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At least 19 recordsLinked to original sources
PubMed1987

Antithrombin III microheterogeneity in antithrombin III deficiency and in the antithrombin III abnormality, "antithrombin III Toyama".

Antithrombin III (AT III) microheterogeneity was investigated in 12 cases of congenital AT III deficiency and 2 cases of congenital AT III abnormality by isoelectric focusing (IEF) and immunofixation. In congenital AT III deficiency, IEF and immunofixation revealed AT III as 8 bands which was indistinguishable from normal control in terms of the number of bands and the isoelectric point (pI) of each band. In the proband of the congenital AT III abnormality, however, IEF and immunofixation showed AT III as 8 bands which shifted slightly but definitely to the acidic side compared to those of normal subjects. This change in pI of the abnormal AT III was considered to reflect the amino acid replacement in the polypeptide chain of the abnormal AT III molecule.

Adult↗
PubMed1992

Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.

The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Antithrombin III↗
PubMed1988

Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow.

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).

Amino Acid Sequence↗
PubMed1982

Distinction of two pathologic antithrombin III molecules: antithrombin III "Aalborg' and antithrombin III "Budapest'.

Plasma from two different thrombophilic families with functional inherited antithrombin III deficiency, i.e., with low antithrombin III activity but normal immunoreactive antithrombin III concentration, were investigated simultaneously in the same laboratory. The experiments (thrombin and Factor Xa inactivation, heparin affinity chromatography, modified two dimensional immunoelectrophoresis and gel filtration) showed a distinct difference between the two antithrombin III anomalies. The antithrombin III "Aalborg' had decreased thrombininactivating activity but normal Factor Xa-inactivating activity. The heparin affinity and the molecule weight are normal. The antithrombin III "Budapest' displays a more profound abnormality with pathologic thrombin and Factor Xa inactivation, decreased heparin affinity and abnormal molecular weight.

Antithrombin III↗
PubMed1987

Formation of a covalent disulfide-linked antithrombin-albumin complex by an antithrombin variant, antithrombin "Northwick Park".

Antithrombin is a major proteinase inhibitor of the blood coagulation system. Its inherited deficiency or abnormality is often associated with thromboembolism. Antithrombin "Northwick Park," a functionally inactive variant antithrombin, has recently been shown by us (Lane, D.A., Flynn, A., Ireland, H., Erdjument, H., Samson, D., Howarth, D., and Thompson, E. (1987) Br. J. Haematol. 65,451-456) to be present in plasma, in part, as a high Mr (approximately 120,000) component which has a characteristic electrophoretic mobility in agarose gels in the absence of denaturing agents. In this communication, we present evidence that this Mr approximately 120,000 variant component is comprised of an antithrombin-albumin covalent disulfide-linked complex. This proposal is supported by results of: (a) fast atom bombardment mass spectrometry of the isolated reduced, S-carboxymethylated, trypsin-digested Mr approximately 120,000 complex; (b) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this complex and its reduced and S-carboxymethylated constituents; (c) immunoblotting of these polyacrylamide gels with antisera specific for antithrombin and albumin; (d) NH2-terminal sequence analysis of one of the isolated, S-carboxymethylated proteins that comprise the Mr approximately 120,000 complex; and (e) fast atom bombardment mass spectrometry of its tryptic peptides.

Antithrombin III↗
PubMed2000

Major structural defects in the antithrombin gene in four families with type I antithrombin deficiency--partial/complete deletions and rearrangement of the antithrombin gene.

The molecular basis of quantitative antithrombin deficiency was investigated in four families predicted to have major antithrombin gene rearrangements. A 1,442 bp deletion and insertion of the sequence 5'T(n = 38-40)GAGACG was characterised in one case. Sequence surrounding the breakpoints contained two perfect, and one imperfect, inverted repeats which may have mediated formation of a stem loop structure on one strand during DNA replication potentiating the deletion. A 9,219 bp deletion spanning introns 2 to 5 was identified in a second family. The identical 6 bp sequence was upstream of each breakpoint and the 5' breakpoint was located in a sequence of the Alu 3 repeat predicted to be susceptible to strand breakage during transcription. This may have promoted misalignment, and deletion, of one of the repeats and the intervening DNA. A novel 1.8 kb antithrombin gene fragment was present in DNA digests from affected members of the third family suggesting a partial antithrombin gene duplication event while in the remaining family, evidence supporting a complete gene deletion was obtained.

Adolescent↗
PubMed1988

Antithrombin Oslo: type Ib classification of the first reported antithrombin-deficient family, with a review of hereditary antithrombin variants.

