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Blood May 1991Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity....
Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III-Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT-III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III-Hamilton is capable of only the first of these events.
Topics: Alleles; Amino Acids; Antithrombin III; Factor Xa; Genetic Variation; Humans; Thrombin
PubMed: 2029579
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1987Antithrombin-III Denver is a mutant protein which differs from the normal in being defective in serine protease binding (Sambrano, J. E., Jacobson, L. J., Reeve, E. B.,...
Antithrombin-III Denver is a mutant protein which differs from the normal in being defective in serine protease binding (Sambrano, J. E., Jacobson, L. J., Reeve, E. B., Manco-Johnson, M. J., and Hathaway, W. E. (1986) J. Clin. Invest. 77, 887-893). It was isolated from the blood of an individual heterozygous for the abnormal gene by: affinity separation on heparin-Sepharose to obtain an antithrombin fraction, and gel filtration of the species present following complexing of the antithrombin fraction with a small excess of thrombin. The reduced, S-carboxymethylated protein formed a mixture of soluble tryptic peptides which was fractionated on Vydac C18. A single, unique peptide not present in a parallel experiment with normal antithrombin-III was isolated. This peptide was identified by sequence analysis and synthesis to correspond to residues 394-399 in the known sequence of the inhibitor, with leucine replacing reactive site P'1 residue Ser394. Although chromatograms of the tryptic peptides from the normal and mutant proteins were otherwise indistinguishable, the existence of additional residue replacements is not excluded. Measurements of the rate of thrombin binding by the mutant protein with p-aminobenzamidine as a fluorescent indicator showed that the second-order rate constant is reduced drastically. Meaningful measurements with the mutant protein could only be made in the presence of heparin and revealed a reduction of about 4000-fold in the rate constant.
Topics: Amino Acid Sequence; Antithrombin III; Binding Sites; Chromatography; Electrophoresis, Polyacrylamide Gel; Genetic Variation; Humans; Kinetics; Peptide Fragments; Thrombin; Trypsin
PubMed: 3805013
DOI: No ID Found -
Annales de Biologie Clinique 1987The in vivo regulation of coagulation is mainly controlled by plasma inhibitors in which antithrombin III (AT III) plays an importance role. AT III is a glycoprotein...
The in vivo regulation of coagulation is mainly controlled by plasma inhibitors in which antithrombin III (AT III) plays an importance role. AT III is a glycoprotein which inhibits all serine proteases, except factor, VIIa, generated during the coagulation process. The proteases are inactivated by formation of an equimolecular complex and this reaction is greatly enhanced in the presence of heparin. A similar catalytic process could occur in vivo, involving heparin-like substances present on the surface of the surface of the endothelial cell. The physiological importance of AT III is clearly demonstrated by the high incidence of thromboembolic disease in patients with congenital AT III défficiency.
Topics: Antithrombin III; Antithrombin III Deficiency; Chemical Phenomena; Chemistry; Drug Synergism; Heparin; Humans; Protease Inhibitors; Thrombin
PubMed: 3619143
DOI: No ID Found -
Clinical and Applied... Jan 2018Thrombate III is a human plasma-derived antithrombin III (AT-III) often utilized in patients on extracorporeal membrane oxygenation (ECMO) with suspected AT-III-mediated...
Thrombate III is a human plasma-derived antithrombin III (AT-III) often utilized in patients on extracorporeal membrane oxygenation (ECMO) with suspected AT-III-mediated heparin resistance. It is supplied as 500-U and 1000-U vials, costing US$4.66 per unit. Literature is limited in describing the clinical value of AT-III in relation to its high cost. The primary objective was to determine conditions of use and associated cost of potentially unnecessary utilization of AT-III at The Johns Hopkins Hospital. Secondary objectives included evaluating the effect of AT-III on anticoagulation parameters and the overall cost utilized and wasted on AT-III. A retrospective cohort study was performed. The primary end point was the total cost associated with potentially unnecessary utilization of AT-III. There were 326 doses of AT-III administered to 65 patients in 2014. There were 177 (54%) potentially unnecessary doses associated with a cost of US$541 634. Antithrombin III repletion significantly increased median AT-III levels in non-ECMO and ECMO patients compared to baseline (non-ECMO: 62% vs 81%, P < .01; ECMO: 63% vs 81%, P < .01); however, 37.3% of ECMO and 49% of non-ECMO patients had therapeutic anticoagulation monitoring parameters prior to administration. A total cost of US$688 478 was spent on administered AT-III and US$417 194 (38%) was wasted. Utilizing restriction criteria and a new dosing strategy potentially results in estimated annual savings of US$556 000. Utilizing restriction criteria and alternative dosing strategies to mitigate waste and unnecessary use has the potential to result in significant cost savings.
