Antithrombin Milano, single amino acid substitution at the reactive site, Arg393 to Cys

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.