Missense changes in the catalytic domain of coagulation factor X account for minimal function preventing a perinatal lethal condition

Abstract

Introduction Inherited deficiencies in the coagulation pathway provide diversified models to investigate the molecular bases of perinatal lethality associated with null‐like variants. Differently from X‐linked haemophilias, homozygous/doubly heterozygous null variants in the rare autosomally inherited deficiency of factor X (FX) might be incompatible with perinatal survival. Aim To provide experimental evidence about the null/close‐to‐null FX function. Methods The residual secreted (ELISA) and functional (thrombin generation assays) protein levels associated with the novel nonsense (c.1382G>A; p.Trp461Ter) and missense (c.752T>C; p.Leu251Pro) variants, found in the proposita with life‐threatening symptoms at birth, were characterized through recombinant (r)FX expression. Results The rFX‐461Ter showed very low secretion and undetectable function. Expression and function of the predicted readthrough‐deriving missense variants (rFX‐461Tyr, rFX‐461Gln) were also severely impaired. These unfavourable features, due to nucleotide and protein sequence constraints, precluded functional readthrough over the 461 stop codon. Differently, the poorly secreted rFX‐251Pro variant displayed residual function that was characterized by anti‐TFPI aptamer‐based amplification or selective inhibition of activated FX function by fondaparinux in plasma and found to be reduced by approximately three orders of magnitude. Similarly to the rFX‐251Pro, a group of catalytic domain missense variants cause poorly secreted molecules with modest function in FX‐deficient patients with life‐threatening symptoms. Conclusions Our data, contributing to the knowledge of the very severe FX deficiency forms, support life‐saving requirement of trace FX function, clearly exemplified by the dysfunctional but not completely inactive rFX‐251Pro variant that, albeit with severely reduced function, is compatible with a residual activity ensuring minimal haemostasis and permitting perinatal survival.

Use of plasma‐derived factor X concentrate in neonates and infants with congenital factor X deficiency

“…The second patient is heterozygous for two novel F10 mutations: c.1188_1200del (p.Phe396Leufs*34) and c.1383G>T (p.Trp461Cys), the first of which is predicted to be pathogenic. Although c.1383G>T has not been previously reported, other variants affecting Trp461 have been associated with FXD 16,17 . Both patients were homozygous for predicted benign variants: c.232‐17T>C and c.792C>T. A third patient is presumably homozygous for c.334T>C (p.Cys112Arg) based on sequencing of an affected relative.…”

The Factor VII Variant p.A354V-p.P464Hfs: Clinical versus Intracellular and Biochemical Phenotypes Induced by Chemical Chaperones

“…Briefly, after incubation for 3 min at 37 • C, FXaG was triggered by 50 pM Tissue Factor (Innovin, Dade Behring, Marburg, Germany) and evaluated by adding a specific FXa fluorogenic substrate (Spectrafluor FXa, American Diagnostica, Stamford, CT, USA). FXaG was also evaluated by inhibition of tissue factor pathway inhibitor (TFPI) by anti-TFPI RNA aptamer (5 -GGAAUAUAdCUUGGdCUdCGUUAGGUGdCGUAUAUAidT-3 ), as previously described [30,31].…”

The carboxyl‐terminal region of coagulation serine proteases: A matter of cut and change

“…The features of this di‐residue are maintained up to chymotrypsin, where a tryptophan‐valine sequence is present. The key role of this boundary tryptophan is suggested by its high degree of conservation in the family of serine proteases, 13 as well as by the detrimental impact of natural variants resulting in its removal or substitution (Figure 1, lower panel) 1,2,11,14‐18 …”

“…The key role of this boundary tryptophan is suggested by its high degree of conservation in the family of serine proteases, 13 as well as by the detrimental impact of natural variants resulting in its removal or substitution (Figure 1, lower panel). 1,2,11,[14][15][16][17][18] Despite the separate evolutionary routes between vitamin K-dependent coagulation factors and FXI, 19 an unusual dimeric serine protease containing the so-called apple domains, 20 these proteins share a trypsin-like catalytic domain (Figure 1, upper panel). 13 In this view, the homology degree of the FXI serine protease domain with that of the PC-FVII-FX-FIX group may allow experimental as well as inferred comparisons.…”