The chaperone-like sodium phenylbutyrate improves factor IX intracellular trafficking and activity impaired by the frequent p.R294Q mutation.

Essentials Missense mutations often impair protein folding, and thus intracellular trafficking and secretion. Cellular models of severe type I hemophilia B were challenged with chaperone-like compounds. Sodium phenylbutyrate improved intracellular trafficking and secretion of the frequent p.R294Q. The increased coagulant activity levels (∼3%) of p.R294Q would ameliorate the bleeding phenotype. SUMMARY: Background Missense mutations often impair protein folding and intracellular processing, which can be improved by small compounds with chaperone-like activity. However, little has been done in coagulopathies, where even modest increases of functional levels could have therapeutic implications. Objectives To rescue the expression of factor IX (FIX) variants affected by missense mutations associated with type I hemophilia B (HB) through chaperone-like compounds. Methods Expression studies of recombinant (r)FIX variants and evaluation of secreted levels (ELISA), intracellular trafficking (immunofluorescence) and activity (coagulant assays) before and after treatment of cells with chaperone-like compounds. Results As a model we chose the most frequent HB mutation (p.R294Q, ~100 patients), compared with other recurrent mutations associated with severe/moderate type I HB. Immunofluorescence studies revealed retention of rFIX variants in the endoplasmic reticulum and negligible localization in the Golgi, thus indicating impaired intracellular trafficking. Consistently, and in agreement with coagulation phenotypes in patients, all missense mutations resulted in impaired secretion (< 1% wild-type rFIX). Sodium phenylbutyrate (NaPBA) quantitatively improved trafficking to the Golgi and dose dependently promoted secretion (from 0.3 ± 0.1% to 1.5 ± 0.3%) only of the rFIX-294Q variant. Noticeably, this variant displayed a specific coagulant activity that was higher (~2.0 fold) than that of wild-type rFIX in all treatment conditions. Importantly, coagulant activity was concurrently increased to levels (3.0 ± 0.9%) that, if achieved in patients, would ameliorate the bleeding phenotype. Conclusions Altogether, our data detail molecular mechanisms underlying type I HB and candidate NaPBA as affordable 'personalized' therapeutics for patients affected by the highly frequent p.R294Q mutation, and with reduced access to substitutive therapy.

Differential functional readthrough over homozygous nonsense mutations contributes to the bleeding phenotype in coagulation factor VII deficiency.

Essentials Potentially null homozygous Factor(F)7 nonsense mutations are associated to variable bleeding symptoms. Readthrough of p.Ser112X (life-threatening) and p.Cys132X (moderate) stop codons was investigated. Readthrough-mediated insertion of wild-type or tolerated residues produce functional proteins. Functional readthrough over homozygous F7 nonsense mutations contributes to the bleeding phenotype. SUMMARY: Background Whereas the rare homozygous nonsense mutations causing factor (F)VII deficiency may predict null conditions that are almost completely incompatible with life, they are associated with appreciable differences in hemorrhagic symptoms. The misrecognition of premature stop codons (readthrough) may account for variable levels of functional full-length proteins. Objectives To experimentally evaluate the basal and drug-induced levels of FVII resulting from the homozygous p.Cys132X and p.Ser112X nonsense mutations that are associated with moderate (132X) or life-threatening (112X) symptoms, and that are predicted to undergo readthrough with (132X) or without (112X) production of wild-type FVII. Methods We transiently expressed recombinant FVII (rFVII) nonsense and missense variants in human embryonic kidney 293 cells, and evaluated secreted FVII protein and functional levels by ELISA, activated FX generation, and coagulation assays. Results The levels of functional FVII produced by p.Cys132X and p.Ser112X mutants (rFVII-132X, 1.1% ± 0.2% of wild-type rFVII; rFVII-112X, 0.5% ± 0.1% of wild-type rFVII) were compatible with the occurrence of spontaneous readthrough, which was magnified by the addition of G418 - up to 12% of the wild-type value for the rFVII-132X nonsense variant. The predicted missense variants arising from readthrough abolished (rFVII-132Trp/Arg) or reduced (rFVII-112Trp/Cys/Arg, 22-45% of wild-type levels) secretion and function. These data suggest that the appreciable rescue of p.Cys132X function was driven by reinsertion of the wild-type residue, whereas the minimal p.Ser112X function was explained by missense changes permitting FVII secretion and function. Conclusions The extent of functional readthrough might explain differences in the bleeding phenotype of patients homozygous for F7 nonsense mutations, and prevent null conditions even for the most readthrough-unfavorable mutations.

Activated factor VII-antithrombin complex predicts mortality in patients with stable coronary artery disease: a cohort study.

