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


METHODS
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.


CONCLUSIONS
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.
Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan; Department of Medical Research, E-DA Hospital/I-Shou University, Kaohsiung, Taiwan.
Hsiang-Chun
HC
Liu
Department of Laboratory Medicine, E-DA Hospital/I-Shou University, Kaohsiung, Taiwan; Department of Medical Research, E-DA Hospital/I-Shou University, Kaohsiung, Taiwan. Electronic address: ltmei@mail.ncku.edu.tw.
Tsun-Mei
TM
Lin
Department of Pathology, Chang Gung Memorial Hospital -Kaohsiung Medical Center, Kaohsiung, Taiwan.
Hock-Liew
HL
Eng
Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Ying-Ting
YT
Lin
Hemophilia Treatment and Thrombosis Center, Changhua Christian Hospital, Changhua, Taiwan. Electronic address: 111710@cch.org.tw.
Ming-Ching
MC
Shen
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005165:Factor V / Q000235:genetics* / Q000378:metabolism*; D005166:Factor V Deficiency / Q000097:blood / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010375:Pedigree; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000378:metabolism*
D005165:Factor V; D013917:Thrombin
Dissociation assay; Factor V deficiency; His147Arg; Missense mutation; Modeling; prothrombinase assay