Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway is Stabilized by an Interdomain Bond Between His 281 and Ser 524 Formed in Factor VIII [Protein Structure and Folding]

April 1st, 2014 by Wakabayashi, H., Monaghan, M., Fay, P. J.

The Factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His281 (A1 domain) with Ser524 (A2 domain), although the resolution of the structure (~4 Å) does not firmly establish this bonding. In order to establish that side chains of these residues participate in an inter-domain bond, we prepared and examined the functional properties of a residue swap variant (His281Ser/Ser524His) where His281 and Ser524 residues were exchanged with one-another and disulfide-bridged variant (His281Cys/Ser524Cys) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced in for His281Ala and Ser524Ala variants in an earlier study. The disulfide-bridged variant showed ~20% increased FVIII stability and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of His281Ser-A1/A3C1C2 dimer with Ser524His-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ~5 and ~10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, Km for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His281 and Ser524 are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.
  • Posted in Journal of Biological Chemistry, Publications
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