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PubMed1994

(ATT) trinucleotide repeats in the antithrombin gene and their use in determining the origin of repeated mutations.

Two (ATT) trinucleotide repeat polymorphisms have been identified in the tails of Alu repeat elements in intron 5 of the antithrombin gene. The frequency and distribution of allele sizes for the Alu 5 and Alu 8 tail polymorphisms have been defined in a sample Caucasian population. The Alu 5 polymorphism has two alleles while that of Alu 8 has 10 alleles with a heterozygosity of 0.83. These polymorphisms have been used in combination with four previously described polymorphisms within the antithrombin gene to construct antithrombin gene haplotypes in the sample Caucasian population. Twenty-two different haplotypes were observed, with the Alu 8 polymorphism being particularly useful in subdividing the core haplotype based on the previously identified polymorphisms. The haplotype data were used to investigate the origin of repeat mutations within the antithrombin locus. We compared the haplotypes associated the mutant antithrombin genes in five families with the mutation 2759C-->T (L99F) and five families with the mutation 5381C-->T (R129Stop). The mutation 2759C-->T (L99F), which occurs within a non-CpG dinucleotide, was carried on a gene associated with an identical haplotype in each of the five families. The mutation 5381C-->T (R129Stop), a single base substitution within a CpG dinucleotide, was associated with at least two different haplotypes. The findings suggest a founder effect in the five families sharing the 2759C-->T (L99F) and at least two independent origins for the CpG dinucleotide mutation 5381C-->T (R129Stop).

Antithrombins↗

PubMed2001

Syndecan-4 as antithrombin receptor of human neutrophils.

Antithrombin inhibits chemokine-induced migration of neutrophils by activating heparan sulfate proteoglycan-dependent signaling. Mechanisms of antithrombin's effects on neutrophils were, therefore, studied by testing function and expression of heparan sulfate proteoglycans in RT-PCR or flow cytometry and cell migration assays, respectively. In vitro effects of antithrombin on human neutrophil migration in modified Boyden chambers were abolished by pretreating cells with heparinase-1, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies. Expression of syndecan-4 mRNA and protein in neutrophils was demonstrated in RT-PCR and anti-syndecan-4 immunoreactivity assay, respectively. In the presence of pentasaccharide, antithrombin lost its activity on the cells. Data suggest that antithrombin regulates neutrophil migration via effects of its heparin-binding site on cell surface syndecan-4.

Antibodies↗

PubMed1993

Crystallization and preliminary X-ray diffraction analysis of two conformations of intact human antithrombin.

Human antithrombin has been crystallized by microdialysis at pH 6.7 using 18% (w/v) polyethylene glycol-4000 as precipitant. Under these conditions two crystal forms grew. The first started growing after ten days, diffracted to 3.0 A resolution and belongs to the monoclinic space group P2(1) with two molecules in the asymmetric unit and unit cell dimensions a = 70.1 A, b = 101.5 A, c = 90.5 A and beta = 105.9 A. The other crystal form took more than three months to appear, diffracted to 5.5 A and belongs to the hexagonal space group of either P6(1) or P6(5) with unit cell dimensions of a = b = 99.3 A and c = 152.9 A and two molecules in the asymmetric unit. The antithrombin redissolved from the monoclinic crystals was shown both by SDS-polyacrylamide gel electrophoresis and by protein sequence analysis to be intact while that from the hexagonal crystals was cleaved in the reactive centre loop between the P'2 and P'3 (i.e. Leu-Asn) residues. Further analysis of the intact inhibitor from the monoclinic crystals indicated that the antithrombin was present in two different conformations; an active form which could inhibit thrombin and form a stable complex with the protease, and a form which was inactive as an inhibitor and which also did not act as a substrate for thrombin. This latter form also had a low affinity for heparin and in these ways resembles latent antithrombin. The active material from the monoclinic crystals had an association rate constant with thrombin in the presence of heparin (kass) of 7.5 x 10(7) M-1 s-1 (kass for native antithrombin = 8.2 (+/- 1.0) x 10(7) M-1 s-1) indicating it still had effective heparin cofactor activity. X-ray diffraction analysis also suggests that two different protein conformations exist within the monoclinic crystals. Whereas the rotation function peak heights are equal for both molecules in the asymmetric unit using the structure of intact ovalbumin as a search model, one of the two molecules gives a much clearer signal than the other when the structures of the two cleaved serpins, alpha 1-antitrypsin and alpha 1-antichymotrypsin are used.

Antithrombins↗

PubMed1998

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Amino Acid Sequence↗

PubMed1991

Expression of human antithrombin III in the cellular slime mould Dictyostelium discoideum.

