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A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa
Author(s) -
WAKABAYASHI H.,
ZHOU Q.,
VARFAJ F.,
FAY P. J.
Publication year - 2007
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2007.02458.x
Subject(s) - residue (chemistry) , protein subunit , chemistry , biochemistry , gene
Summary. 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 ( K d = 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 ( K d = 526 ± 107 n m for E1829A, 144 ± 21 n m 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.