981 ◽  
Author(s):  
T W Barrowcliffe ◽  
C A Eggleton ◽  
M Mahmoud
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Collaborative Study ◽  
Gel Filtration ◽  
Factor Xa ◽  
Heparin Binding ◽  
Predisposing Factor ◽  
Binding Material ◽  
At Iii ◽  
Immunological Assays

Deficiency of antithrombin III (At III), whether hereditary or acquired, is now recognised as a major predisposing factor for the development of venous thromboembolism. Purified At III concentrates are undergoing clinical trials in various conditions associated with At III deficiency; such concentrates may be given in addition to heparin and their potency is usually assessed by heparin co-factor assays. In an international collaborative study, a reference preparation of purified At III had a lower concentration by heparin co-factor than by immunological assays and this was shown to be due to the presence of non-heparin-binding antigens. In the present study we have examined purified At III from several manufacturers by heparin co-factor (amidolytic), progressive antithrombin (clotting) and immunological assays, and their heparin-binding abilities have been studied by crossed immunoelectrophoresis and heparin-agarose affinity chromatography.There was good agreement between progressive antithrombin and immunological assays, but in some concentrates the heparin co-factor assays gave lower activity. The proportions of non-heparin-binding material varied considerably, from less than 5% to as much as 50% of the total At III antigen in some concentrates. The non-binding material isolated from a heparin column had little heparin co-factor activity, but was able to neutralise thrombin and Factor Xa. Gel filtration and polyacrylamide gel electrophoresis showed no major distinction between heparin-binding and non-binding antigens, indicating the absence of At III-protease complexes.These studies show that some At III concentrates contain substantial amounts of partially denatured molecules, in which the heparin-binding ability of the At III has been impaired but its thrombin and Xa neutralising activity left relatively intact.

GAGs-Potentiated Inhibition of Thrombin, Factor Xa and Plasmin in Plasma and in a Purified System Containing Antithrombin III – Correlation with Total Charge Density

1981 ◽  
Vol 46 (04) ◽  
pp. 749-751 ◽  
Author(s):  
E Cofrancesco ◽  
A Vigo ◽  
E M Pogliani
Keyword(s):  
Charge Density ◽  
Chemical Structure ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Total Charge ◽  
Pure System ◽  
At Iii ◽  
Total Charge Density ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryThe ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assaysThere was a good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical.For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma.

Antithrombin ‘DREUX’ (Lys114Glu): A Variant with Complete Loss of Heparin Affinity

2002 ◽  
Vol 88 (09) ◽  
pp. 436-443 ◽  
Author(s):  
James Huntington ◽  
Jacqueline Conard ◽  
Robin Carrell ◽  
Alec Mushunje ◽  
Aiwu Zhou
Keyword(s):  
Factor Xa ◽  
Fold Increase ◽  
Complete Loss ◽  
Factor X ◽  
Heparin Binding ◽  
High Affinity ◽  
The Core ◽  
Factor Xa Inhibition ◽  
Heparin Affinity ◽  
Father And Son

SummaryHere we report the finding of a new natural antithrombin mutation that confirms the critical contribution of lysine 114 to the binding of the core heparin pentasaccharide, with the replacement of lysine 114 by glutamate causing a complete loss in affinity. The variant was identified in a father and son, the father having been investigated for an episode of cerebral ischaemia associated with hypercholesterolaemia. The variant forms SDS-stable complexes with activated factor X (fXa) and its thermal stability and rate of factor Xa inhibition in the absence of heparin are identical to those of normal antithrombin. Normal antithrombin binds to the high affinity heparin pentasaccharide with a Kd of 1nM, as detected by a 45% change in intrinsic fluorescence, resulting in a 230-fold increase in rate of factor Xa inhibition. However, no change in fluorescence was detected for the variant when titrated with heparin or the heparin pentasaccharide, nor was there detectable activation towards factor Xa, indicating a complete loss of heparin binding.

The Heparin Binding Site Of Antithrombin: Identification Of A Critical Tryptophan Residue Within The Amino Acid Sequence

1981 ◽  
Author(s):  
M Blackburn
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Heparin Binding ◽  
High Affinity ◽  
Heparin Binding Site

Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety per molecule of antithrombin III. Heparin protects against tryptophan modification, particularly at low reagent concentrations. Unlike native antithrombin, which has high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti- thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native antithrombin possess progressive antithrombin activity, inactivating these proteases at identical rates. These results indicate that the integrity of a specific tryptophan residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic cleavage techniques have been used to isolate peptides containing this tryptophan from both HNB-labeled and native antithrombin and to identify this critical tryptophan residue within the amino acid sequence of the antithrombin molecule.

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.

Antithrombin III and Venous Thrombosis

1979 ◽  
Author(s):  
Duncan P. Thomas
Keyword(s):  
Venous Thrombosis ◽  
Antithrombin Iii ◽  
Physiological Role ◽  
Factor Xa ◽  
Original Description ◽  
Clear Cut ◽  
Naturally Occurring ◽  
At Iii ◽  
General Studies

Increasing interest in the physiological role of inhibitors of coagulation has highlighting the role of antithrombin III (AT III) as the most important naturally occurring inhibitor of venous thrombosis. Since Egeberg’s original description in 1965, it has been recognized that inherited deficiency of AT III is associated with an increased incidence of venous thromboembolism. The role of acquired deficiency of AT III in the pathogenesis of thromboembolism remainsless clear-cut, partly due to methodological differences. While low values have been reported in groups of patients with thromboembolism, estimations of AT III in individual patients are not allways abnormal. In general, studies which have measured protein concentration rather than functional activity, or cl otting assays which measure total antithrombin activity and not specific anti-Factor Xa activity have failed to demonstrate a clear relationship between AT III and thromboembolism. However, in two groups of patients, namely women on oral contraceptives and patients undergoing total hipreplacement, an acquired deficiency of AT III, particularly when measured by anti-Xa clotting assays, correlates highly with postoperative venous thrombosis. Although venous thrombosis may develop in patients despite normal AT III values, an activity below approximately 80% in an anti-Xa clotting assay has been found to be of predictive value in patients subjected to the stress of trauma or surgery.

Sign in / Sign up

Export Citation Format

Share Document