A Cluster of Basic Residues in the Factor VIIIa A2 Subunit Function in Substrate Binding and Product Release during the Activation of Factor X by Factor Xase.

Factor VIIIa serves as a cofactor for factor IXa in the membrane-dependent conversion of factor X to Xa. Factor VIIIa is a non-covalent trimer of subunits designated A1, A2 and A3-C1-C2. The A2 subunit of factor VIIIa is essential for cofactor activity and extensive evidence suggests it forms an extended interface with the protease domain of factor IXa. A2 residues 484–510 appear important to factor Xase activity inasmuch as this region is a hotspot for inhibitor antibodies that block cofactor function. In an earlier study we showed that individual substitutions of the basic residues Arg489, Arg490, and Lys493 within this segment with alanine yielded little if any effect on activity whereas a mutant where all three residues were replaced showed a significant reduction in the rate for cofactor-dependent conversion of factor X (Jenkins et al., J Thromb Haemost. 2004), suggesting a role for this positive charge density in catalysis. To gain further insights into mechanisms responsible for this effect, we expressed and purified the wild type and the triple mutant (489-3A) as isolated A2 domains in insect cells using a baculovirus expression system. Factor VIIIa reconstituted from purified A1/A3-C1-C2 dimer and the wild type baculovirus (b) A2 yielded cofactor activity that was indistinguishable from that of factor VIIIa reconstituted from BHK cell-derived recombinant factor VIIIa subunits, as judged by factor Xa generation assays. Both wild type and mutant bA2 forms showed similar affinities in forming factor Xase in the presence of A1/A3-C1-C2 dimer, factor IXa and phospholipid vesicles. On the other hand, factor Xase reconstituted from the 489-3A bA2 exhibited an ~25-fold reduced kcat compared with the wild type control. Interestingly, the mutation reduced the Km for substrate factor X by ~10-fold, suggesting that the elimination of the positive charge density enhanced the association of the substrate with factor Xase. However, this enhancement in substrate association was offset by a slow catalytic mechanism that may include turnover and/or release of product. To examine the latter parameter, an active site-modified factor Xa, EGR-factor Xa, was evaluated for its interactions with factor Xase. EGR-factor Xa competitively inhibited substrate factor X binding to factor Xase with a Ki value that was ~100-fold greater for Xase containing the wild type A2 compared with the value for Xase with the mutant A2. This result suggested the mutant A2-containing factor Xase was defective in release of the macromolecular product. In contrast, this mutation in A2 showed little if any effect on the amidolytic activity of factor Xase as judged using a small chromogenic substrate. Overall, these results suggest that this basic cluster within the A2 subunit functions in both macromolecular substrate binding and product release. We speculate that the latter mechanism derives from repulsive forces that are decreased in the 489-3A A2 mutant.