Details

Autor(en) / Beteiligte

• Nogami, Keiji
• Lapan, Kirsty A.
• Zhou, Qian
• Wakabayashi, Hironao
• Fay, Philip J.

Titel

Identification of a Factor Xa-interactive Site within Residues 337–372 of the Factor VIII Heavy Chain

Ist Teil von

• The Journal of biological chemistry, 2004-04, Vol.279 (16), p.15763-15771

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2004

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 μm) and partially blocked cleavage at the A2-B junction (IC50 = 100 μm). 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 ∼50% and an activation rate constant of ∼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.