The Characteristics and Mechanism(s) of Anti-Factor VIII Antibody Developed in Mild Hemophilia A Associated with a Novel Mutation Pro1809Leu.
Authors: Yada Koji , Nogami Keiji , Ogiwara Kenichi , Shima Midori 
Published: October 2019

Abstract
Abstract 2215
The development of inhibitory antibodies to factor (F)VIII in patients with mild hemophilia A is a rare, but significant event. Some reports had described the cases in mild/moderate hemophilia A developing allogeneic but not autogenic inhibitors associated with missense mutations in FVIII including Arg593Cys within the A2 domain (Fijnvandraat et al. Blood 1997) and Arg2150His within the C1 domain (Peerlinck et al. Blood 1999). However, the characteristics of the inhibitors developed in mild/moderate hemophilia A have is poorly understood. We had a patient with mild hemophilia A (FVIII:C 10–15 IU/dl) associated with a Pro1809Leu mutation in the A3 domain of FVIII, exhibiting significant residual FVIII activity (FVIII:C ∼10 IU/dl), despite the development of inhibitor (peak 5.2 BU/ml) after repetitive exposure to FVIII concentrates in replacement therapy for cerebellar hemorrhage. The characteristics and inhibitory mechanism(s) of polyclonal IgG antibody immunopurified from patient's plasmas were examined. FVIII:C in plasma from a healthy volunteer or self plasma from the patient before the development of inhibitor was measured after reaction with patient IgG in a one-stage clotting assay. FVIII:C in normal plasma was decreased by ∼60% at the maximum concentration of patient IgG, whilst was any little affected in self plasma. To directly confirm the allogeneic (wild-type) inhibition by patient IgG, recombinant FVIII (and plasma-derived FVIII) and patient IgG was preincubated, followed by the measurement of FVIII:C. Inactivation of FVIII:C was incomplete by the IgG (maximum by ∼60%), similar to results described above, supporting that the patient IgG behaving as type II inhibitor inhibited allogeneic but not autologous FVIII. The epitope(s) of patient IgG on FVIII was analyzed by SDS-PAGE and Western blot. This reacted with the C2 domain alone, but not with the A2 or A3. To further localize the recognizing epitope, a competitive binding assay between patient IgG with a murine anti-C2 monoclonal antibody ESH8 (type II, epitope 2248–2285) or ESH4 (type I, 2303–2332) on FVIII binding was performed in an ELISA. The patient IgG competitively blocked the FVIII binding of ESH8 by ∼75%, whilst little competed that of ESH4. Furthermore, this IgG any little inhibited the FVIII binding to both VWF and PL, whilst significantly diminished (by ∼60%) the rate of cleavage at Arg1689 in the light chain (LCh) of FVIII by thrombin in a dose-dependent fashion, measured by densitometry following to SDS-PAGE and Western blot. These findings were consistent with the inhibitory mechanism by type II pattern described by our recent report (Matsumoto, Thromb Haemost 2011). We conclude that the patient inhibitor is unique in that they clearly distinguish wild-type from self, mutated FVIII (Pro1809Leu), and behaves as type II inhibitor through disturbance against the cleavage of LCh by thrombin. It is of interest that recognizing epitope(s) within the C2 domain is remote from the mutated site Pro1809 in the A3 domain. A novel mutation Pro1809Leu, not enrolled in the HAMSTeRs database, might alter the conformation of FVIII molecule, resulting in a change of the C2 immunogenicity, while the missense mutation at Pro1809, located within the crucial residues for FIXa binding in the A3 domain (residues 1804–1818) on the tenase complex, would yield a mild phenotype by impairment of interaction with FIXa.

Disclosures:
No relevant conflicts of interest to declare.