The A2 subunit of factor VIIIa modulates the active site of factor IXa

Factor VIIIa, the protein cofactor for factor IXa, is comprised of A1, A2, and A3-C1-C2 subunits. Isolated subunits of factor VIIIa were examined for their ability to accelerate the factor IXa-catalyzed activation of factor X. The A2 subunit enhanced the kcat for this conversion by 100-fold whereas the Km for factor X was unaffected. The apparent Kd for the interaction of A2 subunit with factor IXa was approximately 300 nM. Similar results were obtained using purified A2 expressed as the isolated domain in Chinese hamster ovary cells, although this material was less stable than the factor VIIIa-derived material. Isolated A1 and A3-C1-C2 subunits showed no effect on the rate of factor X conversion. A2 subunit increased the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa (Deltar = 0.015) and markedly increased anisotropy in the presence of factor X (Deltar = 0.057), suggesting that it contributes to the orientation of the factor IXa active site and its relation to substrate. A synthetic peptide to A2 residues 558-565 inhibited the A2-dependent enhancement of factor X activation with an IC50 = 40 microM, a value similar to its Ki for inhibition of the intrinsic factor Xase (105 microM). These results indicate that the isolated A2 subunit modulates the active site of factor IXa and identifies a functional role for this subunit in factor VIIIa.