Abstract
The link between factor (F)VIII and FX is essential for optimum activity of the tenase
complex. The interactive site(s) in FVIII for FX remains to be completely clarified,
however. We investigated the FVIII A2 domain-FX association that was speculated from
inhibitory mechanism(s) by an anti-A2 autoantibody. SDS-PAGE demonstrated that the
purified inhibitor IgG recognizing residues 373–562 blocked FXa cleavage at Arg372
in FVIII, and surface-plasmon resonance (SPR)-based assays showed that intact A2 subunit
directly bound to FX (K
d; 63 nM). The FVIII structure model indicated possible FX-binding site(s) in residues
400–429 in A2. One peptide corresponding to residues 400–409 competitively inhibited
both the A2–FX binding and FVIIIa/FIXa-dependent FXa generation. Covalent cross-linking
was observed between this peptide and FX following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide)
using SDS-PAGE. K408 and S409 were not evident in N-terminal sequence analysis of
the cross-linked product, suggesting that two residues participated in cross-link
formation. SPR-based assays using recombinant FVIII mutants with one or both residues
substituted to alanine demonstrated that K408A and K408A/S409A had approximately fourfold
high K
d values of wild-type (WT-)FVIII. FXa cleavages at Arg372 in both mutants were significantly
delayed, suggesting a contribution of K408 for FXa cleavage at Arg372. Furthermore,
FXa generation assays with these mutants demonstrated that the K
m values were 1.4- to 1.7-fold greater, and overall catalytic efficiency (k
cat/K
m) was 0.49- to 0.89-fold lower than with WT-FVIII, suggesting a significant contribution
of K408 for FVIII–FX interaction in tenase assembly. We concluded that the K408 in
the A2 domain provided an interactive-site for FX.
Keywords
factor VIII - A2 domain - factor X - binding site - tenase complex