Topics: Adolescent; Adult; Antithrombin III; Child; Child, Preschool; Costs and Cost Analysis; Extracorporeal Membrane Oxygenation; Humans; Infant; Male; Middle Aged; Retrospective Studies
PubMed: 28301908
DOI: 10.1177/1076029617693941 -
Rinsho Byori. the Japanese Journal of... Apr 1987
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Nephron 1981Plasma antithrombin-III (AT-III) levels in 18 patients on maintenance haemodialysis were studied. AT-III was measured functionally and immunologically before and after...
Plasma antithrombin-III (AT-III) levels in 18 patients on maintenance haemodialysis were studied. AT-III was measured functionally and immunologically before and after dialysis. Simultaneous counts of platelets were made. Prior to dialysis the average AT-III levels and platelet counts were found to be within the lower part of the normal range. On paired analysis the dialysis was seen to induce small, but significant decreases in AT-III levels and platelet counts. A positive linear correlation was found between levels of functionally and immunologically determined AT-III. Replacement of the intravenous application of heparin with an administration of low-dose, subcutaneous heparin is suggested.
Topics: Antithrombin III; Blood Platelets; Female; Humans; Male; Renal Dialysis
PubMed: 7266723
DOI: 10.1159/000182085 -
Developments in Biological... 1987Inherited antithrombin III (ATIII) deficiency causes a life-long tendency to venous thromboembolism, which is often recurrent and may be life-threatening. In contrast,...
Inherited antithrombin III (ATIII) deficiency causes a life-long tendency to venous thromboembolism, which is often recurrent and may be life-threatening. In contrast, the clinical importance of acquired ATIII deficiency, whether spontaneous or associated with estrogen-containing oral contraceptive treatment, remains uncertain. Moderately reduced ATIII activity before or immediately after surgery is neither sensitive nor specific for a high risk of postoperative venous thromboembolism (VTE), while moderately reduced ATIII activity during heparin treatment for VTE fails to indicate an unusually large heparin requirement or to predict recurrence. In the absence of good clinical trials, the value of ATIII replacement therapy also remains obscure; its use in congenital deficiency is largely based on anecdote, and while it may cause more rapid correction of the hemostatic defect in patients with disseminated intravascular coagulation (DIC), any improvement in morbidity or mortality resulting from ATIII replacement remains to be demonstrated.
Topics: Antithrombin III; Antithrombin III Deficiency; Humans; Postoperative Complications; Thromboembolism
PubMed: 3609485
DOI: No ID Found -
Vojnosanitetski Pregled 1990
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Vox Sanguinis 1983Antithrombin III (AT III) is a plasma protein which acts as the principal inhibitor of thrombin and is a major modulator of intravascular coagulation. Hereditary...
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.
Topics: Antithrombin III; Blood Specimen Collection; Disseminated Intravascular Coagulation; Factor VIII; Fibrinogen; Heparin; Humans
PubMed: 6402859
DOI: 10.1111/j.1423-0410.1983.tb04109.x -
Thrombosis and Haemostasis May 1993During the reaction of antithrombin III (AT III) with target proteases the inhibitor serves as pseudo-substrate and undergoes profound conformational changes, becomes...
During the reaction of antithrombin III (AT III) with target proteases the inhibitor serves as pseudo-substrate and undergoes profound conformational changes, becomes incorporated into a covalent stoichiometric enzyme-inhibitor complex which is, in contrast to native AT III, recognized by monoclonal antibody 4C9. In the absence of the target enzyme thrombin, incubation of AT III with 1-2 M guanidine, 0.6% deoxycholate, heating to 56 degrees C, or buffer at pH 4 resulted in inactivation of the inhibitor with concomitant exposure of the epitope for 4C9 and formation of AT III multimers (from 3.9 S to 7.1-7.4 S). Loss of activity, formation of multimers and exposure of neoepitope(s) of AT III occurred in a concerted fashion and followed second order kinetics with an activation energy of Ea = 31 kcal/mol. AT III-multimerization induced by treatment with 1 M guanidine (mainly AT III-tetramers with M(r) of 250,000) and formation of the binary AT III-thrombin complex revealed similar self-association patterns as judged by gel electrophoresis under non-denaturing conditions. In the presence of heparin, even higher multimers of AT III-thrombin complexes were noted. Moreover, self-association products of the ternary vitronectin-thrombin-AT III complex, which is the ultimate reaction product following thrombin inhibition in the circulation, could be recognized and quantitated due to exposure of the 4C9 epitope on AT III, indicating that AT III exists in multimeric forms within binary and ternary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Antibodies, Monoclonal; Antithrombin III; Humans; Peptide Hydrolases; Polymers; Protein Binding; Protein Conformation
PubMed: 8322264
DOI: No ID Found