A Swedish family with abnormal antithrombin III

An abnormal variant of antithrombin III is reported in a young male with deep vein thrombosis. The heparin cofactor, progressive thrombin inhibition, and factor Xa inactivation are decreased. The abnormality seems to be a mutation which is transmitted in an autosomaldominant way. The half-life and fractional catabolic rate of 125I antithrombin III concentrate is the same in this patient as in patients with the classic type of antithrombin III deficiency and in a control.     

Clinical and genetic aspects of antithrombin III deficiency and abnormal antithrombin III

Antithrombin III (AT III) has been confirmed to play an important role as a serine protease inhibitor in the mechanism of blood coagulation, and its deficiency or abnormality is found to cause thromboembolic disorders by reducing the anticoagulant activity. In this paper AT III gene of patient with congenital AT III deficiency, which was suggested to have qualitative abnormality by isoelectric focusing, was investigated. Analysis of the genomic structure by Southern blot hybridization with a cDNA probe (pAT6) revealed no detectable changes indicating any deletions, rearrangements and translocations. Therefore, we focused to analyze the sequence of exon 6 of AT III gene by polymerase chain reaction (PCR) methods followed by direct sequencing analysis. Nucleotide sequencing of exon 6 of AT III gene showed a G to T transitional mutation resulting in the conversion of arginine-406 to methionine coexisted with normal allele which encodes arginine. The mutation is located near the reactive center of AT III molecule, which region has been proved to be highly conserved during the evolution of serine protease inhibitor (serpin) family. From these results, it is concluded that the new type of mutation at amino acid site 407, which is similar to AT III Utah, is important for maintaining the structural and biological function of this inhibitor.     

Treatment of deep venous thrombosis in congenital antithrombin III deficiency with low molecular weight heparin

A 19-year old male patient with hereditary antithrombin III type I deficiency was admitted for thrombosis of the right iliac, femoral and popliteal veins. Treatment with a low molecular weight heparin resulted in recanalization of the veins confirmed by phlebography. After two weeks of treatment, the drug was replaced by another low molecular weight heparin and this was followed, 3 weeks later, by a documented relapse of thrombosis.     

Comparative effect of heparin and heparan sulphate on two abnormal antithrombin III type 3 variants

Two families were found with an antithrombin III that was unresponsive towards heparin (type 3 AT III variants). The abnormal species were purified using affinity chromatography on Sepharose bound anti-AT III antibodies. This yielded active proteins, as judged by their progressive antithrombin activities. In an attempt to explain the thrombotic tendency observed in this abnormality we compared the effect of heparin and heparan sulphate on these abnormal AT III, since, unlike heparin, heparan sulphate is a naturally occurring anticoagulant in the human. In normal plasma the heparan sulphate used in this study had a heparin-like activity of 50 U/mg by anti-F.XA and anti-F.IIa amidolytic assays. Full expression of the heparin cofactor activity in normal plasma could be obtained at a final concentration of 0.024 mg/ml of heparan sulphate (equivalent to 0.007 mg/ml of heparin). At this concentration of heparan sulphate the two abnormal AT III still exhibit a heparin cofactor activity below 10%. This absence of binding of heparan sulphate to abnormal AT III of type 3 could explain why some patients with this abnormality suffer from thrombo-embolic episodes while their AT III acts normally in the absence of heparin.     

Intravenous nitroglycerin-induced heparin resistance: a qualitative antithrombin III abnormality

An ability of intravenous nitroglycerin to interfere with the anticoagulant properties of intravenous heparin would have profound clinical implications. To investigation nitroglycerin-heparin interactions, the following pilot study was performed. Patients (N = 18) admitted to the coronary care unit with a diagnosis of either acute myocardial infarction or unstable angina were divided into four treatment groups: (1) intravenous nitroglycerin and intravenous heparin; (2) intravenous nitroglycerin alone; (3) intravenous heparin alone; or (4) neither intravenous nitroglycerin nor intravenous heparin. Serial determinations of activated partial thromboplastin time (APTT), serum heparin concentration, antithrombin III (ATIII) antigen (ATA), and ATIII activity (ATC) were obtained over a 72-hour period. Overall, patients receiving intravenous nitroglycerin did not differ significantly from other patients in APTT, heparin dose, heparin concentration, ATA, ATC, or ATA/ATC ratio (ATR). However, patients receiving intravenous nitroglycerin at a rate exceeding 350 micrograms per minute had a lower APTT (p less than 0.05), lower ATC (p = 0.02), higher ATR (p = 0.004), and a larger heparin dose requirement than patients receiving lower infusion rates. ATR correlated directly (r = 0.91; p less than 0.05) and ATC inversely (r = -0.78; p less than 0.05) with the intravenous nitroglycerin dose. Serum heparin concentration did not correlate with the intravenous nitroglycerin dose. Intravenous nitroglycerin-induced heparin resistance occurs at a critical nitroglycerin dose. A nitroglycerin-induced qualitative ATIII abnormality may be the underlying mechanism.     

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.     

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. ©1995 Wiley-Liss, Inc.     

Antithrombins Southport (Leu 99 to Val) and Vienna (Cln 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.     

Purification of antithrombin ''Vicenza'': a molecule with normal heparin affinity and impaired reactivity to thrombin

Antithrombin III (AT) 'Vicenza', a previously described dysfunctional AT associated with familial thrombosis, has been isolated by heparin affinity chromatography. The purified molecule has been investigated by SDS-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis after incubation with different amounts of thrombin. A normal affinity for heparin has been demonstrated. However, evidence is produced that AT 'Vicenza' poorly inhibits thrombin. Present data suggest that AT 'Vicenza' consists of a population of two molecules, half of which does not form a complex with thrombin however and loses its heparin affinity upon thrombin treatment.     

Choice of anticoagulant in a congenital antithrombin III (AT III)-deficient patient with chronic renal failure undergoing regular haemodialysis

Summary We describe a 43-year-old male patient with congenital antithrombin III deficiency requiring haemodialysis due to extension of venous thrombus from recurrent deep vein thrombosis. During dialysis with adequate heparinization, the patient often revealed clot formation in the extracorporeal circuit resulting in unexpected discontinuation of dialysis. When either a combination of antithrombin III concentrate plus heparin or the newly developed synthetic antithrombin preparation, MD805, was infused during dialysis, he could be uneventfully dialysed with either of the two regimens. The functional antithrombin III activity with MD805 increased to the same level as that obtained with antithrombin III concentrate, and it was possible to achieve an antithrombotic effect, as measured from the APTT and thrombin-antithrombin III complex with MD805 during and after dialysis. We thus found that MD805 could be used as an anticoagulant drug when an AT III-deficient patient required anticoagulation in the extracorporeal circulation.     

Immunoelectrophoretic evidence of a thrombin-induced abnormality in a new variant of hereditary dysfunctional antithrombin III (AT III 'Vicenza')

S ummary . An Italian family with thrombosis and hereditary dysfunctional AT III, i.e. reduced biological activity and normal levels of the immunoreactive protein is presented. The abnormal molecule, called AT III 'Vicenza', was characterized by two-dimensional crossed immunoelectrophoresis either in the absence or presence of heparin. In the presence of heparin, a normal pattern was found in plasma, but abnormality was evident in serum. A similar abnormality was found in plasma artificially clotted with thrombin. The role of thrombin in inducing the immunoelectrophoretic abnormality of AT III 'Vicenza' is discussed.     

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.     

Flow and the inhibition of prothrombinase by antithrombin III and heparin

Inhibition of prothrombinase by antithrombin III (ATIII) and heparin was investigated in a continuous-flow system. Phospholipid-coated capillaries, containing phospholipid-bound factor Xa and factor Va, were perfused with 1.0 mumol/L prothrombin and 0.5 nmol/L factor Va. At 25 degrees C and a flow rate of 32 microL/min (shear rate 28 seconds-1) the steady-state rates of prothrombin conversion depended linearly on the surface concentration of prothrombinase up to 2 fmol/cm2. The rate of thrombin generation was 952 +/- 43 (SE) mol/min/mol prothrombinase. When ATIII was included in the perfusate for 10 minutes, the free thrombin concentration at the outlet of the capillary was markedly reduced: a 50% neutralization was obtained at 0.7 mumol/L ATIII. However, the prothrombinase activity was not inhibited, as could be established after a subsequent perfusion with prothrombin and factor Va. At an ATIII concentration typical of normal plasma (2 mumol/L) a slight neutralization of prothrombinase was observed: 10% neutralization following a 10-minute perfusion. During a perfusion with ATIII in the absence of prothrombin, or in its presence with hirudin (2 mumol/L) also included in the perfusate, a more pronounced neutralization of prothrombinase was observed: 40% residual activity was obtained after a 10-minute perfusion. From this observation the suggestion comes forward that thrombin, continuously produced at the surface, consumes ATIII in the boundary layer. In this case the true ATIII concentration in the vicinity of surface-bound prothrombinase will be but a small fraction of the initial ATIII concentration in the bulk fluid. Unfractionated heparin and an ultra-low molecular weight heparin (pentasaccharide) did enhance the ATIII-dependent neutralization of prothrombinase, but to a much lesser extent than observed with small unilaminar phospholipid vesicles as the catalytic sites for prothrombinase assembly. The findings reported here support the notion that regulation of prothrombinase by heparin under in vivo conditions occurs at the stage of its formation, ie, through inhibition of free factor Xa and/or the generation of factor Va, rather than by direct inhibition of the prothrombinase activity.     

Antithrombin III (AT III) Padua2: A “New” congenital abnormality with defective heparin co-factor activities but no thrombotic disease

Summary A “new” antithrombin III (AT III) abnormality is described in five members of the same family. None of the affected members showed thrombotic manifestations and no consanguinity was present in the family. The main laboratory features were: normal routine clotting tests, slightly decreased AT III activities in all assays carried out in the presence of heparin. In the absence of heparin, antithrombin III activities were instead within normal limitis. Progressive AT III activity and AT III antigen were also normal. Crossed immunoelectrophoresis in the absence of heparin showed a normal pattern both in plasma and serum. In the presence of heparin, the propositi's plasma showed a major, less anodal, abnormal peak and a smaller normal peak. Three peaks were present in the propositi's serum as compared with the two normal ones. This AT III abnormality is different from AT III Padua previosly described by us and we propose the toponym of Antithrombin Padua-₂ to define this condition. Supported by grants from the M.P.I. (grant 1592–1981), Rome and from the Veneto Regional Government, Venice     

The antithrombin III content of cryoprecipitate prepared from blood collected with and without heparin

Antithrombin III (AT III) is a plasma protein which acts as the principal inhibitor of thrombin and is a major modulator of intravascular coagulation. Hereditary deficiency of AT III leads to recurrent episodes of thromboembolism. Acquired deficiency of AT III occurs in persons with a variety of conditions, including severe liver disease and disseminated intravascular coagulation. Replacement of AT III may be important in some deficient persons. To determine if cryoprecipitate is a useful source of AT III, we measured the AT III content of cryoprecipitate prepared from citrate phosphate dextrose blood using coagulation and fluorogenic assays and immunoassays. Using the fluorogenic assay, we also determined the effect of adding heparin to blood on the cryoprecipitation of AT III. Functional and antigenic AT III levels were similar to those of normal plasma in all citrate phosphate dextrose blood units tested, indicating that AT III is not concentrated in cryoprecipitate. Heparin had no effect on the cryoprecipitation of AT III.     

Purified radiolabeled antithrombin III metabolism in three families with hereditary AT III deficiency: application of a three-compartment model

Purified human radioiodinated antithrombin III (125I-AT III) was used to study its metabolism in six members from three different families with a known hereditary AT III deficiency. Six healthy volunteers served as a control group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and crossed immunoelectrophoresis (CIE) showed the purified AT III to be homogeneous. Amino acid analysis of the protein revealed a composition identical to a highly purified internal standard. The specific activity was 5.6 U/mg. Analysis of plasma radioactivity data was performed, using a three-compartment model. Neither plasma disappearance half-times nor fractional catabolic rate constants differed significantly between patients and control subjects. The mean absolute catabolic rate in the patient group was significantly lower than that of the control group at 2.57 +/- 0.44 and 4.46 +/- 0.80 mg/kg/day, respectively. In addition, the mean patient alpha 1-phase, flux ratio (k1,2 and k2,1) of the second compartment alpha 2-phase and influx (k3,1) of the third compartment were significantly reduced as compared with control values. It has been tentatively concluded that the observed reduction in the second compartment may be caused by a decrease in endothelial cell surface binding.