A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa

Abstract

Background: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. Objective: Based on our finding of low-specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. Results: The rate of activity decay of E1829A was ∼fourteenfold faster than wild-type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one-stage and two-stage assays were ∼14% and ∼11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A-A3C1C2 was comparable to wt A3C1C2 (Kd = 20.1 ± 3.4 n m for E1829A, 15.3 ± 3.7 n m for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by ∼3.6-fold as a result of the mutation (Kd = 526 ± 107 nM for E1829A, 144 ± 21 nM for wt). Conclusion: As modeling data suggest that Glu1829 is located at the A2-A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa. © 2007 International Society on Thrombosis and Haemostasis.