Patients with classical antithrombin deficiency (Type I) from seven unrelated kindreds were studied by crossed immunoelectrophoresis of plasma in the presence and absence of heparin. The only abnormal pattern was found in the kindred first reported by Egeberg in 1965. An abnormal cathodal peak of antithrombin antigen was found in the presence, but not the absence, of heparin in the first dimension gel. We have named this variant antithrombin Oslo. Such evidence of an abnormal protein, despite equivalent low levels of antithrombin antigen and activity, has been denoted previously by Sas as Type Ib deficiency. In the context of this new report, we review the literature to date on 33 other variants of the Types Ib, II and III subclassifications with a discussion of the value of the classification scheme.

Antithrombins↗
PubMed1992

Prophylactic antithrombin III administration during pregnancy immediately reduces the thrombin hyperactivity of congenital antithrombin III deficiency by forming thrombin-antithrombin III complexes.

We examined the changes of haemostatic molecular markers after antithrombin III (AT III) administration in a 22-year-old woman with congenital AT III deficiency in the third trimester of pregnancy who did not have thrombosis. Various markers including fibrinopeptide A (FPA), thrombin-antithrombin III complex (TAT), prothrombin fragment F1 + 2 (F1 + 2), plasmin-alpha 2antiplasmin, D-dimer, beta-thromboglobulin, and platelet factor 4 were measured before and just after 3,000 U of AT III concentrate, which was given three times per week from the 34 week of pregnancy until delivery. Just after AT III administration, F1 + 2 and FPA levels decreased on most occasions, while TAT sometimes increased. Plasma FPA levels were markedly decreased on all 8 occasions when the plasma FPA levels was above 2.0 ng/ml before AT III administration. Plasma FPA levels were always greater than or equal to 6.4 ng/ml before AT III administration on the 4 occasions when TAT increased to above 115%. The changes of plasma F1 + 2 levels were significantly correlated with the AT III level. These results suggest that prophylactic AT III administration in the third trimester immediately inactivates intravascular thrombin to form TAT and reduce the plasma FPA level. Thus, the transient TAT elevation following AT III administration may not only be due to extraction of thrombin from the fibrin clots of thrombi but also to intravascular thrombin which is not attached to thrombi. FPA is the best molecular marker for thrombin hyperactivity and it should be monitored in AT III-deficient pregnant women in the third trimester.

Adult↗
PubMed1989

Clinical experience with antithrombin III concentrate in treatment of congenital and acquired deficiency of antithrombin. The Antithrombin III Study Group.

Phase I clinical studies of antithrombin III (ATIII) concentrate demonstrated a mean in vivo incremental recovery of functional activity of 1.4 percent per unit/kg administered, an initial 50 percent disappearance time of 22 hours, and a biologic half-life of 3.8 days. Based on these observations, a treatment regimen designed to maintain plasma ATIII levels between 75 and 120 percent of normal has been developed. None of 10 subjects with congenital ATIII deficiency treated prophylactically had evidence of thromboembolism, including four pregnant women at the time of delivery. Five subjects treated for acute thrombosis and/or thromboembolism, four of whom were pregnant, recovered without further thrombotic extension or recurrence. Heparin resistance was reversed in two subjects, both pregnant. Nine subjects with acquired ATIII deficiency also received ATIII treatment for venous or arterial thrombosis or disseminated intravascular coagulation, all with low plasma ATIII levels. Two subjects with disseminated intravascular coagulation demonstrated improvement, one clinically, the other biochemically. All patients with congenital ATIII deficiency survived, but only five of nine with acquired deficiency survived, highlighting the importance in acquired ATIII deficiency of the underlying disease in prognosis. Survival rate was especially poor in subjects with arterial thrombosis in the setting of low plasma ATIII. Administration of ATIII concentrate was well tolerated. None of the subjects who received ATIII concentrate demonstrated evidence of an infectious transmissible agent. These studies demonstrate that it is now feasible to safely replace the deficient protein in congenital ATIII deficiency, either prophylactically or therapeutically.

Antithrombin III↗
PubMed1991

Effective prophylaxis of thrombosis by antithrombin III concentrate in a pregnant woman with congenital antithrombin III deficiency: relations between plasma antithrombin III activity and the plasma levels of hemostatic molecular markers.

The value of antithrombin III (AT III) concentrate and a standard criterion for its use were examined in a pregnant woman with congenital AT III deficiency by continuous monitoring of plasma AT III activity and the plasma levels of hemostatic molecular markers. The rates of improvement of various markers after AT III administration (frequency of improvement/frequency of administration) were as follows: fibrinopeptide A (FPA) 82%, D-dimer 70%, fibrinopeptide B beta 15-42 73%, beta-thromboglobulin 60%, and platelet factor 4 50%. There was no significant correlation between the plasma AT III activity and all plasma FPA values, but FPA values of over 3.9 ng/ml showed a significant negative correlation with AT III activity: AT III activity (%) = -6.59 x FPA (ng/ml) + 125, r = -0.851, p less than 0.02. We therefore recommend continuous monitoring of the plasma FPA level and administration of AT III concentrate when the FPA level is elevated. According to the regression line shown above, plasma AT III activity should be raised to 100% to keep the FPA level below 6.0 ng/ml with 95% confidence limit.