In order to test the biotechnological potential of the cellular slime mould Dictyostelium discoideum the cDNA coding for human antithrombin III was expressed in this microorganism. The 1392-bp antithrombin III cDNA was fused to the N-terminal coding part of the D. discoideum actin 6 gene. In constructs carrying this artificial N-terminal coding region only low amounts of antithrombin III were detected. However, constructs from which all actin coding nucleotides were removed produced significant amounts of anti-thrombin III, most of which was secreted into the culture broth. Stationary cultures (1.5 x 10(7) cells/ml) of certain stable transformants accumulated up to 1.0 microgram antithrombin III/ml culture medium within 24 h. The recombinant protein has a slightly smaller molecular weight in sodium dodecyl sulphate-polyacrylamide gels than authentic plasma antithrombin III and it is glycosylated, as determined by concanavalin A labelling.

Amino Acid Sequence↗

PubMed1989

Homocysteine, factor VII and antithrombin III in subjects with different gene dosage for cystathionine beta-synthase.

Cystathionine beta-synthase deficiency results in severe homocysteinaemia, precocious arteriosclerosis and frequent thromboembolism. In addition, antithrombin III activity and factor VII are low. Arteriosclerosis seems to be increased in heterozygotes as well (cystathionine beta-synthase gene dosage 50%) but rare in Down syndrome (cystathionine beta-synthase gene dosage 150%). In the present study total plasma homocysteine was high in three homozygotes, slightly increased in 20 obligate heterozygotes but not reduced in nine subjects with Down syndrome when compared to controls. After methionine loading, increases of homocysteine were pathologically high in 14 of 20 heterozygotes but was not, as expected, low in subjects with Down syndrome. Antithrombin III activity and factor VII antigen tended to be low in homozygotes but were normal in heterozygotes. In Down syndrome antithrombin III activity was reduced and factor VII antigen normal. There were no correlations between levels of homocysteine, antithrombin III activity and factor VII antigen. Thus, subjects with Down syndrome seem not to exhibit the expected gene dosage effect on homocysteine metabolism which could explain their reduced proneness to develop arteriosclerosis, nor do antithrombin III activity or factor VII antigen seem to be related to homocysteine metabolism.

Antithrombin III↗

PubMed1984

Distribution of antithrombin III and glucosylceramide in human plasma lipoproteins and lipoprotein deficient plasma.

We have investigated the distribution of antithrombin-III and glucosylceramide (Glc-Cer) in human plasma, plasma lipoproteins and lipoprotein-deficient plasma. Antithrombin III activity was measured employing immunochemical and biological assays. Glc-Cer was quantified by gas liquid chromatography (GLC). Whole plasma contained 145 micrograms antithrombin III/ml plasma, all of which was associated with the lipoprotein-deficient plasma (d greater than 1.25 g/ml). Whereas, most if not all the plasma GlcCer was associated with plasma low density lipoproteins (LDL) (d-1.022-1.055 g/ml) and high density lipoproteins (HDL) (d-1.063-1.25). GlcCer was not found in the lipoprotein-deficient plasma. We conclude that GlcCer on lipoproteins does not contribute to antithrombin III activity. Moreover, the absence of GlcCer in lipoprotein-deficient plasma does not impair antithrombin-III activity.

Antithrombin III↗

PubMed1988

Antithrombin III concentrate in the treatment of fulminant hepatic failure.

Twenty-six patients with fulminant hepatic failure were treated with daily infusions of antithrombin III concentrate until recovery of consciousness or death. Seven patients were alive (group A), 7 survived 17 to 47 days after treatment (group B), and 12 died within 9 days (group C). Decreased plasma antithrombin III levels increased on the day after treatment, irrespective of the pretreatment levels in all patients. Continuous or temporary normalization was seen in all patients in groups A and B, but in only 5 in group C patients whose bleeding was extensive (p less than 0.05). An abrupt drop in peripheral platelet counts occurred when plasma antithrombin III levels were below normal. General bleeding accompanied this drop. These results suggest that maintained normal plasma antithrombin III levels are beneficial for prolonged survival time in fulminant hepatic failure, probably through controlling intravascular coagulation, and that antithrombin III infusion may be useful for such treatment.

Antithrombin III↗

PubMed1984

Antithrombin III levels in preeclampsia correlate with maternal and fetal morbidity.

In 57 patients with pregnancy-induced or aggravated hypertension, antithrombin III levels correlated inversely with maternal morbidity. Morbidity was determined by the maximal diastolic blood pressure, disturbance of renal and liver function, and thrombocytopenia. Antithrombin III levels and platelet counts correlated inversely with the degree of placental infarction. Proteinuria (grams per 24 hours) was most predictive of fetal outcome, which was considered to be either favorable if a healthy baby could be discharged with its mother or unfavorable in case of perinatal death or a prolonged stay in the neonatal intensive care unit. Plasma antithrombin III and serum glutamic oxaloacetic transaminase levels, in that order, augmented the number of correct predictions. Antithrombin III inhibits blood coagulation by forming irreversible complexes with activated clotting enzymes, notably with factor Xa and thrombin. Evidence is presented which suggests that antithrombin III levels in preeclampsia are depressed as a result of increased consumption in the maternal vascular tree, rather than decreased synthesis or increased urinary loss.

