Antithrombin Milano, single amino acid substitution at the reactive site, Arg393 to Cys
ABSTRACT Antithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; antithrombin Milano peak 1 of Mr approximately 60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr approximately 120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the approximately 120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr approximately 60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the approximately 120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxymethylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, Northwick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J. Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.
Full-textDOI: · Available from: Hediye Erdjument-Bromage, Jun 01, 2015
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ABSTRACT: HE PRESENCE OF activated coagulation factors T within the circulation would result in life-threatening intravascular clot formation were it not for the presence of circulating inhibitors of coagulation in the plasma. The most important of these is antithrombin-I11 (AT-III).I,2 Human AT-I11 is a single-chain glycopeptide of 432 amino acids that specifically inhibits several activated serine pro- teases of the coagulation cascade, including factors XIIa,3 XIa; Xa: and IXa6 as well as a-thrombin. The inhibition of a-thrombin, which is relatively slow under physiologic conditions but greatly accelerated in the presence of heparin, is probably the most important of these.' Elucida- tion of the mechanism of action of AT-I11 requires that the specific amino acids that interact with a-thrombin and heparin be defined clearly. The reactive center of AT-I11 has been identified as the Arg393-Ser394 bond near the carboxy terminal end of the protein.'.'' When a serine protease such as a-thrombin interacts with AT-111, it
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ABSTRACT: 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.
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ABSTRACT: Inherited defects of antithrombin III, protein C, protein S, heparin cofactor II, plasminogen and the fibrinogens are thought to be responsible for between 10 and 15% of all patients presenting with recurrent venous thrombosis. The structure, function and expression of these genes and the nature of the gene lesions underlying the deficiency states are reviewed in detail.Blood Reviews 03/1991; 5(1):55-70. DOI:10.1016/0268-960X(91)90009-2 · 5.57 Impact Factor