Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.
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.
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.
Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.
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.
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