Natural and engineered carboxy-terminal variants: decreased secretion and gain-of-function result in asymptomatic coagulation factor VII deficiency

Abstract

We report 2 asymptomatic homozygotes for the nonsense p.R462X mutation affecting the carboxy-terminus of coagulation factor VII (FVII, 466 aminoacids). FVII levels of 3-5% and 2.7 ± 0.4% were found in prothrombin time-based and activated factor X (FXa) generation assays with human thromboplastins. Noticeably, FVII antigen levels were barely detectable (0.7 ± 0.2%) which suggested a gain-of-function effect. This effect was more pronounced with bovine thromboplastin (4.8 ± 0.9%) and disappeared with rabbit thromboplastin (0.7 ± 0.2%). This suggests that the mutation influences tissue factor/FVII interactions. Whereas the recombinant rFVII-462X variant confirmed an increase in specific activity (~400%), a panel of nonsense (p.P466X, p.F465X, p.P464X, p.A463X) and missense (p.R462A, p.R462Q, p.R462W) mutations of the FVII carboxy-terminus resulted in reduced secretion but normal specific activity. These data provide evidence for counteracting pleiotropic effects of the p.R462X mutation, which explains the asymptomatic FVII deficiency, and contributes to our understanding of the role of the highly variable carboxy-terminus of coagulation serine proteases.

Figure 1.

Studies in plasma. (A) Thrombin generation curves in plasma from PFVII-R462X (closed squares) and PFVII-A354V-P464fs† (closed triangles). Serial dilutions of pooled normal plasma (% of PNP) in FVII-deficient plasma have been used as reference and are reported above curves (open squares). (Inset) Relationship between lag time and percentage of PNP. Lag times obtained in plasma from PFVII-R462X (closed squares) and PFVII-A354V-P464fs† (closed triangles) are reported within the reference curve. (B) FXa generation activity (expressed as Rfu/sec) in plasma from PFVII-R462X (closed squares) and PFVII-A354V-P464fs† (closed triangles), and in serial dilution (%) of PNP in FVII-deficient plasma. Results are expressed as % of PNP and are reported as mean ± standard deviation from 3 independent experiments. (C) FVII coagulant (white bar) and FXa generation (gray bars) activity as well as FVII antigen (black bar) levels measured in plasma from PFVII-R462X. Results are expressed as % of PNP and are reported as mean ± standard deviation from 3 independent experiments. Hum_1 and Hum_2 are referred to RecombiPlasTin 2G and Innovin, respectively. (Inset) Western blotting analysis of FVII molecules in plasma from PFVII-R462X (2 independent samples) and PFVII-A354V-P464fs†. Diluted PNP (1%) and plasma-derived FVII (pdFVII, 50 ng/mL) have been used as controls. The Supersignal® West Femto reagent (Thermo Scientific, Rockford, IL, USA) was exploited for detection. The films were exposed overnight.

Figure 2.

Expression studies. (A) Schematic representation of the expression vector with the mutations inserted in the human FVII cDNA. CMV: cytomegalovirus; PP: prepro-leader sequence; GLA: γ-carboxyglutamic rich domain; EGF1 and EGF2: epidermal growth factor like domains 1 and 2; AD: activation domain. (B) FXa generation activity (Rfu/sec) in FVII deficient plasma in relation to FVII antigen (ng/mL) levels of rFVII-wt (open squares) and rFVII-462X (closed squares). (Inset) Western blotting analysis of recombinant FVII molecules in conditioned media. The presence of the rFVII-462X has been assessed before (−) and upon concentration (+). rFVII-wt in medium was loaded at the concentration of 40 ng/mL. (C) Antigen (striped bars) and FXa generation activity (black bars) levels of the rFVII-wt and of the nonsense (rFVII-462X, rFVII-463X, rFVII-464X, rFVII-465X, rFVII-466X) and missense (rFVII-462A, rFVII-462Q, rFVII-462W) variants in conditioned medium. Results are expressed as % of rFVII-wt and are reported as mean ± standard deviation from 3 independent experiments.