Identification of a factor Xa-interactive site within residues 337-372 of the factor VIII heavy chain.
We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC50 = 11 microm) and partially blocked cleavage at the A2-B junction (IC50 = 100 microm). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of approximately 50% and an activation rate constant of approximately 30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.
Nogami
Keiji
K
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Lapan
Kirsty A
KA
Zhou
Qian
Q
Wakabayashi
Hironao
H
Fay
Philip J
PJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D005169:Factor VIII / Q000032:analysis / Q000378:metabolism*; D015951:Factor Xa / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D005169:Factor VIII; D015951:Factor Xa