Antithrombin Sheffield: amino acid substitution at the reactive site (Arg393 to His) causing thrombosis.

A Sheffield family with a predisposition towards thrombosis has been shown to have a functional abnormality of antithrombin. The abnormality was detected as reduced heparin cofactor activity, with normal antigenic levels of antithrombin. Crossed immunoelectrophoresis performed in the absence and presence of heparin was normal. The antithrombin was isolated by heparin Sepharose affinity chromatography. It had normal mobility on SDS polyacrylamide gel electrophoresis. However, the second order rate constant of inhibition of thrombin was about half that of normal, and this was compatible with a heterozygous abnormality involving the reactive site. The antithrombin was further purified by chromatography on thrombin-Sepharose (to remove the normal component), reduced, S-carboxymethylated and fragmented with cyanogen bromide. A pool containing the reactive site region was digested with trypsin and the molecular size of peptides generated determined by fast atom bombardment mass spectrometry. The two peptides adjacent to the Arg393-Ser394 bond of mass 2290 and 700 were almost absent from the mass spectrum, but an additional peptide of mass 2952 was present. Subdigestion with V8 protease reduced the mass of this peptide to 1748. These peptides generated by trypsin and V8 protease were almost identical to those obtained when another variant, antithrombin Glasgow, was treated in the same way (Erdjument et al, 1988). It is concluded that the molecular abnormality of antithrombin Sheffield is identical to that of antithrombin Glasgow, Arg393 to His.