Adult↗

PubMed1991

Oral contraceptives and antithrombin III: variations by dosage and ABO blood group.

A randomized clinical trial of oral contraceptives evaluated 67 women on a regimen of 50 micrograms ethinyl estradiol and 1.0 mg norethindrone, 61 women on a regimen of 35 micrograms ethinyl estradiol and 1.0 mg norethindrone, and 64 women on a regimen of 35 micrograms ethinyl estradiol and 0.5 mg norethindrone. ABO blood group type was determined in all women. At baseline and at 3, 6, and 9 months, plasma antithrombin III levels, by both an immunologic and an activity method, and selected plasma levels of the contraceptive steroids were measured. Antithrombin III levels for all oral contraceptive groups combined decreased from baseline by 19.7% and 28.8% for the immunologic and activity methods, respectively. Analysis of interrelationships among antithrombin III by activity method, oral contraceptive type, and ABO blood group showed larger declines in antithrombin III for type O women using the highest estrogen dose preparation (31.6%) and for non-type O women using the lowest progestin dose preparation (38.9%). Plasma levels of contraceptive steroids also were related to changes in the most extreme levels of antithrombin III.

ABO Blood-Group System↗

PubMed1984

Measurement of human activated factor X-antithrombin complex by an enzyme-linked differential-antibody immunosorbent assay.

An enzyme-linked immunoabsorbent assay (ELISA) has been developed for the measurement of the complex of human antithrombin and Factor Xa. Rabbit anti-human Factor X antibodies are adsorbed to ELISA plates, and samples containing Xa-antithrombin complex are added. This is followed by the addition of F(ab')2 fragments of rabbit antibodies against human antithrombin, previously labeled with alkaline phosphatase, and subsequent measurement of the bound labeled antibody by hydrolysis of p-nitrophenylphosphate. The minimum level of complex detectable in a sample is ca. 0.1 nM. The assay has been used to follow the generation of Xa-antithrombin complex in kinetic situations by the addition of 1 microM Ile-Glu-Gly-Arg-chloro- methylketone to the ELISA sampling buffer, and it has also been used in plasma systems, where a 20-fold reduction in the sensitivity of the assay is observed. This reduction was shown to be entirely caused by the plasma Factor X. The assay has been used to follow generation of the Xa-antithrombin complex in defibrinated plasma upon activation of the clotting system with the Factor X-activating protein of Russell's Viper venom, and has been compared with the total generation of Factor Xa, measured by a radiopeptide assay of Factor X activation in the same mixtures.

Antibodies↗

PubMed1983

Localization of the disulfide bond in human antithrombin III required for heparin-accelerated thrombin inactivation.

Heparin accelerates the rate of inhibition of thrombin by antithrombin III. Reduction of one of the three antithrombin disulfide bonds with dithiothreitol under mild conditions abolishes this rate-enhancing effect without affecting the rate of reaction in the absence of heparin. Alkylation of mildly reduced antithrombin III with [3H]iodacetic acid followed by digestion with cyanogen bromide yielded two major labeled peptides. The smaller peptide, containing Cys-422, was identified as extending from Gly-414 to the C-terminus, Lys-424. Our data are consistent with the larger labeled peptide being the one extending from Glu-104 to Met-243 and containing Cys-239. Cys-422 has been shown by others to be linked to Cys-239. These data indicate that the sensitive disulfide bond in antithrombin III extends between Cys-239 and Cys-422; the site at which thrombin cleaves the antithrombin III is between these two half-cystines.

Antithrombin III↗

PubMed1984

The role of tryptophan residues in heparin-antithrombin interactions.

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl) sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of "heparin cofactor" activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent "activation" of the macromolecule.

Antithrombins↗

PubMed1977

Binding of heparin to human antithrombin III as studied by measurements of tryptophan fluorescence.

Corrected fluorescence excitation and emission spectra of human antithrombin III have been determined. The fluorescence observed originates almost entirely from tryptophan residues. Reduction of the disulfide bonds followed by carboxymethylation did not change the fluorometric properties of the protein. The binding of heparin to antithrombin III caused a marked fluorescence enhancement by about 30% of the intrinsic protein emission intensity. Various samples of heparin yielded different binding curves. Heparin fractionated by gel filtration seemed to be bound to two sites on antithrombin III with association constants of 0.6-10(6)m-1 and 0.2-10(6)M-1 respectively. Heparin, prepared by affinity chromatography on matrix-bound antithrombin III appeared to be bound to only one site with an association constant of 2.3-10(6)M-1. Under similar conditions heparin caused no increase of the intrinsic protein emission intensity when added to reduced and carboxymethylated antithrombin III. The implications of these findings are discussed.

Alpha-Globulins↗

PubMed1992

Purification and characterization of an antithrombin III inactivating enzyme from the venom of the African night adder (Causus rhombeatus).

A serine proteinase was isolated from the venom of the night adder (Causus rhombeatus) by fast protein liquid chromatography (anion-exchange, gel filtration and hydrophobic interaction). The protein (termed CR-serpinase) had an estimated mol. wt of 45,500 as determined by SDS-PAGE, pI of 4.7 and a carbohydrate content of 18.9%. Incubation of CR-serpinase with purified human antithrombin III at a molar ratio of 1:66 resulted in a loss of more than 90% of the initial AT III activity within 10 min. The reaction was dependent on heparin. In SDS-PAGE inactivation of human antithrombin III was correlated with the occurrence of two cleavage products. The cleavage site in the antithrombin III molecule was determined to be Arg 393-Ser 394 by amino-terminal sequencing. CR-Serpinase had no thrombin-like activity since no fibrinogen conversion was induced and had no procoagulant activity. CR-Serpinase activity was not inhibited by antithrombin III-heparin and was not decreased by a 10-min preincubation in normal human plasma. Inactivation of antithrombin III by CR-serpinase appeared to be very specific.

Amino Acid Sequence↗

PubMed1982

Specific binding of thrombin-antithrombin III complex to hepatocytes.

Thrombin-antithrombin III complex binds selectively to isolated hepatocytes, whereas antithrombin III alone does not. The binding is time and concentration dependent at 37 degrees C: the apparent Km value is 0.8/microM. The rate of binding is approximately 1.6 X 10(5) molecules h-1 cell-1 at this concentration. At 4 degrees C there is no measurable interaction between the complex and the hepatocytes. The binding is also prevented by pretreatment of cells with trypsin. On the other hand, about 80% of the thrombin-antithrombin III complex bound to hepatocytes is releasable by trypsin digestion. NaF or carboxyatractyloside does not inhibit the process. The interaction of thrombin-antithrombin III complex with hepatocytes seems to be specific, since the complexes of antithrombin III with other proteinases, like trypsin or plasmin, are not bound at the concentrations used. Based on these data, a mechanism for the binding of the inactive complexed form of thrombin to hepatocytes is suggested.

Animals↗

PubMed1987

Regulation of factor VIIa activity in plasma: evidence that antithrombin III is the sole plasma protease inhibitor of human factor VIIa.

The inhibition of human factor VIIa by antithrombin III and normal human plasma was studied in the presence and absence of heparin. In the absence of heparin, no apparent inhibition of factor VIIa was observed in either system. In the presence of heparin, factor VIIa activity was inhibited 50% by purified antithrombin III and plasma in 90 min and 75 min, respectively. No inhibition of factor VIIa was observed in heparinized plasma previously depleted of antithrombin III by immunoaffinity adsorption. Incubation of factor VIIa with antithrombin III-heparin or heparinized plasma resulted in the formation of a covalent complex with an apparent molecular weight of 100 kilodaltons. These data indicate that antithrombin III appears to be the sole plasma protease inhibitor of human factor VIIa, and the expression of its inhibitory activity against factor VIIa is absolutely dependent upon the presence of exogenous heparin.

Antithrombin III↗

PubMed1988

Physiological coagulation inhibitors (protein S, protein C and antithrombin III) in severe preeclamptic states and in users of oral contraceptives.

Protein C, protein S and antithrombin III were evaluated in normal pregnancy, severe preeclampsia and chronic hypertension with superimposed severe preeclampsia. The same study was performed on a group of 10 normal women using oral contraceptives. In normal pregnancy a significant decrease in the level of free and total PS was observed in the 2nd trimester of pregnancy and was sustained throughout the remaining months. No significant changes in the levels of protein C and antithrombin III were observed during normal pregnancy. In preeclamptic states a significant decrease in protein C was observed. It was more evident in severe preeclampsia when compared with the normal pregnancy group at similar gestational age. No statistically significant differences in protein S were found when the normal and pathological groups were compared. Antithrombin III decreased only slightly in the severe preeclamptic group. The decrease in protein C and antithrombin III levels in severe preeclampsia could be related with the microthrombotic state that these patients may present. However, the role played by protein S, which decreases during normal pregnancy and in preeclampsia, is not clear. A decrease in the level of total protein S was observed in the group of women using oral contraceptives. No significant changes in protein C and antithrombin III levels were observed in this group.

Adult↗