title: 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 approximately fourteen fold 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 approximately 14% and approximately 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 nM for E1829A, 15.3 +/- 3.7 nM for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by approximately 3.6-fold as a result of the mutation (K(d) = 526 +/- 107 nM for E1829A, 144 +/- 21 nM for wt).
  
  
  CONCLUSIONS
  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.
authors: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY 14642, USA.
authors: H
authors: H
authors: Wakabayashi
authors: Q
authors: Q
authors: Zhou
authors: F
authors: F
authors: Varfaj
authors: P J
authors: PJ
authors: Fay
mesh_terms: D015944:Factor VIIIa / Q000737:chemistry / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D018698:Glutamic Acid; D015944:Factor VIIIa