Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan;20(1):69-81.
doi: 10.1111/jth.15552. Epub 2021 Oct 24.

F9 missense mutations impairing factor IX activation are associated with pleiotropic plasma phenotypes

Alessio Branchini  1 , Massimo Morfini  2 , Barbara Lunghi  1 , Donata Belvini  3 , Paolo Radossi  4 , Loredana Bury  5 , Maria Luisa Serino  6 , Paola Giordano  7 , Dorina Cultrera  8 , Angelo Claudio Molinari  9 , Mariasanta Napolitano  10 , Elisabetta Bigagli  11 , Giancarlo Castaman  12 , Mirko Pinotti  1 , Francesco Bernardi  1 ; GePKHIS Study Group of AICE
Collaborators, Affiliations

F9 missense mutations impairing factor IX activation are associated with pleiotropic plasma phenotypes

Alessio Branchini et al. J Thromb Haemost. 2022 Jan.

Abstract

Background: Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia 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 the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK).

Methods: We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an 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 results 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. Pharmacokinetics 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 h, 95% confidence interval 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 = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX.

Conclusions: FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment.

Keywords: factor IX activation; hemophilia B; pharmacogenetics; pharmacokinetics; recombinant proteins.

PubMed Disclaimer

Conflict of interest statement

A.B. reports grants and personal fees from Pfizer, grants from Bayer, and grants and non‐financial support from Grifols, outside the submitted work. M.M. reports personal fees from Kedrion, Novo Nordisk, SOBI, Bayer, Bioverativ, Octapharma, CSL Behring and grants from Pfizer, outside the submitted work. A.C.M. has acted as a paid consultant to Bayer, CSL, Kedrion, Novo Nordisk, Pfizer, Roche, Shire, and Sobi, and as a paid invited speaker for Bayer, CSL, Novo Nordisk, Shire, and Sobi, outside the submitted work. G.C. reports personal fees from Roche, Bayer, Shire, Uniqure, Kedrion, Novo Nordisk, and Werfen; grants and personal fees from CSL Behring and Sobi; and grants from Pfizer, outside the submitted work. M.P. received grants from Novo Nordisk and Pfizer and personal fees from Pfizer, outside the submitted work. F.B. reports grants from Bayer, outside the submitted work, and the financial support of GePKHIS by an unconditioned Investigator Initiated Research grant from Pfizer. The remaining authors state that they have no conflicts of interest to declare.

