Antithrombin III Toyama: replacement of arginine-47 by cysteine in hereditary abnormal antithrombin III that lacks heparin-binding ability.

Structural analyses of a hereditary abnormal antithrombin III, antithrombin III Toyama, which has normal progressive antithrombin activity but no heparin cofactor activity, have been carried out to elucidate the molecular abnormality causing recurrent thrombophlebitis of a patient and to identify an amino acid residue essential for the binding with heparin. Abnormal antithrombin III was reduced, S-pyridylethylated, and treated with cyanogen bromide. Eleven fragments were isolated by the combination of Sephadex G-50 gel filtration and reversed-phase HPLC and compared with those from normal antithrombin III. One large fragment (CN-III) that appeared to have a different amino acid composition from that of the corresponding fragment from normal antithrombin III was digested with trypsin, and the digests were separated by HPLC. The abnormal peptide was identified by comparing the peptide map with that from normal antithrombin III. Amino acid sequence analysis of the abnormal peptide indicated that the arginine-47 of normal antithrombin III had been replaced by cysteine in antithrombin III Toyama. One base mutation, C leads to T, in the 5' terminal position of the arginine-47 genetic codon (CGT) is probably responsible for this substitution. These results also suggest that arginine-47 is an essential amino acid residue for the binding with heparin.     

Characterization of antithrombins produced by active site mutagenesis of human alpha 1-antitrypsin expressed in yeast

Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.     

Further investigations on antithrombin III in the plasmas of patients with the abnormality of antithrombin III Budapest.

Antithrombin III (AT-III) was studied in a thrombophilic family with an abnormal AT-III molecule (antithrombin III Budapest) using a modified crossed immunoelectrophoresis technique, gel filtration, 'rocket' immunoelectrophoresis and a heparin cofactor assay. When agarose was applied in the first phase of the crossed immunoelectrophoresis, the normal and the pathological AT-III revealed identical electrophoretic mobility. However, when heparin was mixed with agarose in the first phase of electrophoresis, the propositus' plasma displayed a different AT-III pattern from normal plasma. His plasma contained the first component of the normal plasma (Immune Antithrombin III1, IAT-III) in a concentration of only 5% of normal, and a protein in high concentration which although immunoreactive to AT-III antisera, had an electrophoretic mobility similar (but not identical to that of IAT-III2. This abnormal protein had no heparin cofactor activity and a molecular size greater than normal plasma AT-III. Unlike AT-III, the addition of heparin did not change the molecular size of the pathogic AT-III molecule significantly. The abnormal protein was present in lower concentrations in the patient's children and at the time of study they had no clinical or laboratory evidence of intravascular coagulation.     

A heparin-binding synthetic peptide of heparin/heparan sulfateinteracting protein modulates blood coagulation activities

We have previously identified and characterized a heparin-binding cell surface protein (heparin/heparan sulfate-interacting protein, or HIP) present on epithelial and endothelial cells. A synthetic peptide mimicking a heparin-binding domain of HIP is now shown to bind a small subset of heparin molecules with high affinity and, therefore, presumably recognizes a specific structural motif in the heparin molecule. Further analyses revealed that the heparin molecules exhibiting a high affinity for the HIP peptide also show an extremely high affinity for antithrombin III (AT-III), a cofactor required for heparin’s anticoagulant activity. The HIP peptide was shown to compete with AT-III for binding to heparin and to neutralize the anticoagulant activity of heparin in blood plasma assays. Furthermore, the heparin subfraction that binds to the HIP peptide with high affinity exhibits an extremely high anticoagulant activity. We conclude that although the HIP peptide shows no sequence similarity with AT-III, the two proteins recognize the same or similar structural motifs in heparin.     

Antithrombin "Chicago": a functionally abnormal molecule with increased heparin affinity causing familial thrombophilia

A family with a high incidence of spontaneous thromboembolism over four generations has been investigated. The propositus is a 21-yr-old male with a history of thrombophlebitis. Medical histories of 46 family members were obtained. Twelve of these individuals have experienced deep venous thromboses and/or pulmonary emboli. Seven members of the kindred, with a prior history of thrombotic phenomena, were investigated in detail. These subjects were found to have normal plasma concentrations of immunoreactive antithrombin (mean 96%), decreased plasma levels of progressive antithrombin activity (mean 50%), and greatly reduced amounts of plasma heparin cofactor activity (mean 42%). The abnormal antithrombin ("Chicago") was found to elute from heparin- Sepharose at a higher ionic strength than normal inhibitor. The functionally defective antithrombin molecules exhibit a reduced ability to neutralize thrombin in the presence or absence of heparin (approximately 10%-20% of normal). The molecular defect of this protease inhibitor thus appears to be distinct from those of previously described abnormal antithrombins.     

