Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cellexpressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K d for A2. The second assay determined the K d for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K d and k cat values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K d values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K d for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k cat for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.Factor VIIIa is an essential blood coagulation protein that acts as a cofactor for the serine protease factor IXa in the conversion of factor X to Xa during the propagation phase of coagulation. Defects or deficiencies in factors VIII and IX result in hemophilia A and B, respectively. Factor VIII is synthesized as a multidomain, single chain precursor with a molecular mass of ϳ300 kDa and domain structure A1-A2-B-A3-C1-C2. It circulates primarily as a heterodimer resulting from proteolysis at the B-A3 junction wherein the heavy chain (A1A2B) and light chain (A3C1C2) are associated by metal ion-dependent and independent linkages (see Ref. 1 for review). Factor VIII is activated by limited proteolysis catalyzed by thrombin or factor Xa to the active cofactor, factor VIIIa. These cleavages convert the heterodimer or single chain precursor to the heterotrimer (2, 3) with the heavy chain-derived A1 subunit and light chain-derived A3C1C2 subunit maintaining stable association, whereas the A2 subunit is weakly associated with the A1/A3C1C2 dimer through electrostatic interactions (4, 5). Factor VIIIa can be reconstituted from the isolated A2 subunit and A1/A3C1C2 dimer to regenerate high levels of cofactor activity (3, 5).Association of factors VIIIa and IXa on a phospholipid surface forms the intrinsic factor Xase complex, which increases the cataly...