Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex

Author:

Jenkins P. Vincent1,Freas Jan1,Schmidt Kyla M.1,Zhou Qian1,Fay Philip J.1

Affiliation:

1. From the Department of Biochemistry and Biophysics and the Department of Medicine, University of Rochester Medical Center, NY.

Abstract

AbstractThe 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIIIWT) and B-domainless FVIII containing the hemophilia A–associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [Kd] values ∼5 nM-20 nM and ∼100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A–associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIIIWT and FVIIIArg562Ala showed catalytic rate constant (kcat) values of approximately 60 minute−1 in the presence of phospholipid, whereas the hemophilia A–associated mutants showedkcat values ranging from 3.3 minute−1 to 7.5 minute−1. In the absence of phospholipid, all kcat values were reduced but FVIIIWT and FVIIIArg562Ala retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa.

Publisher

American Society of Hematology

Subject

Cell Biology,Hematology,Immunology,Biochemistry

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