Distinct roles of Ser-764 and Lys-773 at the N terminus of von Willebrand factor in complex assembly with coagulation factor VIII

Abstract

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.

FIGURE 1.

The N terminus of VWF is differentially modified by TMT in the presence of FVIII. White bars, 35 nm VWF was incubated with TMT-126 in the presence of 70 nm FVIII and with TMT-127 in the presence of 70 nm FVIIIa as described under “Experimental Procedures.” Gray bars, as a control, 35 nm VWF was modified with TMT-126 and TMT-127 in the absence of FVIII or FVIIIa. The modified proteins were mixed at a 1:1 molar ratio, cleaved by chymotrypsin (A) or trypsin (B), and analyzed by nano-LC-MS. Shown is the mean TMT-127:TMT-126 ratio plus S.D. of lysine-containing peptides for which this ratio was determined three or more times.

FIGURE 2.

Changing the molar ratio of FVIII and VWF affects TMT modification of Ser-764 and Lys-773. Increasing concentrations of VWF were incubated with TMT-126 in the presence of 70 nm FVIII and with TMT-127 in the presence of 70 nm FVIIIa as described under “Experimental Procedures.” The indicated molar ratios of FVIII and VWF were employed in the experimental setup. The labeled proteins were mixed, proteolyzed, and analyzed by nano-LC-MS. A, TMT-127:TMT-126 ratio of peptide Ser-764–Arg-782. B, TMT-127:TMT-126 ratio of peptide Ser-766–Leu-774. The modified residues Ser-764 and Lys-773 are indicated by asterisks.

FIGURE 3.

VWF(S764A) is more effective and VWF(K773A) is less effective than WT VWF in competing with immobilized VWF for binding FVIII. Increasing concentrations of VWF(S764A) (▴), WT VWF (●), and VWF(K773A) (♦) were incubated with the FVIII light chain (A) or the FVIII heterodimer (B) in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm CaCl₂, 2% human serum albumin, and 0.1% Tween 20. Residual FVIII or FVIII light chain binding to immobilized VWF was assessed using peroxidase-labeled monoclonal antibody CLB-CAg 12 as described under “Experimental Procedures.” C, multimer pattern of the purified proteins that were employed in the analysis, with WT VWF (I), VWF(K773A) (II), and VWF(S764A) (III).

FIGURE 4.

VWF(S764A) exhibits a higher affinity and VWF(K773A) exhibits a lower affinity for the FVIII light chain compared with WT VWF. 2000 response units of WT VWF (A), VWF(S764A) (B), and VWF(K773A) (C) were immobilized on the surface of a CM5 sensor chip. Increasing concentrations of the FVIII light chain were subsequently perfused over the immobilized VWF variants in buffer containing 20 mm HEPES (pH 7.4), 150 mm NaCl, 5 mm CaCl₂, and 0.005% Tween 20. Binding is expressed in response units. The insets show the estimated equilibrium binding responses as a function of the employed FVIII light chain concentration. Half-maximum binding reflects the equilibrium dissociation constant (K_D) of the FVIII light chain-VWF variant complex.

FIGURE 5.

VWF(S764A) exhibits a higher affinity and VWF(K773A) exhibits a lower affinity for the FVIII heterodimer compared with WT VWF. 2000 response units of WT VWF (A), VWF(S764A) (B), and VWF(K773A) (C) were immobilized on the surface of a CM5 sensor chip. Increasing concentrations of the FVIII heterodimer were subsequently passed over the immobilized VWF variants in buffer containing 20 mm HEPES (pH 7.4), 150 mm NaCl, 5 mm CaCl₂, and 0.005% Tween 20. Binding is expressed in response units. The insets show the estimated equilibrium binding responses as a function of the employed FVIII heterodimer concentration. Half-maximum binding reflects the equilibrium dissociation constant (K_D) of the FVIII-VWF variant complex.

FIGURE 6.

VWF variants offer a differential protection of FVIII against proteolytic activation by FXa. 0.3 nm FVIII was incubated with 200 nm FX, 0.3 nm FIXa, and 25 μm phospholipid vesicles comprising 15% phosphatidylserine, 20% phosphatidylethanolamine, and 65% phosphatidylcholine in the presence of 15 nm VWF(K773A) (□), WT VWF (○), or VWF(S764A) (▵). FVIII was activated by 1 nm FXa, and the newly formed FXa was assessed as a function of time.

FIGURE 7.

Molecular model of the N-terminal part of the D′ domain. A model of the first 64 residues of the D′ domain, i.e. the TIL domain, was constructed by comparative homology modeling with Modeller 9v3. The NMR structure of AMCI-1 (Protein Data Bank code 1CCV) was used as a template (32, 33). Shown are side and front views of the model. S–S bridges between the cysteine residues (yellow) are shown in red. Lys-773 is indicated in blue, and Ser-764 in green. The primary sequence alignment of the TIL domain of VWF and AMCI-1 is shown below the model. Replacements of amino acids that are associated with VWF type 2N disease are indicated above the sequence (see the ISTH-SSC VWF Database).