title: Localization of a factor X interactive site in the A1 subunit of factor VIIIa.
abstract: The protein cofactor, factor (F) VIIIa, is required for the efficient conversion of the substrate FX to FXa by the serine protease FIXa. The interaction between human FVIII (and its constituent subunits) and FX was characterized using a solid phase binding assay performed in the absence of phospholipid and FIXa. Saturable binding of FX to heterodimeric FVIII, the FVIII heavy chain (contiguous A1-A2 domains), the FVIIIa-derived A1/A3-C1-C2 dimer, and the isolated A1 subunit was observed with estimated Kd values ranging from approximately 1 to 3 microM. The interaction of FX with FVIII was inhibited by moderate ionic strength and was Ca2+-dependent, consistent with the salt sensitivity observed in a phospholipid-independent FXa generation assay. Negligible binding to FX was observed for the isolated A2 and A3-C1-C2 subunits of FVIIIa, suggesting that the A1 subunit of FVIII contains a primary binding site for FX. A synthetic peptide to the COOH-terminal acidic region of the A1 subunit, designated FVIII337-372, bound FX and effectively competed with A1 for FX binding (Ki = approximately 16 microM). Cross-linking between the FVIII337-372 peptide and the FX heavy chain was observed following reaction with 1-ethyl-3-[(diethylamino)propyl]carbodiimide. The presence of FX reduced the rate of activated protein C-catalyzed cleavage at Arg336 by approximately 5-fold. These results identify a primary FX interactive site on the cofactor of the intrinsic FXase.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Lapan
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D015944:Factor VIIIa / Q000737:chemistry* / Q000378:metabolism; D005170:Factor X / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009994:Osmolar Concentration; D011487:Protein Conformation
chemical_list: D015944:Factor VIIIa; D005170:Factor X