Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase

Abstract

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.

Figure 1. Binding of FVIII LC to FX

A) Binding of FVIII LC to FX by SPR. Maximum RU values at equilibrium for association of FVIII LC were plotted as a function of FVIII LC, and the data were fitted using the Equation 1 according to a single-site binding model. Experiments were performed at least three separate times and mean values are shown. Inset: Curves 1–7 show representative association/dissociation curves for FVIII LC (0, 50, 100, 200, 500, 1000 and 2000 nM, respectively. B) Binding of FVIII LC to FX by ELISA-based assays. Various concentrations of FVIII LC were reacted with FX (50 nM) immobilized onto microtiter wells. Bound FVIII LC was detected using a biotinylated anti-FVIII LC antibody, 10104. Data were fitted using the Equation 1 (single-site biding model). Inset: Mixtures of FVIII LC (500 nM) and various concentrations of FX were incubated with immobilized FX. Absorbance value corresponding to FVIII LC binding to FX in the absence of competitor was defined as 100%. The data were fitted by non-linear least squares regression using Equation 2 (competitive inhibition model). Experiments were performed at least three separate times and mean values are shown. C) EDC cross-linking of FVIII LC and FX. FVIII LC (200 nM) was reacted with FX (200 nM) in the presence of various concentrations of EDC (lanes 1–4; 0, 1, 2, and 5 mM, respectively) at 23 °C for 2 hours, followed by immunoblotting using the anti-FVIII LC antibody, 2D2 (panel a) or the anti-FX antibody (panel b). Experiments were performed at least three separate times and typical results are shown.

Figure 1. Binding of FVIII LC to FX

A) Binding of FVIII LC to FX by SPR. Maximum RU values at equilibrium for association of FVIII LC were plotted as a function of FVIII LC, and the data were fitted using the Equation 1 according to a single-site binding model. Experiments were performed at least three separate times and mean values are shown. Inset: Curves 1–7 show representative association/dissociation curves for FVIII LC (0, 50, 100, 200, 500, 1000 and 2000 nM, respectively. B) Binding of FVIII LC to FX by ELISA-based assays. Various concentrations of FVIII LC were reacted with FX (50 nM) immobilized onto microtiter wells. Bound FVIII LC was detected using a biotinylated anti-FVIII LC antibody, 10104. Data were fitted using the Equation 1 (single-site biding model). Inset: Mixtures of FVIII LC (500 nM) and various concentrations of FX were incubated with immobilized FX. Absorbance value corresponding to FVIII LC binding to FX in the absence of competitor was defined as 100%. The data were fitted by non-linear least squares regression using Equation 2 (competitive inhibition model). Experiments were performed at least three separate times and mean values are shown. C) EDC cross-linking of FVIII LC and FX. FVIII LC (200 nM) was reacted with FX (200 nM) in the presence of various concentrations of EDC (lanes 1–4; 0, 1, 2, and 5 mM, respectively) at 23 °C for 2 hours, followed by immunoblotting using the anti-FVIII LC antibody, 2D2 (panel a) or the anti-FX antibody (panel b). Experiments were performed at least three separate times and typical results are shown.

Figure 2. Inhibition of FVIII LC and FX binding by DEGR-APC

FVIII LC (500 nM) was incubated with FX-coated wells (50 nM/well) in the presence of various concentrations of DEGR-APC. Bound FVIII LC was detected using a biotinylated anti-FVIII LC antibody, 10104. The absorbance value corresponding to FVIII LC bound to FX in the absence of DEGR-APC was defined 100%. The percentage of FVIII LC was plotted as a function of DEGR-APC concentration, and the data were fitted by nonlinear least squares regression using Equation 2 (competitive inhibition model). Experiments were performed at least three separate times and mean values are shown.

Figure 3. Effect of FX on A1 cleavage at Arg³³⁶ by APC

Mixtures of FVIIIa (130 nM) and various concentrations of FX were incubated with APC (2 nM) and PSPCPE (100 μM) in HBS-buffer containing 2 mM CaCl₂ for 10 min. Samples were run on 8% gels followed by Western blotting using an anti-A1 monoclonal antibody, 58.12. Inset: Lanes 1–6 show the A1 cleavage of FVIIIa in the presence of FX (0, 20, 50, 100, 200, and 400 nM, respectively). The graph shows quantitative densitometry of the A1³³⁶ product. Density values of A1³³⁶ generated by APC cleavage in the absence of FX were used to represent the 100% level. Data were fitted by nonlinear least squares regression using Equation 2 (competitive inhibition model) and shown as a dashed line. Experiments were performed at least three separate times and typical results are shown.

Figure 4

(A) Effect of 2007–2016 peptide on FVIII LC binding to FX. FVIII LC (200 nM) was mixed with various concentrations of the 2007–2016 peptide (open circles) or scrambled sequence peptide (closed circles) and mixtures were then incubated with immobilized FX (50 nM). Bound FVIII LC was detected using a biotinylated anti-FVIII LC antibody. The absorbance value corresponding to FVIII LC binding to FX in the absence of competitor was defined as 100%. The data were fitted (dashed line) by non-linear least squares regression using Equation 2 (competitive inhibition model). Experiments were performed at least three separate times and mean values are shown. (B) Direct binding of 2007–2016 peptide to FX as determined by SPR. Various amounts of the 2007–2016 peptide (open circles) or scrambled sequence peptide (closed circles) were injected onto a sensor chip containing immobilized FX for 2 minutes, followed by a change to running buffer for 2 minutes. Maximum values for association of the peptide were plotted as a function of peptide concentration, and the data were fitted using the Equation 1 (single-site binding model). Experiments were performed at least three separate times and mean values are shown. (C) EDC cross-linking of 2007–2016 peptide and FX. Panel a) FX (200 nM) was incubated with biotinylated 2007–2016 peptide (200 μM) in the presence of indicated concentrations of EDC at 23 °C for 2 hours, followed by immunoblotting using streptavidin. Panel b) Cross-linked products formed with FX (200 nM) and indicated concentrations of the 2007–2016 peptide with EDC (2 mM). Panel c) FX (150 nM) and biotinylated 2007–2016 peptide (100 μM) reacted with EDC (2 mM) in the presence of indicated concentrations of the unlabeled 2007–2016 peptide and were immunoblotted. Experiments were performed at least three separate times and typical results are shown.

Figure 5. Michaelis-Menten Analysis of FXase formed using FVIII LC mutants

FVIII (1 nM) was activated by thrombin (30 nM) for 1 minute in the presence of 20 μM PSPCPE. FXa generation was initiated by the addition of FIXa (40 nM) and various concentrations of FX (0–500 nM) as described in Methods. The symbols used are as follows; wild type (open circles), Thr2012Ala (closed circles), Phe2014Ala (open squares), Thr2012Ala/Phe2014Ala (closed squares), and Thr2012Ala/Leu2013Ala/Phe2014Ala (open triangles). Experiments were performed at least three separate times and mean values are shown.