Antithrombin III deficiency: decreased synthesis of a biochemically normal molecule

A 29-yr-old white female has suffered from recurrent venous thromboses over the last 12 yr. Plasma antithrombin III (AT-III) levels were 48% of normal by immunoelectrophoresis and 56% by chromogenic assay. Three of four siblings and the father had similar AT-III levels without associated venous thromboses. Heparin-Sepharose chromatography demonstrated normal behavior of the patient's AT-III. Her purified AT- III could not be distinguished from AT-III purified from a normal control either by SDS polyacrylamide gel electrophoresis or by crossed immunoelectrophoresis, and the heparin cofactor activity and the progressive antithrombin activity of both AT-III samples were identical. Turnover studies were made in the patient using her own purified AT-III labeled with 131I, (*I). The results did not differ significantly from studies made with autologous *I-AT-III in two normal control women. Her fractional breakdown rate of 0.54 total plasma AT- III per day compared with 0.45 and 0.52 in the controls. These studies indicate that the patient synthesizes a normal AT-III molecule at half normal rates.     

The anticoagulant effect of heparin + antithrombin III using purified fibrinogen as substrate

The anticoagulant effect of heparin in test systems composed of purified factors [antithrombin III (AT-III), fibrinogen and thrombin] was studied. As expected, the sensitivity to heparin depended on the concentrations of AT-III and thrombin, whereas the fibrinogen level was less decisive. In addition, qualitative differences proved important. Thus, the sensitivity to heparin was greater with crude bovine thrombin than with highly purified thrombin from the same species. Further, the sensitivity to heparin increased following removal of cold-insoluble material from the fibrinogen preparation. Finally, during storage of purified AT-III at +4 degrees C for more than 4 weeks, the sensitivity to heparin decreased more rapidly than expected from the amidolytic AT-III assays. Smaller amounts of heparin were required to give a prolonged thrombin clotting time than in whole plasma, indicating that components of normal platelet-poor plasma (besides AT-III and fibrinogen) interfere with the anticoagulant effect of heparin. The present test systems may prove suitable for the detection and evaluation of factors of importance for the so-called heparin tolerance.     

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.     

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.     

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

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.     

Management with antithrombin III concentrate in a pregnant woman with hereditary antithrombin III deficiency

Pregnant women with hereditary antithrombin III (AT-III) deficiency are frequently associated with thromboembolic disorders. We have treated a pregnant woman with hereditary AT-III deficiency, who had suffered from thromboembolic disorders at her past three gestations, with AT-III concentrate. Dosage of AT-III concentrate to maintain plasma AT-III activity over 80% was 3,500 units per week during second and third trimesters, but more frequent administration was necessary around delivery. In recent reports, pregnant women with hereditary AT-III deficiency had been treated with heparin or warfarin except for during abortion and delivery, in which time AT-III concentrate was widely utilized. But the use of heparin or warfarin during gestation is occasionally harmful, AT-III concentrate should be chosen for management in pregnancy in women with hereditary AT-III deficiency.     

The anticoagulant activation of antithrombin by heparin

Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-Å structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin–protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.     

Investigations on Antithrombin III in Normal Plasma and Serum

Studies on antithrombin III (AT-III) were made by a modification of the two dimensional crossed immunoelectrophoresis technique and gel filtration. Mixing various quantities of heparin with agarose in the first phase of electrophoresis, AT-III from normal human plasma and serum revealed a heterogeneity which depended on the heparin concentration in the agarose gel. At heparin concentrations higher than 16 u/ml, AT-III displayed three components with different electrophoretic mobilities. The component with the highest mobility (designated immunoantithrombin III1 : IAT-III1) dominated in plasma. In normal serum, however, the quantity of this component was decreased and the two other peaks with a slower electrophoretic mobility (IAT-III2 and IAT-III3) became more evident. Normal human plasma and serum were filtered on Sephadex G-200 and the AT-III concentration measured in the fractions by rocket immunoelectrophoresis. The peaks of AT-111 were found in the same fractions for both plasma and serum and were coincident with the albumin peak of the plasma proteins. However, in the case of serum the AT-III concentration decreased less sharply in those fractions with higher molecular weight than in the corresponding plasma fractions. Analysis of these fractions by crossed immunoelectrophoresis revealed that the two components with slower electrophoretic mobility (IAT-III2 and IAT-III3) had higher molecular size than IAT-III1, that the concentration of IAT-III2 and IAT-III3 was significantly higher in serum, and that the high molecular weight components in plasma and serum were qualitatively identical. It is concluded that high molecular weight complexes between AT-III and activated coagulation factors may be present in normally circulating blood and that their detection and possibly quantitation can be achieved using the heparin/agarose crossed immunoelectrophoresis system.     

Clinical and biochemical characterization of antithrombin III Franconville, a variant with Pro 41 Leu mutation

We describe a familial study of AT III, a type III antithrombin III variant which was identified in the propositus by gene analysis as Pro 41 Leu heterozygous mutation. None of the four members of the family who presented with defective heparin cofactor (hep-cofactor) activity, and therefore probably carried the mutation, had experienced deep venous thrombosis. The abnormal AT III was purified from the propositus' plasma, taking advantage of the difference in NaCl concentrations required to elute variant and normal AT III from heparin-Sepharose. The antithrombin and anti-Xa activities of the purified variant AT III were comparable to those observed for normal AT III, but hep-cofactor activity was strikingly reduced. The enhancement by heparin of thrombin and F Xa inhibition by normal and variant AT III was compared in the absence of NaCl and in the presence of normal NaCl concentrations. The difference between the degrees of inhibition by normal and variant AT III was maximal at physiological ionic strength (i.e. at a concentration of 0.15 M). The quantification of heparin AT III interaction with both normal and variant purified proteins in a double reciprocal plot yielded similar dissociation constants but a 9-fold decrease in the maximal pseudo-first order constant. This suggests that Pro 41 is more involved in the molecular changes induced by heparin than in the primary binding of the activator.     