Figures

FIGURE 1
FIGURE 1
Natural and designed amino acid substitutions at factor IX (FIX) activation sites. A, Schematic representation of human coagulation FIX showing the pre‐ and pro‐peptides (PP), and light and heavy chains (LC and HC, respectively). Hemophilia B (natural) variants affecting the R191 and R226 sites are reported above the FIX scheme (n, patient number in the EAHAD FIX Variant Database https://f9‐db.eahad.org/), with the prevalent HB phenotypes associated with mutations on top. Blue and red variants, patients also available in the GePKHIS study. Designed recombinant variants are indicated below the FIX scheme. Color code: blue, red, and black, natural variants; orange, designed variants. The alignment and conservation of residues in the two activation sites (red arrows) is also shown. B, FIX variants available in the GePKHIS study. Missense (with amino acid changes not involving activation sites) and null (nonsense, splicing, and deletion) variants are reported above and below the FIX scheme, respectively
FIGURE 2
FIGURE 2
Characterization of factor IX (FIX) variants at the activation sites. A, Activity (left panel) and antigen (right panel) levels of the 191 and 226 variants estimated in GePKHIS patients, reported in the international EAHAD FIX variant database (https://f9‐db.eahad.org/) or upon recombinant expression. Reference for plasma or recombinant values were pooled normal plasma or recombinant FIX wild type (rFIX‐WT), as appropriate. B, Comparative expression of equivalent amino acid substitutions at 191 and 226 positions, after transient expression in HEK293 cells. The S411P variant (chymotrypsin numbering S195) was expressed as an additional control for inactive FIX with high antigen values. C, Left panel. Western blotting analysis of the activation profile of natural (red) and designed (orange) recombinant 226 variants in the presence (+) or absence (–) of FXIa. Protein forms, resulting from FIX activation, as well as relative molecular weights (kDa), are indicated on left and right of the blot, respectively. Serial dilutions of rFIX‐WT were loaded as control and reference. rFIX variants with similar FIX antigen levels (226P/L/A/D/E/K, range 70%–100%) were tested after equal dilution (1:10) in phosphate buffered saline (PBS), while those with antigen levels exceeding wild‐type rFIX (226Q/W), prior to being diluted in PBS, were first normalized to ~100% by dilution in medium from untransfected cells. Zym, zymogen FIX; FXIa, activated factor XI. Right panel. FIX antigen and activity levels, as well as activity/antigen ratio, of the designed 226K variant. D, Comparative analysis of activity and antigen levels of FIX variants bearing natural missense changes found in hemophilia B (HB) patients (p, red squares; left panel) and after recombinant expression (r, blue circles; right panel). E, Relation between mean activity levels and activity/antigen ratio of FIX variants bearing natural (blue circles) or designed (orange circles) amino acid substitutions. Inset, Analysis of the relation with the 191K variant included. Results, indicated as % of reference, are reported as mean ± standard error of the mean (panels A, patients bar, and E) or mean ± standard deviation of n = 6 replicates (panels A, recombinant bar, B and C). *P < .05; **P < .01; ***P < .001; ****P < .0001
FIGURE 3
FIGURE 3
Analysis of female carriers of the p.R226W variant. Plasma factor IX (FIX) antigen and activity levels, as well as activity/antigen ratio, estimated in female carriers (n = 3) of the p.R226W variant. Results are reported as mean ± standard error of the mean. *P < .05
FIGURE 4
FIGURE 4
Factor IX (FIX) pharmacokinetic (PK) parameters, FIX antigen, and F9 genotypes. A, Alpha HL parameter, F9 genotypes and FIX antigen levels. Relation between mean antigen levels of expressed FIX missense variants of the hemophilia B (HB) cohort and Alpha HL parameter. Alpha HL of the R191C (n = 3 patients), R226Q (n = 3), and G236D (n = 2) variants is reported as mean of PK values from each patient. B, Alpha HL, Beta HL, and mean residence time (MRT) and F9 genotypes. Comparative plot of selected PK parameters (Alpha HL, upper panel; Beta HL, middle panel; MRT, lower panel) among FIX variants categorized by three mutation groups: null (nonsense, splicing, deletion), missense variants affecting (act. sites) or not (no act. sites) the FIX R191 or R226 activation sites. Circles, activation site variants (blue, R191C; red, R226Q/W); gray squares, nonsense variants; gray triangles, no activation site variants
See this image and copyright information in PMC

References

    1. Monroe DM, Hoffman M. What does it take to make the perfect clot? Arterioscler Thromb Vasc Biol. 2006;26:41‐48. - PubMed
    1. Lesk AM, Fordham WD. Conservation and variability in the structures of serine proteinases of the chymotrypsin family. J Mol Biol. 1996;258:501‐537. - PubMed
    1. Yousef GM, Elliott MB, Kopolovic AD, Serry E, Diamandis EP. Sequence and evolutionary analysis of the human trypsin subfamily of serine peptidases. Biochim Biophys Acta. 2004;1698:77‐86. - PubMed
    1. Di Scipio RG, Kurachi K, Davie EW. Activation of human factor IX (Christmas factor). J Clin Invest. 1978;61:1528‐1538. - PMC - PubMed
    1. Bajaj SP, Rapaport SI, Russell WA. Redetermination of the rate‐limiting step in the activation of factor IX by factor XIa and by factor VIIa/tissue factor. Explanation for different electrophoretic radioactivity profiles obtained on activation of 3H‐ and 125I‐labeled factor IX. Biochemistry. 1983;22:4047‐4053. - PubMed

Publication types