Characterization of mild coagulation factor VII deficiency: activity and clearance of the Arg315Trp and Arg315Lys variants in the Cys310-Cys329 loop (c170s).
OBJECTIVE
Arginine 315 in factor VII (FVII) belongs to a solvent-exposed loop involved in direct interaction with the co-factor (tissue factor, TF), in transmission of TF-induced effects and potentially in FVIIa inactivation. Natural FVII variants at position 315 provide peculiar models for structure-function studies.


METHODS
We characterized a mild coagulation FVII deficiency associated with reduced FVII activity (26%) and antigen (67%). Mutations were searched by FVII gene sequencing. FVII variants were created by mutagenesis of FVII cDNA and characterized through expression in HEK293 cells followed by functional studies. FVII antigen in media was estimated by immunoassay while FVII activity was assessed by prothrombin-time based and FXa generation assays. FVII variants were injected into mice to investigate their recovery and half-life. One-way ANOVA was used to test statistical significance.


RESULTS
The patient was double heterozygous for a novel R315W mutation and for the R304Q substitution (FVII Padua) previously demonstrated to impair TF binding. The recombinant 315W-FVII was normally expressed in medium but showed a markedly reduced coagulant function (52%) and activity towards factor X (FX) in plasma (34%). Moreover, the 315W-FVII showed significantly decreased recovery of the protein (20%) and a slightly shorter half-life (8.6 min) as compared to wt-FVII (50% and 10.7 min). We also studied the conservative R315K change that was responsible for low recovery (20%) and a decreased half-life (7 min) of a FVII variant with virtually normal FVII antigen and activity levels.


CONCLUSIONS
These findings suggest a dual role of R315 for FVII function and clearance, and indicate that substitutions at this position have appreciable effects on human FVII biology, compatible with residual FVII function and thus with mild FVII deficiency.
Furlan Freguia
Christian
C
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy.
Toso
Raffaella
R
Pollak
Eleanor S
ES
Arruda
Valder R
VR
Pinotti
Mirko
M
Bernardi
Francesco
F
D000328:Adult; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005167:Factor VII / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005168:Factor VII Deficiency / Q000235:genetics*; D005170:Factor X / Q000378:metabolism; D005260:Female; D006207:Half-Life; D006579:Heterozygote; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics / Q000502:physiology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D003062:Codon; D018076:DNA, Complementary; C080432:Factor VII Padua; D011993:Recombinant Fusion Proteins; D005167:Factor VII; D005170:Factor X