Factor VIII A2 Domain Contains a Binding Site for Factor X
Authors: Takeyama Masahiro , Nogami Keiji , Matsumoto Tomoko , Shima Midori 
Published: October 2019

Abstract
Acquired hemophilia A (AHA) is a rare hemorrhagic disease in which autoantibodies against coagulation factor (F) VIII impair the coagulation system. The inhibitors developed in AHA are polyclonal autoantibodies and the majority of FVIII inhibitors bind to the A2, A3, or C2 domains. Depending on the location of the epitope, different mechanisms of action for the anti-FVIII antibodies have been reported. Anti-A3 antibodies neutralize the procoagulant activity of FVIII by preventing its interaction with FIXa. Anti-C2 antibodies inhibit the binding of FVIII to phospholipid membrane and/or von Willebrand factor, whereas A2 and A3 inhibitors block the binding of FVIII to FIXa and FX, respectively, and obstruct the formation of the Xase complex. We have a case of AHA whose inhibitor recognizes only A2 domain and attempted several approaches to determine the mechanism of neutralizing FVIII. Thrombin and plasmin generation assay using patient’s plasma showed that the thrombin and plasmin generation in this AHA patient were decreased compared with that in congenital severe hemophilia A patient. Furthermore, FX generation (Coatest) in this AHA was also decreased compared with that in congenital severe hemophilia A patient (p<0.05). These results indicated that this inhibitor impaired the generation of Xase complex and might cause the severe bleeding disorder in this patient. The IgG subclass of inhibitor in our case was IgG1 and IgG4. Western blotting analysis using FVIIIa revealed that the inhibitor IgG recognized only A2 domain. Furthermore, western blotting analysis using FVIII A2 fragment, digested by activated protein C, showed that the inhibitor IgG bound to FVIII A2N (residue 372-562) fragment. It is known that FVIII A2 domain contains FIXa and thrombin binding sites. Western blotting analysis revealed that the inhibitor IgG inhibited Arg336 cleavage in FVIIIa by FIXa and Arg372 cleavage in FVIII by thrombin. However, the FXa-catalyzed cleavage at Arg372 in FVIII was inhibited by this inhibitor IgG. ELISA-based assay showed that the inhibitor IgG inhibited FX binding to FVIII A2. These results suggest that FX(a) binds to FVIII A2 domain. Therefore, to determine the direct binding of FX and FVIII A2 domain, ELISA-based assay was employed to assess this interaction. ELISA-based assay showed that FVIII A2 fragment bound FX in a dose-dependent manner with moderate affinity (Kd = 338 nM). FX inhibited FVIII A2 fragment binding to immobilized FX up to 70% with an inhibition constant (Ki = 254 nM) similar to the affinity constant. It is known that the residue 484-509 in the A2 domain interacts with FIXa. We hypothesized that FX binding site in the A2 domain might be in the opposite side of FIXa binding site in the A2 domain. According to the 3-D model of FVIII molecule, we prepared synthetic peptides corresponding to FVIII A2 residues 400-409, 409-419, and 420-429. To determine the specificity of these sequences for FX interaction, we examined the effects of these peptides on FVIII A2 binding to FX using ELISA-based assay. The 400-409 peptide inhibited the A2 and FX interaction up to 70%. In contrast, the 410-419 and the 420-429 peptides inhibited the interaction up to 30%. Covalent cross-linking was observed between the 400-409 peptide and FX following reaction with EDC using SDS-PAGE. These results indicate that FVIII A2 domain contains the binding site for FX(a), and the 400-409 region in the FVIII A2 domain contributes to a unique FX(a)-interactive site.

Disclosures
No relevant conflicts of interest to declare.