The inhibition of high and low molecular weight urokinase in plasma

The rates of inhibition of high molecular weight (HMW) and low molecular weight (LMW) urokinase (UK) incubated in plasma or with purified antithrombin III (AT-III) were compared. Using a fibrinolytic assay system to determine residual biologic activity, polyacrylamide gel electrophoresis to demonstrate the formation of complexes, and selective immunoprecipitation techniques to identify the plasma inhibitors participating in the neutralization process, it was established that: (A) HMW-UK is inhibited more rapidly than LMW-UK, both in plasma and with purified AT-III; (B) heparin (3--10 U/ml accelerates the neutralization process in both systems, but only slightly; and (C) in plasma, several inhibitors, alpha 2-macroglobulin, alpha 1-antitrypsin, and antithrombin III, neutralize the activity of HMW-UK and LMW-UK.     

Formation of the antithrombin heterodimer in vivo and the onset of thrombosis.

Antithrombin is shown to undergo a slow spontaneous conversion to its inactive latent conformation with readily discernible amounts present in plasma on incubation at 37 degrees C for 72 hours. More rapid conversion occurs on incubation of isolated antithrombin at 41 degrees C or 50 degrees C, but the appearance on electrophoresis of free latent antithrombin is preceded by the formation, in reciprocal proportions, of a new slow band. This slow component is shown to be a heterodimer of active and latent antithrombin. It can be isolated as a single stable band either by incubation of antithrombin or by mixing equimolar proportions of active and latent antithrombin under the same conditions that give overnight crystallization of the active/latent antithrombin heterodimer. Similarly, equimolar addition of latent antithrombin to plasma results electrophoretically in a quantitative shift to the slower heterodimer mobility. Clinically, the presence of latent antithrombin is potentially deleterious, because its linkage to form the heterodimer results in inactivation of the otherwise normal molecule linked to the latent antithrombin. In the case of alpha-antithrombin, because the dimer readily dissociates, there is only a 11% additive loss of activity, but with beta-antithrombin the dimer appears more stable, with the additive loss of activity from the normal beta component being 21%, increasing to 33% on stabilization of the dimer with heparin. This linked and selective loss of activity of beta-antithrombin provides an explanation for the unexpected severity of thrombotic episodes in heterozygotes with conformationally unstable antithrombins.     

Antithrombin activity of intact human platelets.

Antithrombin activity has been identified in intact washed human platelets. An apparent activity was demonstrated at platelet concentrations above 0.31 X 10(9)/ml, when platelet suspensions were incubated with 2.0 NIH units/ml of thrombin. Neither red cells nor white cells revealed antithrombin activity. No significant loss of the platelet antithrombin activity was observed after ten successive washings or after treatment of platelets with antibodies to antithrombin III or alpha2-macroglobulin. Almost the same amount of antithrombin activity as normal platelets was demonstrated in the platelets from an afibrinogenemic patient. Pre-treatment of platelets with trypsin, papain, and neuroaminidase reduced the activity significantly, whereas lipase was without effect. The platelet antithrombin reacted with thrombin in less than 3 seconds, and this rapid reaction of platelet antithrombin was different from that of plasma antithrombin III or fibrinogen. The thrombin-like clotting activity of ancrod was inhibited by fibrinogen but not platelets. Also, unlike plasma antithrombin III or fibrinogen, brief exposure to heat (56 degrees C or 60 degrees C) reduced considerable amounts of platelet antithrombin activity. These results suggest that platelets possess a specific antithrombin with different characteristics from other known antithrombins.     

Treatment of familial antithrombin-III deficiency with danazol

3 individuals from 2 unrelated families with recurrent thromboses and quantitative deficiencies of antithrombin III (AT-III) were treated with danazol, 600 mg daily for 4 months. Significant increases of AT-III (p less than 0.025) measured as heparin cofactor activity were noted in 1 female and 1 male patient. Failure to augment levels in the other male patient may have been due to poor absorption of the drug following small bowel resection for mesenteric infarction. Side effects of estrogen deficiency necessitated dosage reduction in the female patient. The 2 males experienced no adverse side effects except for prolongation of the prothrombin time in 1 who was receiving oral anticoagulants. We conclude that danazol causes a significant increase in some individuals with familial AT-III deficiency. Additional studies are necessary to determine whether this form of therapy may prove to be a suitable alternative to long-term anticoagulation and to assess the long-term clinical benefits in individuals with recurrent thrombosis.