Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain.

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X.

Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity.

Generation of factor VIII cofactor activity requires divalent metal ions such as Ca2+ or Mn2+. Evaluation of cofactor reconstitution from isolated factor VIIIa subunits revealed the presence of a functional Ca2+ binding site within the A1 subunit. Isothermal titration calorimetry demonstrated at least two Ca2+ binding sites of similar affinity (K(d) = 0.

Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex.

The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIII(WT)) and B-domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately 5 nM-20 nM and approximately 100 nM-250 nM in the presence and absence of phospholipid, respectively).

Cofactor activities of factor VIIIa and A2 subunit following cleavage of A1 subunit at Arg336.

Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K(d) = 1.