Abstract

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. It is poor correlation between FV levels in plasma and the severity of bleeding tendency. In the present study, we identified 5 mutations in the FV gene (F5) in 5 unrelated Japanese patients with reduced plasma FV activities associated with inherited FV deficiency. Their bleeding tendencies varied in severity from asymptomatic to severe. We hypothesized that the severity of bleeding symptoms with severe FV deficiency is correlated with FV levels in platelets, and performed recombinant mutant proteins expression experiments and analyzed of platelet FV. The data concerning 5 patient’s FV levels in plasma and the bleeding symptoms are given in Table. The F5 mutations in 5 Japanese patients were identified by direct sequencing. One of the 5 patients was a compound heterozygote for FV mutations and carried a 5-base pair (bp) deletion in exon 22 (del.ACCCT) and V1813M. The other 4 patients were homozygous for a D68H, N468S, V1813M, and R2174L mutations, respectively. Four mutations except V1813M are newly identified mutations. These mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA, and the wild-type and mutant FV proteins were expressed in HEK293 cells. In the conditioned media, FV specific activities of the FV-D68H, FV-N468S, FV-V1813M, FV-R2174L, and 5bp del. mutants were an approximately 22%, 81%, 28%, 40%, and 19% of wild-type, respectively. On the other hand, analysis of platelet from patient by using RT-PCR showed that platelet F5 mRNA of FV-R2174L and FV-N468S was equal amount to that of normal subjects, although the amount of FV-V1813M platelet F5 mRNA was reduced. Platelet FV protein from patients was analyzed by western blotting and ELISA. Although the amount of platelet FV-R2174L protein was equal to that of normal platelets, platelet FV-V1813M protein was considerably reduced. In addition, the amount of FV-N468S protein in platelet was observed between that of normal subjects and FV-V1813M. These results indicate that the fact that sufficient amounts of FV are stored in platelet is required for local hemostasis. The results of FV-R2174L suggest that both functionality and amount of FV-R2174L in platelet is enough to cope with local bleeding, resulting very mild bleeding tendency. On the basis of present findings, we conclude that the severity of bleeding due to severe FV deficiency is correlated with not only plasma FV level, but also platelet FV.

Five patient’s FV levels in plasma and the bleeding symptoms

Patient No.MutationFV activity (%)FV antigen (%)Bleeding symptoms
D68H asymptomatic 
N468S asymptomatic 
V1813M & 5-bp deletion <1 severe 
V1813M <1 moderate 
R2174L very mild 
Patient No.MutationFV activity (%)FV antigen (%)Bleeding symptoms
D68H asymptomatic 
N468S asymptomatic 
V1813M & 5-bp deletion <1 severe 
V1813M <1 moderate 
R2174L very mild 

Author notes

Disclosure: No relevant conflicts of interest to declare.