Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase.
Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having μM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 μM) and directly bound to FX (K(d) = 7.7 μM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, United States.
Masahiro
M
Takeyama
Hironao
H
Wakabayashi
Philip J
PJ
Fay
D020134:Catalytic Domain / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics; D005170:Factor X / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D009363:Neoplasm Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D005169:Factor VIII; D005170:Factor X; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant