The carboxyl-terminal region of coagulation factors: role in biosynthesis and function of FVII and FX

-BACKGROUND- Factor VII (FVII), factor IX (FIX), factor X (FX) and protein C (PC), belonging to the family of coagulation vitamin K-dependent serine proteases, share high sequence and structural homology at the gene and protein level. However, their carboxyl-terminal region displays striking differences in length and aminoacid composition. While this region of FIX and PC has been demonstrated to be essential for efficient biosynthesis and secretion, little is shown for FVII and FX. -AIMS- The main aim was to investigate the carboxyl-terminal region of FVII and FX as determinant of biosynthesis/secretion and/or function. In the study we took advantage of the characterization of i) a natural variant of FVII characterized by a nonsense mutation (R402X) leading to a slightly truncated protein (-4 residues), and ii) natural anti-FVII inhibitory antibodies developed in a patient with an altered carboxyl-terminal region. The study was approached both by assays in patient’s plasma and by expression of the recombinant FVII variants in eukaryotic cells. To address the issue of the role of the carboxyl-terminal region of FX, a panel of progressively truncated FX variants has been expressed and characterized. -MAIN RESULTS AND CONCLUSIONS - The main results of the studies can be summarized as follows. i) Demonstration that the truncated FVII-402X molecule, albeit poorly secreted, possesses an increased specific activity, which explains the association of the R402X nonsense mutation with an asymptomatic phenotype; ii) identification of an anti-FVII inhibitory antibody in a patient homozygous for a frequent FVII frameshift mutation (11125delC) and data supporting the FVII carboxyl-terminal region as the main epitope of this antibody; iii) demonstration that the carboxyl-terminal region of FVII is essential for efficient biosynthesis and secretion, and the presence of an inverse relationship between the extent of the deletion of the carboxy-terminus and secretion levels. The deleted variants however possess a normal specific activity, thus not supporting a functional role for the very last residues of FVII; iv) demonstration that efficient secretion of FX, at variance from its highly homologous FVII, FIX and PC proteins, is not affected by short deletions (up to 21 residues) of the carboxyl-terminal region, which seems to have a functional role. Taken together these data indicate a differential role of the carboxyl-terminal region of FVII and FX, which might have contributed to divergence and evolution of these serine proteases from the common ancestor enzyme. -METHODOLOGICAL APPROACHES- Production and expression of recombinant proteins, ELISA-based and functional assays have been exploited in this study to investigate the expression and the activity of different natural or recombinant variants of coagulation FVII and FX

Comparative Analysis Of Residual Factor VIII Expression from Recurrent F8 Nonsense Mutations Indicates that Localization in the B- domain Favours Readthrough- mediated Protein Output

Background: Nonsense mutations, inserting premature termination codons (PTCs), might undergo, with low frequency (<0.01%), spontaneous suppression (readthrough) with production of full-length proteins upon amino acid insertion at the PTC. This process, dictated by nucleotide/protein sequence features, might have implications for hemophilia A (HA) patients. Aims: To investigate residual factor VIII (FVIII) expression through complementary studies in HA patients’ plasma and exploiting a sensitive in-vitro expression platform. Methods: Detection of plasma FVIII levels (ELISA, aPTT), and expression studies (HEK293 cells) with a highly-sensitive naturally-secreted luciferase (Gaussia, GL) fused to FVIII (FVIII-GL). Results: Plasma samples from HA patients affected by six nonsense mutations (p.R446X, p.R814X, p.K1289X, p.W1726X, p.R1985X, p.R2135X) revealed traces of FVIII. Strikingly, the two B-domain variants (p.R814X, p.K1289X) showed the highest FVIII levels, suggesting a position-dependent effect. Expression studies with the FVIII-GL variants showed that those of the B-domain produced the highest luciferase activity levels, thus supporting in vivo findings. Accordingly, the predicted readthrough-deriving amino acid changes (R446W, R814W, K1289Q/Y, W1726Y, R1985W, R2135W) showed a minor impact for those affecting the B-domain. To verify further our hypothesis, the panel of F8 mutations was rationally expanded to be representative of the majority of patients with nonsense mutations (60%), including the most frequent (50% of patients) in the B-domain. Through our sensitive platform we observed that all F8 nonsense variants led to detectable luciferase activity (0.4-6%). Strikingly, when categorized in two groups (B-domain, n=21; other domains, n=26), secreted luciferase activity of B-domain variants was significantly higher (p<0.0001) as compared with variants located in the other FVIII domains. Conclusions: Our findings for the first time indicate that nonsense mutations in the B-domain, known to tolerate missense changes as those potentially arising from readthrough, are favoured in terms of readthrough-mediated protein output, which might have pathophysiological implications for HA patients

F9 Missense mutations impairing factor ix activation are associated with pleiotropic plasma phenotypes

Background: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in haemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. Objectives: To investigate i) expression of a complete panel of missense mutations at FIX activation sites and ii) contribution of F9 genotypes on the FIX pharmacokinetics (PK). Methods: FIXag and activity assays in plasma and after recombinant expression of FIX variants. Analysis of infused FIX PK parameters in patients (n=30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). Results: The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants result in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. PK analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 hrs, 95% CI 44.3-114.5) in patients (n=7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, p=0.004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. Conclusions: FIXag levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients’ phenotypes and substitutive treatment

Do residual FVIII levels associated with F8 nonsense mutations reduce the inhibitor risk in hemophilia A patients?

Finanziamento - Grifols Martin Villar Haemostasis Award - Clinical Research Awar

Improved intracellular processing of protein variants as a personalized therapeutic approach for Haemophilia

Severe bleeding disorders such as Haemophilia B are mainly caused by missense mutations that may affect protein folding, thus leading to the production of structurally-altered proteins associated with low or very low circulating levels. The research activity was aimed at characterizing the mechanism underlying the defective intracellular biosynthesis (misfolding), premature degradation and/or stress of the endoplasmic reticulum in cellular models of sever Haemophilia B and to evaluate the effects of small molecules or drugs acting as chemical/pharmacological chaperones in restoring the molecular defect. Reference: Pignani S, Todaro A, Ferrarese M, Marchi S, Lombardi S, Balestra D, Pinton P, Bernardi F, Pinotti M, Branchini A. The chaperone-like sodium phenylbutyrate improves factor IX intracellular trafficking and activity impaired by the frequent p.R294Q mutation. J Thromb Haemost. 2018 Oct;16(10):2035-2043

Factor IX variants with superior pharmacokinetics as next-generation therapeutics for Haemophilia B

Finanziamento - Early Career Investigator Award (Bayer Hemophilia Awards Program

Hemophilia A patients with nonsense mutations: residual factor VIII expression levels and development of inhibitory antibodies

Finanziamento - Progetto finanziato da AICE (Associazione Italiana Centri Emofilia