Abstract

The intrinsic factor Xase complex, comprised of the cofactor, factor VIIIa and serine proteinase, factor IXa assembled on a phospholipid membrane, catalyzes the conversion of factor X to factor Xa. Factor VIIIa, a heterotrimer comprised of A1, A2 and A3C1C2 subunits, is labile due to the weak affinity association of the A2 subunit with the A1/A3C1C2 dimer. However, this interaction is partially stabilized by association of factor VIIIa within the Xase complex and likely results from protein-protein interactions between residues in the A2 subunit and protease domain of factor IXa. One region of A2 implicated in direct interaction with factor IXa is the 558-loop based upon peptide inhibition studies, molecular modeling and evaluation of point mutations in the Hemophilia A database. In order to gain insights into the contributions of individual residues within this region to inter-molecular affinity and activity, we prepared a series of point mutations in the 558-loop where residues were individually replaced with Ala. These mutants included V559A, D560A, R562A, G563A, N564A and D569A, and were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90% as judged by SDS-PAGE. Affinity parameters were determined by two reconstitution assays using purified components. In one assay, factor VIIIa was reconstituted from varying concentrations of the isolated A2 form and a fixed level of A1/A3C1C2 dimer, and subsequent activity generated used to determine the Kd for the inter-factor VIIIa subunit interaction. In a second assay, factor Xase was reconstituted from varying concentrations of the A2 form and fixed concentrations of A1/A3C1C2 dimer, factor IXa and phospholipid vesicles. Activity generated from the reconstituted factor Xase was used to determine and inter-molecular Kd value for A2. Evaluation of wild type A2 in the two assays showed a marked reduction in the Kd values for factor Xase compared with factor VIIIa (Table) consistent with the stabilizing effect of assembly of factor VIIIa in the enzyme complex. Kd values obtained for the A2 variants in the factor VIIIa reconstitution assay yielded values within 2-fold of the wild type value suggesting minimal effects of these residues on the association of A2 with the A1/A3C1C2 dimer. However, we observed marked increases in the Kd parameter for Xase reconstitutions for A2 variants V559A (∼30-fold), D560A (∼9-fold) and R562A (∼5-fold), suggesting these residues directly participate in the A2-factor IXa interface. Affinity parameters for the G563A and D569A variants could not be determined because of low levels of reconstituted activities, although saturating levels of the V559A and D560A A2 variants showed significant reductions in activity following reconstitution of factor VIIIa or Xase, suggesting an important role for these residues in contributing to cofactor function. Results from these studies identify critical residues within the 558-loop that likely contribute to stabilizing the association of the A2 subunit within factor Xase as well as residues that impact and/or are required for cofactor activity.

Affinity and activity parameters for A2 in reconstituting factor (F) Xase and VIIIa

A2 variantFXaseFVIIIaFXaseFVIIIa
 Kd for A2 (nM) Kd for A2 (nM) Vmax(/min) Vmax(/min) 
WT 15.9 ± 1.2 102 ± 7 155 ± 3 96 ± 2 
V559A 476 ± 150 187 ± 26 35.0 ± 7.6 16.7 ± 1.2 
D560A 135 ± 7 72 ± 8 6.7 ± 0.2 7.8 ± 0.3 
R562A 81 ± 12 111 ± 13 164 ± 10 73 ± 3 
N564A 50 ± 6 61 ± 11 118 ± 6 198 ± 14 
A2 variantFXaseFVIIIaFXaseFVIIIa
 Kd for A2 (nM) Kd for A2 (nM) Vmax(/min) Vmax(/min) 
WT 15.9 ± 1.2 102 ± 7 155 ± 3 96 ± 2 
V559A 476 ± 150 187 ± 26 35.0 ± 7.6 16.7 ± 1.2 
D560A 135 ± 7 72 ± 8 6.7 ± 0.2 7.8 ± 0.3 
R562A 81 ± 12 111 ± 13 164 ± 10 73 ± 3 
N564A 50 ± 6 61 ± 11 118 ± 6 198 ± 14 

Author notes

Disclosure: No relevant conflicts of interest to declare.