Functional characterization of a novel missense mutation, His147Arg, in A1 domain of FV protein causing type II deficiency

摘要

Introduction Congenital factor V (FV) deficiency is a rare inherited disorder. Three compound heterozygous missense mutations, Asp68His, His147Arg, and Arg2074Cys, were observed in a Taiwanese patient with moderately severe FV deficiency. Method The novel His147Arg mutation in the A1 domain was investigated by protein modeling, followed by in vitro expression studies in COS-1 cells, to elucidate the molecular pathology associated with FV deficiency. Results The His147Arg mutation was associated with normal antigen levels, both in cell lysates and conditioned media, whereas FV activity was significantly reduced to 63.5 ± 17.0%. These observations correspond to a type II FV deficiency mutation. Protein modeling by short-duration molecular dynamics (MD) simulation showed that the His147Arg mutation was associated with a conformational change, which could disrupt the stability of FVa by interfering with His1817 coordination of the copper ion. In functional activation assays, the His147Arg mutation did not affect FV protein activation by thrombin; however, reduced cofactor activity of the FVa protein, due to an increased rate of dissociation of heavy and light chains, was observed. Conclusion Our results show that the His147Arg mutation in the A1 domain of FV does not impair synthesis or procoagulant activity. Instead, the His147Arg mutation appears to disrupt the stability of FVa, providing a potential explanation for the functional deficiency.