Reduced Affinity To VWF Induced By a Novel Mutation At Pro1809Leu (factor VIII-Tenri) Is The Cause Of Mild Hemophilia A Developing Anti-Factor VIII Inhibitor
Authors: Yada Koji , Nogami Keiji , Takeyama Masahiro , Minami Hiroaki , Fay Philip J. , Shima Midori 
Published: October 2019

The development of inhibitory antibodies to factor (F)VIII in patients with mild hemophilia A is a rare, but significant event. We reported the mild hemophilia A associated with Pro(P)1809Leu(L) mutation in FVIII gene (FVIII-Tenri), developing the type II inhibitor (peak 5.6BU/ml), which inhibited allogeneic but not autologous FVIII recognizing the epitope(s) overlapping with that of anti-FVIII monoclonal antibody (mAb) ESH8 in the C2 domain remote from the mutated site in the A3 domain, at the 54th ASH meeting (#52283). However, the haemostatic characteristics of FVIII-Tenri remain unclear. In this study, to elucidate the characteristics of FVIII-Tenri, we prepared and stably expressed a recombinant, B-domainless FVIII mutant, P1809L. FVIII:C of the mutant reconstituted with FVIII-deficient plasma (0.3 nM) was 31.5 IU/dl, whilst that of the wild-type (WT) (0.3 nM) was 88.6 IU/dl, similar to the level of patient’s plasma. The polyclonal IgG immune-purified from this patient’s plasma decreased FVIII:C of WT by ∼60% at the maximum concentration of IgG, whilst any little affected in the mutant. The mutant was activated by thrombin and FXa, showing ∼16- and 7-fold increase in FVIII:C compared to baseline control, respectively, similar to WT. Furthermore, purified FXa generation assays showed that the mutant was any little different in Km for FIXa compared to WT (0.74 ± 0.07 and 0.72 ± 0.13 nM, respectively), supporting that this mutation little affected the association with FIXa. Interestingly, the mutant showed an ∼3-fold weak binding affinity (Kdapp 0.92 ± 0.23nM) to VWF compared to WT (Kdapp 0.33 ± 0.01nM), whilst any little difference was observed in binding affinity to phospholipid between the mutant and WT, in an ELISA. To further investigate the haemostatic mechanism(s) associated with less binding affinity to VWF, the residual FVIII:C of the mutant or WT (0.05-0.25nM) was measured in a one-stage clotting assay, after incubation with several anti-FVIII mAbs including anti-C2 mAbs ESH8 (residues 2248-2285) and ESH4 (2303-2332) with individual epitope overlapping the VWF-binding site(s) in the C2 domain. ESH8 conferred the ∼60% lower inhibition of FVIII:C in the mutant compared to that in WT, whilst ESH4 showed the similar inhibition of FVIII:C between in the mutant and WT. Anti-A2 mAb JR8 showed any little difference in the inhibition between in the mutant and WT. These results demonstrated that the mutation P1809L in the A3 domain did little affect upon activation by thrombin and FXa as well as association with FIXa and phospholipid, but attenuated the binding to VWF through the conformational change in the C2 domain. Taken together with the development of the allogeneic inhibitor associated with this mutation, these results strongly suggested that the conformational change occurred in the C2 domain remote from the mutated site in the A3 domain hampered the binding to VWF, resulting in the haemostatic impairment due to fragility of the mutant FVIII molecule as well as the change of the immunogenicity in the epitopes overlapping with the FVIII-VWF binding site (residue 2248-2285) presented on allogeneic FVIII.

Disclosures:

No relevant conflicts of interest to declare.