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PubMed1989

A novel amino acid substitution in the reactive site of a congenital variant antithrombin. Antithrombin pescara, ARG393 to pro, caused by a CGT to CCT mutation.

Antithrombin is a plasma protein inhibitor that can be grouped within a serine proteinase inhibitor superfamily. Antithrombin Pescara is a functional variant of antithrombin found in a family with a high incidence of thrombosis. Preliminary functional analysis has suggested that the abnormality resides in the reactive site rather than in the heparin binding domain of the molecule. Accordingly, we have isolated the variant from plasma using heparin-Sepharose chromatography, followed by chromatography upon thrombin-Sepharose to remove the normal antithrombin that is present (the propositus is heterozygous for the variant). The variant protein was reduced, S-carboxy-methylated, and fragmented with CNBr. A pool ("CNBr pool 4") containing the reactive site region was isolated by reverse-phase high performance liquid chromatography and sequentially treated with trypsin and V8 protease. Fast atom bombardment-mass spectrometric analysis of this subdigest identified a novel peptide of mass 1708. Four steps of Edman degradation together with further analysis by fast atom bombardment-mass spectroscopy identified the NH2-terminal sequence of this peptide as Ala-Ala-Ala-Ser. The mass of the novel peptide and its changing mass in response to Edman degradation are only compatible with its identity as Ala382-Arg399, with the reactive site Arg393 replaced by Pro. Using specific oligonucleotide hybridization, we demonstrated that the molecular defect of antithrombin Pescara is caused by a CGT to CCT mutation in codon 393. These findings may be of broad interest, as other members of the serine protease inhibitor superfamily contain arginine at their reactive sites and may be expected to undergo a similar mutation.

Amino Acid Sequence↗

PubMed1994

Prothrombin fragment 1 + 2, thrombin-antithrombin III-complexes and fibrinopeptide A in spontaneously clotting whole blood in vitro. Effects of heparin addition and antithrombin III deficiency.

Previous in vitro studies using spontaneously clotting whole blood revealed thrombin formation and high fibrinopeptide A (FPA) concentrations measured during incubation time. This occurred in spite of normal concentrations of thrombin antagonists present in the blood of the healthy subjects examined. However, there are several reports showing that in vivo increased thrombin-antithrombin III-complex (TAT) concentrations and relatively low FPA concentrations may occur e.g. in patients with (pre)thrombotic disorders. These in vivo findings indicate more effective thrombin inhibition by antithrombin III, with almost no fibrin formation. To find an explanation for the differences observed in vitro and in vivo, we extended the in vitro studies by measuring concentrations of prothrombin fragment 1 + 2 (F1 + 2), TAT and FPA at several time points until 30 min. Our goal was to test whether thrombin at least initially is neutralized by antithrombin III, resulting in a lack of fibrin formation, either in the absence or in the presence of heparin (0.2 and 0.5 U/ml whole blood, respectively). In the absence of heparin a simultaneous increase in the concentrations of F1 + 2, TAT and FPA was observed. Thrombin was only partially neutralized by antithrombin III and large amounts of fibrin were formed. The addition of heparin virtually suppressed thrombin formation since the F1 + 2 concentration remained low. Moreover, the small amounts of thrombin formed were neutralized by antithrombin III to a greater extent than in the absence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2001

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin.

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.

Allosteric Regulation↗

PubMed1981

[Anticoagulating activity and lytic action of the heparin-antithrombin III and antithrombin III-heparin-thrombin complexes on non-stabilized fibrin].

The heparin-antithrombin III and antithrombin III-heparin-thrombin complexes were prepared by rapid mixing of the components at different weight ratios (1:1, 1:3, 1:4, 1:6, 1:7, 3:1 - heparin/antithrombin III), pH 7.0-7.2. The reaction mixture was incubated for 15 min at 37 degrees. The complexes were precipitated at pH 4.5-4.8. All the heparin-antithrombin III complexes possess high anticoagulating and lytic activities. The antithrombin III-heparin-thrombin complex prepared at the weight ratio of 1:1:6 has a high lytic activity. The antithrombin III-heparin-thrombin complex prepared at the weight ratio of 1:1:1 reveals a high anticoagulating activity and a strong lytic action with respect to the non-stabilized fibrin.

Antithrombin III↗

PubMed1994

Thrombin-based antithrombin assays show overestimation of antithrombin III activity in patients on heparin therapy due to heparin cofactor II influence.

An extensive comparison has been performed on the clinical chemistry automate Hitachi 717 between thrombin- and Factor Xa-based methods for determination of antithrombin III activity. In 460 patients who did not receive any heparin therapy the agreement between assays was in general close although the thrombin-based methods resulted in slightly higher assignments of 0.3-2.6% antithrombin III activity. The discrepancy was, however, substantial in plasmas from patients receiving heparin of > or = 20000 IU/day, resulting in plasma levels of heparin of 0.8-1.2 IU/ml. Thus, analysis of 102 patients showed that the thrombin-based methods resulted in, on average, 7-16% higher assignment of antithrombin III activity as compared to the Factor Xa-based method used. Addition of antibodies to antithrombin III and heparin cofactor II revealed that the discrepancy was primarily due to contribution of heparin cofactor II activity in the thrombin-based methods. The results thus suggest that the Factor Xa-based antithrombin III activity method provides more valid results in patients on heparin therapy.

Antithrombin III↗

PubMed1983

[Perioperative plasma antithrombin activity with "low-dose" heparin prophylaxis. Perioperatively acquired antithrombin III deficiency as a cause for the failure of heparin prophylaxis?].

Antithrombin III activity was estimated using a chromogenic substrate perioperatively in the plasma of 200 patients undergoing major elective abdominal surgery during low-dose heparin prophylaxis. With an initial value of 10.9 +/- 2 IU/ml there was a mean postoperative lowering of plasma antithrombin activity in all patients with a minimum on the second postoperative day. In patients with empirically established increased risk of thromboembolism the postoperative depression of antithrombin was significantly more pronounced (P less than 0.01) than in the control groups. An increased risk prevailed particularly in patients with malignant diseases and in major surgery, i.e. of prolonged duration. In such cases with increased risk of thromboembolism routine assessment of antithrombin III is recommended.

Abdomen↗

PubMed2006

Antithrombin Nagasaki (Ser 116 to Pro): a rare antithrombin variant with abnormal heparin binding presenting during pregnancy.

Quantitative antithrombin deficiency constitutes an important risk factor for venous thromboembolism, stillbirth, and other complications of pregnancy. Studies suggest, however, that individuals heterozygous for missense mutations involving the heparin-binding site of antithrombin do not have a significantly increased thrombotic risk. Owing to the rarity of such mutations, it remains unclear whether any specific heparin-binding site defects might be associated with thrombotic potential. We report here the case of a pregnant woman with an exceptionally rare Type II heparin-binding site antithrombin variant. This case highlights the difficult issues that are associated with the management of Type II antithrombin deficiency during pregnancy.

Adult↗

PubMed1987

Prophylactic use of antithrombin III concentrate following surgery in congenital antithrombin III deficiency.

Antithrombin III is the major physiological inhibitor of thrombin, and congenital deficiency of antithrombin III is associated with increased risk of venous thrombosis either spontaneously or following trauma, surgery or pregnancy. The successful use of antithrombin III concentrate during and following surgery to prevent venous thrombosis is described in a previously asymptomatic man with familial antithrombin III deficiency.

Antithrombin III↗

PubMed1993

Acquired antithrombin III deficiency: replacement with antithrombin III concentrates in a patient with protein S deficiency accelerates response to therapy.

A 33-year-old man with inherited protein S deficiency and a 20-year history of deep and superficial venous thrombosis was admitted for treatment of extensive, acute, superficial venous thrombosis of the left lower extremity. Clinical response to intravenous heparin was very slow. After discovery of acquired antithrombin III deficiency superimposed on the protein S deficiency, he was given antithrombin III concentrates with the intravenous heparin. His response to therapy was accelerated. The development of acquired antithrombin III deficiency in patients with venous thrombosis may slow the response to standard anticoagulant therapy. Therapy of the thrombosis may be improved with administration of antithrombin III concentrates.

Adult↗

PubMed1986

Abnormal antithrombin III with defective serine protease binding (antithrombin III "Denver").

A hereditary (three family members) deficiency of antithrombin III (AT-III) in which AT-III antigen (AT-III ag) is normal in spite of low heparin cofactor and antithrombin activity is described. Plasma levels were: AT-III ag, 0.92-0.96 U/ml; AT-III heparin cofactor activity, 0.54-0.62 U/ml; progressive antithrombin activity index, 0.13-0.18; anti-Xa activity, 0.50-0.56 U/ml. Plasma crossed immunoelectrophoresis (CIE) patterns performed with and without added heparin were normal, but serum CIE revealed a decreased complex peak. Purification of the patient's plasma AT-III by heparin-sepharose affinity chromatography showed a normal protein recovery and elution profile, but the purified AT-III fraction showed only 50% of the normal progressive thrombin neutralization and anti-Xa activity. When thrombin-antithrombin (TAT) complexes were formed by incubating with excess thrombin, SDS-polyacrylamide gel electrophoresis (PAGE) analysis revealed that half the patient AT-III formed TAT complexes while the remainder migrated as free AT-III. All the control AT-III formed TAT complexes. The patient's nonreacting AT-III (AT-III "Denver"), isolated by affinity chromatography, showed CIE and SDS-PAGE migration patterns characteristic of normal AT-III but failed to bind thrombin or Xa. Calculations from turnover studies in one patient and normal subjects with autologous 131I-AT-III suggested that AT-III "Denver" is removed from the plasma slightly more rapidly than normal. These studies indicate that the patients' variant AT-III molecule was characterized by normal heparin interaction but defective binding and inhibition of thrombin and Xa. These characteristics allow isolation of the nonreactive variant molecule by heparin-sepharose affinity chromatography.

Antithrombin III↗

PubMed1988

Use of synthetic oligonucleotides in the characterization of antithrombin III Northwick Park (393 CGT----TGT) and antithrombin III Glasgow (393 CGT----CAT).

Antithrombin III (ATIII) Northwick Park is caused by a single amino acid substitution, Arg 393---Cys and antithrombin III Glasgow is caused by Arg 393----His. Examination of the genetic code and the sequence of normal antithrombin III revealed that these amino acid substitutions could arise from the substitution of either two nucleotides or a single nucleotide at codon 393 of the antithrombin III gene. In two families, detection of the ATIII variants by genetic linkage analysis was not possible owing to lack of informative RFLP markers. Consequently, we synthesized two 22-base-long oligonucleotides specific for the single-base substitutions in the region of codon 393 and demonstrated by oligonucleotide hybridization that the molecular defect of ATIII Northwick Park is caused by the CGT----TGT mutation at codon 393 and that ATIII Glasgow is caused by the CGT----CAT mutation at codon 393. These oligonucleotide probes should prove useful as an alternative method for early detection of the ATIII variants.

Alleles↗

PubMed1974

Abnormal antithrombin III (antithrombin III "Budapest") as a cause of a familial thrombophilia.

A family with a high incidence of spontaneous thromboembolism has been investigated and those members affected were found to have significantly depressed levels of plasma and serum heparin cofactor activity; i.e., antithrombin III and anti-Xa activity. Further studies revealed that despite a marked diminution of antithrombin III activity in these patients measurement of antithrombin III by immunological techniques showed the levels to be normal. It is concluded that this anomaly represents a defect in the synthesis of the antithrombin III molecule. The abnormality appeared to be inherited but the mode of inheritance could not be determined with the available data.

Adsorption↗

PubMed1995

Antithrombin III Kumamoto II; a single mutation at Arg393-His increased the affinity of antithrombin III for heparin.

Abnormal antithrombin III (AT III) was found in a 30-year-old woman who suffered from recurrent thrombosis during pregnancy and the postpartum period. Among her family members, only her father had recurrent episodes of deep vein thrombosis of the lower extremities, from his youth. The antithrombin and antifactor Xa heparin cofactor activities of the proposita's plasma were 61% and 42% of normal, respectively. The progressive antithrombin and antifactor Xa activities were also decreased to 55% and 58% of normal, respectively. The immunoreactive level of AT III was within the normal range (23.1 mg/dl). Analysis of the proposita's plasma by crossed immunoelectrophoresis in the presence or absence of heparin and by affinity chromatography on heparin-Sepharose revealed that the proposita's AT III had apparently normal affinity for heparin. Nucleotide sequencing of 7 exons of the proposita's AT III gene amplified by polymerase chain reaction (PCR) disclosed that the second base of codon 393 comprised both G and A, indicating Arg393-His conversion. The base sequences of exons 1, 2, 3a, 3b, 4, and 5 were normal, excluding any other mutation. These findings indicated that the proposita's AT III was a variant of AT III at the thrombin binding site and that the proposita was a heterozygote for the abnormality. Heparin affinity of purified abnormal AT III from the proposita's plasma was demonstrated to be increased upon affinity chromatography using heparin-Sepharose, suggesting that the mutation (Arg393-His) per se could possibly increase the affinity of antithrombin III for heparin. For this variant AT III (Arg393-His), the name AT III Kumamoto II is proposed.

Adult↗

PubMed2000

Molecular bases of antithrombin deficiency in French families: identification of seven novel mutations in the antithrombin gene.

We have investigated the molecular bases of familial antithrombin deficiency in eight French families. Eight mutations in the antithrombin coding exons were identified, seven of which were novel mutations. In all cases, individuals were heterozygous for the mutation. We found two small frameshift deletions in exon 3a, leading to type I deficiency. Five missense mutations in exons 3b or 5 also caused type I deficiency and their potential consequences on the antithrombin three-dimensional structure were analysed. The last mutation in exon 4 was associated with a type II 'reactive site' deficiency: a dysfunctional antithrombin that is affected in its interaction with thrombin was present in circulation.

Antithrombin III Deficiency↗

PubMed1998

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin.

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

Antithrombin III↗

PubMed1995

Antithrombins Southport (Leu 99 to Val) and Vienna (Gln 118 to Pro): two novel antithrombin variants with abnormal heparin binding.

We report the characterization of three variant antithrombins with reduced heparin binding as the primary abnormality. Two of these variants, antithrombin Southport (Leu 99 to Val, 2759 C to G) and antithrombin Vienna (Gln 118 to Pro, 5349 A to C) were novel, whereas the third, Pro 41 to Leu, has been previously described as antithrombin Basel. All three variants exhibited reduced binding for heparin on crossed immunoelectrophoresis and in a quantitative monoclonal antibody-based assay. The mutations were characterized by direct sequence analysis of enzymatically amplified genomic DNA and all affected individuals were heterozygous for the mutations. These three mutations do not occur at the sites of the basic amino acids directly involved in heparin binding nor do they result in a change in charge of the affected residue. It seems probable that they reduce heparin affinity either by perturbing the initial contact site involved in the heparin-binding domain (Arg 47, Arg 129 and possibly Arg 24), or by preventing the subsequent heparin-induced conformational change.

Adult↗

PubMed1990

Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors.

Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release mediated by fluid-phase thrombin with their activity against the clot-bound enzyme. Incubation of thrombin with citrated plasma results in concentration-dependent FPA generation, which reaches a plateau within minutes. In contrast, there is progressive FPA generation when fibrin clots are incubated with citrated plasma. Heparin, hirudin, hirudin dodecapeptide (hirugen), and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) produce concentration-dependent inhibition of FPA release mediated by fluid-phase thrombin. However, heparin is much less effective at inhibiting thrombin bound to fibrin because a 20-fold higher concentration is necessary to block 70% of the activity of the clot-bound enzyme than is required for equivalent inhibition of fluid-phase thrombin (2.0 and 0.1 U/ml, respectively). In contrast, hirugen and PPACK are equally effective inhibitors of fluid- and solid-phase thrombin, while hirudin is only 50% as effective against the clot-bound enzyme. None of the inhibitors displace bound 125I-labeled thrombin from the clot. These studies indicate that (a) clot-bound thrombin is relatively protected from inhibition by heparin, possibly because the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin, and (b) clot-bound thrombin is susceptible to inactivation by antithrombin III-independent inhibitors because the sites of their interaction are not masked by thrombin binding to fibrin. For these reasons, antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings.

Amino Acid Chloromethyl Ketones↗

PubMed1989

[Study of serum level of antithrombin-III antigen, antithrombin activity, and its ratio of AT-III-heparin complex formation in maintenance hemodialysis patients].

The plasma level of Antithrombin-III (AT-III) antigen, antithrombin activity and the ratio of AT-III-heparin complex formation have been studied in 20 patients with chronic glomerulonephritis on regular hemodialysis at two occasions with 2 year interval (in 1986 and 1988), and studied as to each variation in each plasma levels, and compared with the period of length of hemodialysis-history of individual cases. A significant decrease was found not only in plasma level of AT-III antigen (25.7 +/- 4.1 mg/dl to 23.4 +/- 3.9 mg/dl), but also in antithrombin activity (90.8 +/- 11.8% to 85.2 +/- 16.8%). Moreover and ratio of AT-III-heparin complex formation was significantly reduced during two years, too (50.7 +/- 3.0% to 53.0 +/- 1.5%). Similarly, a significant negative correlation could be found on the comparison between the duration of hemodialysis and each plasma levels of AT-III antigen (r = -0.27), antithrombin activity (r = -0.33), and as well as ratio of AT-III-heparin complex formation (r = -0.34). Therefore, it was interestingly suggested the possibility that prolonged hemodialysis treatment induced a functional loss of AT-III, especially in a capacity to form a AT-III-heparin complex due to partial loss of the affinity for heparin.

Adult↗