Adult↗
PubMed1989

Monoclonal antibodies against antithrombin III. Identification of their epitopes and effects on antithrombin III activities.

Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.

Animals↗
PubMed2003

Structure of beta-antithrombin and the effect of glycosylation on antithrombin's heparin affinity and activity.

Antithrombin is a member of the serpin family of protease inhibitors and the major inhibitor of the blood coagulation cascade. It is unique amongst the serpins in that it circulates in a conformation that is inactive against its target proteases. Activation of antithrombin is brought about by a conformational change initiated upon binding heparin or heparan sulphate. Two isoforms exist in the circulation, alpha-antithrombin and beta-antithrombin, which differ in the amount of glycosylation present on the polypeptide chain; beta-antithrombin lacks the carbohydrate present at Asn135 in alpha-antithrombin. Of the two forms, beta-antithrombin has the higher affinity for heparin and thus functions as the major inhibitor in vivo even though it is the less abundant form. The reason for the differences in heparin affinity between the alpha and beta-forms have been shown to be due to the additional carbohydrate changing the rate of the conformational change. Here, we describe the most accurate structures of alpha-antithrombin and alpha-antithrombin+heparin pentasaccharide reported to date (2.6A and 2.9A resolution, respectively, both re-refinements using old data), and the structure of beta-antithrombin (2.6A resolution). The new structures have a remarkable degree of ordered carbohydrate and include parts of the antithrombin chain not modeled before. The structures have allowed a detailed comparison of the conformational differences between the three. They show that the structural basis of the lower affinity for heparin of alpha-antithrombin over beta-antithrombin is due to the conformational change that occurs upon heparin binding being sterically hindered by the presence of the additional bulky carbohydrate at Asn135.

Antithrombins↗
PubMed1993

High molecular weight kininogen potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin: role for antithrombin in the regulation of kallikrein.

The effects of previously characterized interactions of high molecular weight kininogen (H-kininogen) with plasma kallikrein and with heparin on the regulation of kallikrein by the heparin-activated inhibitor, antithrombin, were investigated. H-kininogen, at levels sufficient to fully complex kallikrein, greatly potentiated the acceleration of antithrombin inhibition of kallikrein produced by heparin with high affinity for antithrombin. At I = 0.15, pH 7.4, 25 degrees C, kininogen thus maximally increased the heparin enhancement of the second-order rate constant for the antithrombin-kallikrein reaction from 13-fold (1.6 x 10(2) M-1 s-1 to 2.1 x 10(3) M-1 s-1) to 1200-fold (1.9 x 10(5) M-1 s-1). In contrast, H-kininogen had no effect on the antithrombin-kallikrein reaction in the absence of heparin, nor did the protein enhance the rate constants of 1.7 x 10(4) and 3.4 x 10(4) M-1 s-1 for kallikrein reactions with its primary plasma inhibitors C1-inhibitor and alpha 2-macroglobulin, respectively, in the absence or presence of heparin. Consistent with these results, SDS gel electrophoresis of the 125I-labeled kallikrein-inhibitor complexes formed in a mixture of these kallikrein inhibitors at their relative plasma concentrations indicated that antithrombin effectively competed with C1-inhibitor and alpha 2-macroglobulin for kallikrein, accounting for 54% of the total kallikrein complexes, only when both heparin and H-kininogen were present. Similarly, the presence of therapeutic levels of heparin (approximately 1 unit/mL) in normal, factor XII-deficient, and prekallikrein-deficient plasmas enhanced the rate of inactivation of added kallikrein by 2.3-fold and significantly altered the partitioning of radiolabeled kallikrein from predominantly C1-inhibitor and alpha 2-macroglobulin complexes (86-92%) to mostly antithrombin complexes (50-53%). Experiments in antithrombin-deficient and H-kininogen-deficient plasmas confirmed that the enhanced kallikrein inactivation rate and predominant formation of antithrombin-kallikrein complexes in heparinized plasma were dependent on antithrombin and H-kininogen. The contribution of antithrombin to kallikrein inhibition in plasma remained significant (approximately 40-70%) at optimal concentrations of unfractionated or size- and antithrombin affinity-fractionated heparin, in the presence of plasma levels of calcium and zinc ions, at 37 degrees C, and with minimal plasma dilution. These results suggest that antithrombin and H-kininogen may play important roles in the regulation of kallikrein activity in the presence of heparin or heparin-like glycosaminoglycans.

Antithrombins↗
PubMed2000

Salmon antithrombin has only three carbohydrate side chains, and shows functional similarities to human beta-antithrombin.

