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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2002-06-1928.
Submitted June 28, 2002
Accepted August 6, 2002
R2074C 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*
Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy
IRCCS Maggiore Hospital, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, Department of Internal Medicine University of Milan, Milan, Italy
* Corresponding author; email: marialuisa.tenchini{at}unimi.it.
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 the mechanism underlying the deficiency was 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 (R2074C) in the C2-domain of the protein. The effect of the R2074C 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 R2074C 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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