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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2002-06-1928.
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Blood, 1 January 2003, Vol. 101, No. 1, pp. 173-177
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Arg2074Cys missense mutation in the C2 domain of factor V causing
moderately severe factor V deficiency: molecular characterization by
expression of the recombinant protein
Stefano Duga,
Maria
Claudia Montefusco,
Rosanna Asselta,
Massimo Malcovati,
Flora Peyvandi,
Elena Santagostino,
Pier Mannuccio Mannucci, and
Maria Luisa Tenchini
From the Department of Biology and Genetics for Medical
Sciences, University of Milan, and the Angelo Bianchi Bonomi Hemophilia
and Thrombosis Center and Fondazione Luigi Villa, Department of
Internal Medicine, University of Milan and Istituto di Ricovero e Cura
a Carattere Scientifico (IRCCS) Maggiore Hospital, Italy.
Factor V (FV) deficiency is a rare bleeding disorder whose genetic
basis has been described in a relatively small number of cases. Among a
total of 12 genetic defects reported in severely or moderately severe
deficient patients, 3 were missense mutations and in no case was the
mechanism underlying the deficiency explored at the molecular
level. In this study, a homozygous missense mutation at cDNA
position 6394 in exon 23 of the FV gene was identified in a 22-year-old
Italian patient. This mutation causes the replacement of arginine 2074 with a cysteine residue (Arg2074Cys) in the C2 domain of the
protein. The effect of the Arg2074Cys mutation on FV secretion,
stability, and activity was investigated. Site-directed mutagenesis of
FV cDNA was used to introduce the identified mutation, and wild-type as
well as mutant FV proteins were expressed by transient transfection in
COS-1 cells. An enzyme immunoassay detected low FV antigen levels both
in the conditioned media of cells expressing the mutant protein and in
cell lysates. Metabolic labeling and pulse-chase experiments confirmed
that the mutation caused an impaired secretion of FV associated with
rapid intracellular degradation. In addition, evaluation of wild-type
and mutant coagulant activity demonstrated that the FV molecules
carrying the Arg2074Cys mutation have reduced activity. These findings,
beside confirming the structural and functional importance of the
arginine 2074 residue, demonstrate that its substitution with a
cysteine impairs both FV secretion and activity.
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