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J Biol Chem. 2014 May 16;289(20):14020-9. doi: 10.1074/jbc.M114.550566. Epub 2014 Apr 1.

Cofactor activity in factor VIIIa of the blood clotting pathway is stabilized by an interdomain bond between His281 and Ser524 formed in factor VIII.

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  • 1From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642.
  • 2From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642 philip_fay@urmc.rochester.edu.

Abstract

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His(281) (A1 domain) with Ser(524) (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His(281) and Ser(524) residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) 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 for H281A and S524A 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 H281S-A1/A3C1C2 dimer with S524H-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 His(281) and Ser(524) are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

KEYWORDS:

Blood Coagulation Factors; Factor VIII; Mutagenesis; Protein Stability; Protein Structure

PMID:
24692542
[PubMed - indexed for MEDLINE]
PMCID:
PMC4022872
Free PMC Article
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