Biochemical and Functional Study of Antithrombin III in Newborn Infants

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.

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The Thrombin Protecting Function of a Complement Inhibitor in Human Plasma

10.1055/s-0039-1684681 ◽

1979 ◽

Author(s):

E.R. Podack ◽

J.G. Curd ◽

J.H. Griffin ◽

H.J. Müller-Eberherd

Keyword(s):

Complex Formation ◽

Antithrombin Iii ◽

Membrane Attack Complex ◽

Protein S ◽

Thrombin Time ◽

Two Dimensional ◽

Complement Inhibitor ◽

Functional Studies ◽

Sds Page ◽

At Iii

S-protein (S) is a newly discovered 80,000 MW plosma glycoprotein. It functions as an inhibitor of the membrane attack complex of complement. We now wish to report that S also functions as thrombin protecting factor in coagulation; S forms a reversible complex with thrombin which is more resistant to inactivation by antithrombin III (AT III) than thrombin alone. An S-thrombin complex and on S-throm-bin-AT III complex were formed in clotted plasma and with isolated proteins as demonstrated by two dimensional Immunoelectrophoresis. Functional studies measuring the esterolytic or clotting activity of thrombin showed that S in the presence and absence of heparin decreased the rate of inactivation of thrombin by AT III. Similar results were observed using plasma. For example, in the presence of 0.04 u/ml heparin and 1.6 u/ml thrombin, the thrombin time of plasma depleted in S was 150 sec. as opposed to 15 sec. when the plasma was reconstituted with purified S. That this effect of S was due to a decreased inactivation of thrombin by AT III was demonstrated directly by SDS-PAGE analysis of plasma containing 125l-thrombin. In the presence of S the rate of formation of the 95,000 dalton 125I-thrombin-AT III complex was markedly decreased compared to the rate of complex formation in the S-depleted plasma. These data suggest that S may modulate the interactions of thrombin and AT III.

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Antithrombin III Avranches, a New Variant with Defective Serine-Protease Inhibition - Comparison with Antithrombin III Charleville

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647642 ◽

1988 ◽

Vol 60 (01) ◽

pp. 094-096 ◽

Cited By ~ 3

Author(s):

M Aiach ◽

M Roncato ◽

G Chadeuf ◽

P Dezellus ◽

L Capron ◽

...

Keyword(s):

Antithrombin Iii ◽

Factor Xa ◽

Kinetic Studies ◽

Exchange Chromatography ◽

Protease Inhibition ◽

Sds Page ◽

Recurrent Venous Thromboembolism ◽

Factor Xa Inhibition ◽

New Variant ◽

At Iii

SummaryA decreased plasma antithrombin activity in presence or in absence of heparin was discovered in a 47-year-old patient presenting with recurrent venous thromboembolism. The immunoreactive material (AT ΠΙ-IR) was normal. The same biological abnormalities were found in two relatives of the patient, leading to the diagnosis of hereditary qualitative AT III deficiency.The propositus’ AT III was coeluted with normal AT III from an heparin-sepharose column. An additional step of ion-exchange chromatography on a Mono Q column using a FPLC system (Pharmacia, St-Quentin en Yvelines, France) allowed the purification of a protein which was homogenous in SDS-10% polyacrylamide electrophoresis gel (PAGE). AT III purified from propositus’ plasma, normal plasma and the plasma of the patient known to have an AT III variant with defective protease binding (AT III Charleville) were compared. The specific activities measured as heparin cofactor anti thrombin or factor Xa inhibition in absence of heparin were decreased to half the normal value.Kinetic studies confirmed a decreased rate of thrombin inhibi-tion for both abnormal AT III preparations. SDS-PAGE experi-ments performed in purified system and immunoblots obtained from plasma showed that the two variants have different behaviour: in the case of AT III Charleville thrombin induced an apparent 5 Δ increase in molecular mass, probably due to a conformational change. AT III Avranches did not form stoechiometric complexes with thrombin, but was unmodified by the protease.

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Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa

Blood ◽

10.1182/blood.v77.10.2185.bloodjournal77102185 ◽

1991 ◽

Vol 77 (10) ◽

pp. 2185-2189

Author(s):

RC Austin ◽

RA Rachubinski ◽

FA Ofosu ◽

MA Blajchman

Keyword(s):

Complex Formation ◽

Serine Proteases ◽

Antithrombin Iii ◽

Factor Xa ◽

Biological Behavior ◽

Form Complex ◽

Free System ◽

Reactive Center ◽

Sds Page ◽

At Iii

Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III- Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT- III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III- Hamilton is capable of only the first of these events.

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Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa

Blood ◽

10.1182/blood.v77.10.2185.2185 ◽

1991 ◽

Vol 77 (10) ◽

pp. 2185-2189 ◽

Cited By ~ 20

Author(s):

RC Austin ◽

RA Rachubinski ◽

FA Ofosu ◽

MA Blajchman

Keyword(s):

Complex Formation ◽

Serine Proteases ◽

Antithrombin Iii ◽

Factor Xa ◽

Biological Behavior ◽

Form Complex ◽

Free System ◽

Reactive Center ◽

Sds Page ◽

At Iii

Abstract Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III- Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT- III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III- Hamilton is capable of only the first of these events.

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

Blood ◽

10.1182/blood.v79.6.1428.bloodjournal7961428 ◽

1992 ◽

Vol 79 (6) ◽

pp. 1428-1434 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v79.6.1428.1428 ◽

1992 ◽

Vol 79 (6) ◽

pp. 1428-1434 ◽

Cited By ~ 10

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.

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Immunologic Studies of Antithrombin III Heparin Cofactor in the Newborn

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646736 ◽

1978 ◽

Vol 39 (03) ◽

pp. 624-630 ◽

Cited By ~ 21

Author(s):

W E Hathaway ◽

L L Neumann ◽

C A Borden ◽

L J Jacobson

Keyword(s):

Gestational Age ◽

Antithrombin Iii ◽

Newborn Infants ◽

Term Infant ◽

Sick Newborn ◽

At Iii ◽

Thrombotic Complications ◽

Low Levels ◽

Collection Methods ◽

Made In

SummarySerial quantitative immunoelectrophoretic (IE) measurements of antithrombin III heparin cofactor (AT III) were made in groups of well and sick newborn infants classified by gestational age. Collection methods (venous vs. capillary) did not influence the results; serum IE measurements were comparable to AT III activity by a clotting method. AT III is gestational age-dependent, increasing from 28.7% of normal adult values at 28-32 weeks to 50.9% at 37-40 weeks, and shows a gradual increase to term infant levels (57.4%) by 3-4 weeks of age. Infants with the respiratory distress syndrome (RDS) show lower levels of AT III in the 33-36 week group, 22% vs. 44% and in the 37-40 week group, 33.6% vs. 50.9%, than prematures without RDS. Infants of 28-32 week gestational age had only slight differences, RDS = 24%, non-RDS = 28.7%. The lowest levels of AT III were seen in patients with RDS complicated by disseminated intravascular coagulation and those with necrotizing enterocolitis. Crossed IE on representative infants displayed a consistent pattern which was identical to adult controls except for appropriate decreases in the amplitude of the peaks. The thrombotic complications seen in the sick preterm infant may be related to the low levels of AT III.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650091 ◽

1980 ◽

Vol 44 (02) ◽

pp. 092-095 ◽

Cited By ~ 8

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.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653468 ◽

1981 ◽

Vol 46 (04) ◽

pp. 749-751 ◽

Cited By ~ 3

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.

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Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657178 ◽

1982 ◽

Vol 47 (03) ◽

pp. 244-248 ◽

Cited By ~ 47

Author(s):

D P Thomas ◽

Rosemary E Merton ◽

T W Barrowcliffe ◽

L Thunberg ◽

U Lindahl

Keyword(s):

Antithrombin Iii ◽

Specific Activity ◽

High Specific Activity ◽

Factor Xa ◽

Blood Levels ◽

Thrombin Time ◽

High Affinity ◽

Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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