BACKGROUND: Plasma concentration of activated factor VII (FVIIa)-antithrombin (AT) complex has been proposed as an indicator of intravascular exposure of tissue factor. OBJECTIVES: The aims of this observational study were to evaluate (i) FVIIa-AT plasma concentration in subjects with or without coronary artery disease (CAD) and (ii) its association with mortality in a prospective cohort of patients with CAD. METHODS: FVIIa-AT levels were measured by elisa in 686 subjects with (n = 546) or without (n = 140) angiographically proven CAD. Subjects with acute coronary syndromes and those taking anticoagulant drugs at the time of enrollment were excluded. CAD patients were followed for total and cardiovascular mortality. RESULTS: There was no difference in FVIIa-AT levels between CAD (84.8 with 95% confidence interval [CI] 80.6-88.2 pmol L(-1) ) and CAD-free subjects (83.9 with 95% CI 76.7-92.8 pmol L(-1) ). Within the CAD population, during a 64-month median follow-up, patients with FVIIa-AT levels higher than the median value at baseline (≥ 79 pmol L(-1) ) had a two-fold greater risk of both total and cardiovascular mortality. Results were confirmed after adjustment for sex, age, the other predictors of mortality (hazard ratio for total mortality: 2.05 with 95% CI 1.22-3.45, hazard ratio for cardiovascular mortality 1.94 with 95% CI 1.01-3.73, with a slight improvement of C-statistic over traditional risk factors), FVIIa levels, drug therapy at discharge, and even patients using all the usual medications for CAD treatment. High FVIIa-AT levels also correlated with increased thrombin generation. CONCLUSIONS: This preliminary study suggests that plasma concentration of FVIIa-AT is a thrombophilic marker of total and cardiovascular mortality risk in patients with clinically stable CAD.

The carboxyl-terminal region is NOT essential for secreted and functional levels of coagulation factor X.

BACKGROUND: The homologous coagulation factor X (FX), VII (FVII), IX (FIX) and protein C (PC) display striking differences in the carboxyl-terminus, with that of FX being the most extended. This region is essential for FVII, FIX and PC secretion. OBJECTIVES: To provide experimental evidence for the role of the FX carboxyl-terminus. METHODS: Recombinant FX (rFX) variants were expressed in multiple eukaryotic cell systems. Protein and activity levels were evaluated by ELISA, coagulant and amidolytic assays. RESULTS AND DISCUSSION: Expression of a panel of progressively truncated rFX variants in HEK293 cells revealed that the deletion of up to 21 residues in the carboxyl-terminus did not significantly affect secreted protein levels, as confirmed in HepG2 and BHK21 cells. In contrast, chimeric rFX-FVII variants with swapped terminal residues showed severely reduced levels. The truncated rFX variants revealed normal amidolytic activity, suggesting an intact active site. Intriguingly, these variants, which included that resembling the activated FXß form once cleaved, also displayed remarkable or normal pro-coagulant capacity in PT- and aPTT-based assays. This supports the hypothesis that subjects with nonsense mutations in the FX carboxyl-terminus, so far never identified, would be asymptomatic. CONCLUSIONS: For the first time we demonstrate that the FX carboxyl-terminal region downstream of residue K467 is not essential for secretion and provides a modest contribution to pro-coagulant properties. These findings, which might suggest an involvement of the carboxyl-terminal region in the divergence of the homologous FX, FVII, FIX and PC, help to interpret the mutational pattern of FX deficiency.

Asymmetric processing of mutant factor X Arg386Cys reveals differences between intrinsic and extrinsic pathway activation.

Alterations in coagulation factor X (FX) activation, mediated by the extrinsic VIIa/tissue factor (FVIIa/TF) or the intrinsic factor IXa/factor VIIIa (FIXa/FVIIIa) complexes, can result in hemorrhagic/prothrombotic tendencies. However, the molecular determinants involved in substrate recognition by these enzymes are poorly defined. Here, we investigated the role of arginine 386 (chymotrypsin numbering c202), a surface-exposed residue on the FX catalytic domain. The naturally occurring FX386Cys mutant and FX386Ala variant were characterized. Despite the unpaired cysteine, recombinant (r)FX386Cys was efficiently secreted (88.6±21.3% of rFXwt) and possessed normal clearance in mice. rFX386Cys was also normally activated by FVIIa/TF and displayed intact amidolytic activity. In contrast, rFX386Cys activation by the FIXa/FVIIIa complex was 4.5-fold reduced, which was driven by a decrease in the kcat (1.6∗10(-4) s(-1) vs 5.8∗10(-4) s(-1), rFXwt). The virtually unaltered Km (70.6 nM vs 55.6nM, rFXwt) suggested no major alterations in the FX substrate exosite. Functional assays in plasma supplemented with rFX386Cys indicated a remarkable reduction in the thrombin generation rate and thus in coagulation efficiency. Consistently, the rFX386Ala variant displayed similar biochemical features suggesting that global changes at position 386 impact the intrinsic pathway activation. These data indicate that the FXArg386 is involved in FIXa/FVIIIa-mediated FX activation and help in elucidating the bleeding tendency associated with the FX386Cys in a rare FX deficiency case. Taking advantage of the unpaired cysteine, the rFX386Cys mutant may be efficiently targeted by thiol-specific ligands and represent a valuable tool to study FX structure-function relationships both in vitro and in vivo.