The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex

• Published in: The Journal of biological chemistry Vol. 284; no. 6; pp. 3379 - 3388
• Main Authors: Soeda, Tetsuhiro, Nogami, Keiji, Nishiya, Katsumi, Takeyama, Masahiro, Ogiwara, Kenichi, Sakata, Yoichi, Yoshioka, Akira, Shima, Midori
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.02.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Enzyme Activation - physiology; Factor IXa - chemistry; Factor IXa - genetics; Factor IXa - metabolism; Factor VIIIa - chemistry; Factor VIIIa - genetics; Factor VIIIa - metabolism; Factor X - chemistry; Factor X - genetics; Factor X - metabolism; Humans; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Phospholipids - chemistry; Phospholipids - genetics; Phospholipids - metabolism; Protein Binding - physiology; Protein Structure, Tertiary - physiology; Surface Plasmon Resonance - methods
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M804955200

Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 domain of factor VIIIa and the Gla domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla domain of factor IXa inhibited binding by ∼95%, and Gla domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 domain interacted with the Gla domain of factor IXa. A fragment designated C2-(2182–2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182–2259, demonstrated that peptide 2228–2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228–2240 in the factor VIIIa C2 domain constitutes an interactive site for the Gla domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.