The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

Abstract

SummaryWe report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild‐type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (Kd from 4·4 nmol/l for FVIIaWT to 17·3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (kcat/Km from 2·3 × 107/mol/l s for FVIIaWT to 8·7 × 105/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen‐like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF‐catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the Km value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.

A novel compound heterozygous variant linked to hematuria in a family with hereditary factor VII deficiency

“…Q100R, F328S, and P303T changed the conformation of FVII, weakening its protease activity and diminishing affinity for TF. [32][33][34] In the present study, FVII mRNA levels and protein activity of the R53fs variant were barely detectable as a result of premature termination of translation, which would induce nonsense-mediated mRNA decay. Moreover, the R53fs truncated mutant completely lost the functional domain, including the The two variants p.Gly403Ser and p.Arg53fs in the present study were not reported in related pathogenic studies, and they were found to significantly deplete the protease activity of FVII.…”

“…Although the expression and secretion of this mutant protein was normal, the binding of FVII to TF was impaired, which in turn reduced its proteolytic activity. 32 The present study found that the activity of p.…”

“…There is a site‐directed variant, P303T, in exon 8 of the F7 gene. Although the expression and secretion of this mutant protein was normal, the binding of FVII to TF was impaired, which in turn reduced its proteolytic activity 32 . The present study found that the activity of p.Gly403Ser FVII, also originating from exon 8, significantly decreased.…”

Molecular characterization of iranian patients with inherited coagulation factor VII deficiency

“…Mutation detection in patient 2 revealed the 10824C>A homozygous substitution that causes the P303T defect in FVII protein. This mutation was previously detected in an Iranian patient [ 16 ]. It has been shown that residues P303, L305 and M306 are involved in tissue factor (TF) binding [ 17 ].…”

Factor VII Gene Defects: Review of Functional Studies and Their Clinical Implications

“…Protein structure analysis by X-ray crystallography has displayed that the region that contains this residue plays an important role in the interaction of EGF1 with TF [54] . In the same way, the Q100R mutation may affect the protein expression and cause defective FVIIa/TF complex formation [59] . Peyvandi et al [60] have studied Pro303Thr variant in an Iranian patient with relatively severe hemorrhage.…”