Antithrombin, a major coagulation inhibitor in mammals, has for the first time been cDNA cloned from a fish species. The predicted mature liver antithrombin of Atlantic salmon (Salmo salar) consists of 430 amino acids and shows about 67% sequence identity to mammalian and chicken antithrombins. Due to a single nucleotide replacement, Asn135 of the antithrombin in higher vertebrates is substituted by Asp in the salmon homolog. Hence, in contrast to the vertebrate antithrombins known so far, salmon antithrombin lacks the potential glycosylation site located close to the heparin binding site. The existence of only three N-linked side chains is evidenced by the sequential removal of three carbohydrate chains from salmon antithrombin during timed-digestion with N-glycosidase F. The high heparin binding affinity of the salmon inhibitor, Kd of 2.2 and 48 nM at I = 0.15 and 0.3, respectively, is very similar to that of the minor human isoform beta-antithrombin, which is not glycosylated at Asn135. Furthermore, the invariant third-position Ser137 at this glycosylation site of mammalian and chicken antithrombins is substituted by Thr in the salmon, a replacement that has been shown to induce full glycosylation in human antithrombin. Thus a rapidly reacting pool of antithrombin may have evolved in two different ways: absence of a glycosylation site in lower vertebrates vs. incomplete glycosylation of a part of the circulating antithrombin in higher vertebrates. Salmon antithrombin appears to have three complex oligosaccharide side chains containing sialic acid terminally linked alpha(2-3) to galactose, while trace amounts of Galbeta(1-4)GlcNAc suggest microheterogeneity due to partial loss of sialic acid.

Amino Acid Sequence↗
PubMed1995

Mechanism of acceleration of antithrombin-proteinase reactions by low affinity heparin. Role of the antithrombin binding pentasaccharide in heparin rate enhancement.

The role of the sequence-specific pentasaccharide region of high affinity heparin (HAH) in heparin acceleration of antithrombin-proteinase reactions was elucidated by determining the accelerating mechanism of low affinity heparin (LAH) lacking this sequence. LAH was shown to be free of HAH (< 0.001%) from the lack of exchange of added fluorescein-labeled HAH into LAH after separating the polysaccharides by antithrombin-agarose chromatography. Fluorescence titrations showed that LAH bound to antithrombin with a 1000-fold weaker affinity (KD 19 +/- 6 microM) and 5-6-fold smaller fluorescence enhancement (8 +/- 3%) than HAH. LAH accelerated the antithrombin-thrombin reaction with a bell-shaped dependence on heparin concentration resembling that of HAH, but with the bell-shaped curve shifted to approximately 100-fold higher polysaccharide concentrations and with a approximately 100-fold reduced maximal accelerating effect. Rapid kinetic studies indicated these differences arose from a reverse order of assembly of an intermediate heparin-thrombin-antithrombin ternary complex and diminished ability of LAH to bridge antithrombin and thrombin in this complex, as compared to HAH. By contrast, LAH and HAH both accelerated the antithrombin-factor Xa reaction with a simple saturable dependence on heparin or inhibitor concentrations which paralleled the formation of an antithrombin-heparin binary complex. The maximal accelerations of the two heparins in this case correlated with the inhibitor fluorescence enhancements induced by the polysaccharides, consistent with the accelerations arising from conformational activation of antithrombin. 1H NMR difference spectroscopy of antithrombin complexes with LAH and HAH and competitive binding studies were consistent with LAH accelerating activity being mediated by binding to the same site on the inhibitor as HAH. These results demonstrate that LAH accelerates antithrombin-proteinase reactions by bridging and conformational activation mechanisms similar to those of HAH, with the reduced magnitude of LAH accelerations resulting both from a decreased antithrombin affinity and the inability to induce a full activating conformational change in the inhibitor.

Antithrombins↗
PubMed1987

Interaction of antithrombin III and thrombin-antithrombin III complex with cultured aortic endothelial cells.

The binding of antithrombin III, thrombin, thrombin-antithrombin III complex to endothelial cells was investigated. While the rate of the binding of thrombin to these cells was very rapid, that of antithrombin III was relatively slow and the thrombin-antithrombin III complex was intermediate. Binding kinetics indicated that antithrombin III, like thrombin, showed high affinity to endothelial cells; with a Kd of 3 X 10(-8) M and with 5 X 10(4) binding sites per cell. The dissociation of the inhibitor molecule was also rapid, i.e., approximately 70% bound antithrombin III was released in 2 minutes. Heparin, in a 100-fold molar excess to antithrombin III, or the modification of lysine residues of the inhibitor involved in the interaction with heparin, did not influence the association of antithrombin III with endothelial cells. In addition, antithrombin III did not compete with thrombin blocked in its active center for binding to endothelial cells. It is suggested that the binding sites of endothelial cells are different for thrombin and antithrombin III, and antithrombin III does not bind to these cells through its heparin binding domain.

Animals↗