Summary
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;
anti thrombin Milano peak 1 of Mr ∼60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr ∼120,000 which was inactive. The latter component also reacted with antisera to both
antithrombin and albumin on immunoblotting. Under reducing conditions, the ∼120,000
Mr component migrated on SDS-PAGE as two distinct bands with Mr ∼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 ∼120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex.
The variant antithrombin was isolated, following reduction and S-carboxy-methylation,
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, North-wick 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.
Keywords
Antithrombin - Albumin complex - Antithrombin variants - Functional antithrombin deficiency
- Familial thrombosis
