Page 238 - Haematologica - Vol. 105 n. 6 - June 2020

M. A.Przeradzka et al.
B.10 Because of limited proteolysis of the B domain, FVIII is heterogeneous in size with molecular weights ranging from 160 kDa to 330 kDa.11,12
For effective binding to FVIII, VWF requires the pres- ence of a short acidic amino acid region at the start of the FVIII A3 domain. This region, which includes sulphated tyrosine residues, is referred to as the a3 region.13,14 Next to this VWF binding region, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and previous site-directed mutagenesis studies have identified binding sites for VWF in the C1 and C2 domain of FVIII as well.15–19 During acti- vation of FVIII, the a3 region is removed from FVIII lead- ing to the dissociation of the FVIII-VWF complex. Additional cleavages by thrombin generates activated FVIII that can perform its role in the coagulation cascade as a cofactor for activated factor IX ultimately leading to fibrin formation.20
It has previously been established that the N-terminal D’-D3 domains of VWF comprise the binding site for FVIII. In 1987, limited proteolysis studies of VWF revealed that a VWF fragment comprising the residues 764-1036 harbors the interaction site for FVIII.21 Based on cryoelec- tron microscopy (cryo-EM) structures of FVIII in complex with D’-D3, it has later been shown that the main interac- tive region for FVIII resides in the D’ domain.19 Recently, we have found that the presence of the VWD3 subdomain of the D3 domain is required to optimally support the interaction between D’ and FVIII.22 Using a primary amine-directed chemical foot printing approach combined with mass spectrometry analysis, we have further demon- strated that Lys773 contributes to FVIII binding.23 In the present study, we have employed HDX-MS combined with site-directed mutagenesis and protein binding stud- ies to further explore the FVIII binding regions on VWF. The combined results show that the D’ domain region Arg782-Cys799 is part of the FVIII binding interface.
Methods
Materials
Tris-HCl was from Invitrogen (Breda, the Netherlands), NaCl was obtained from Fagron (Rotterdam, the Netherlands) and HEPES was from Serva (Heidelberg, Germany), FreeStyle 293 expression medium was obtained from Gibco (Thermo Fisher Scientific). Tween-20 and D2O was from Sigma-Aldrich (St Louis, MO, USA). Human serum albumin (HSA) was obatined from the Division of Products at Sanquin (Amsterdam, the Netherlands). All other chemicals were from Merck (Darmstadt, Germany), unless indicated otherwise.
Proteins
Antibody CLB-EL14 (EL14), CLB- Rag20, CLB-CAg12 (CAg 12) and HPC4 have been described before.22,24,25 D’-D3 fragment, FVIII lacking the B domain residues 746-1639 (referred to as FVIII throughout this paper) and VWF were obtained essentially as described before.26–28 Purified proteins were dialyzed against a buffer with 50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM CaCl2, 50% (v/v) glycerol and stored at -20°C. Site-directed mutagenesis of the D’-D3 fragment was employed using Quik Change (Agilent Technologies) according to the manufacturer's instructions.
HDX-MS
D’-D3 was pre-incubated in presence or absence of FVIII in 1:1 molar ratio for 5 min at 4˚C. Samples were subsequently diluted
ten times in deuterated binding buffer (98% D2O) or standard binding buffer and incubated for 10 sec, 100 sec or 1,000 sec at 24˚C. A detailed description is available in the Online Supplementary Materials and Methods.
Solid-phase competition assays
Recombinant VWF (1 μg/mL) was immobilized overnight at 4˚C in a buffer containing 50mM NaHCO3 pH 9.8 in a 96-wells microtiter plate (Nunc Maxisorp). Increasing concentrations (0.3- 900 nM) of D’-D3 and variants with single mutations were pre- incubated with 0.3 nM FVIII in a buffer containing 50 mM Tris, 150 mM NaCl, 2% human serum albumin, 0.1% Tween 20, pH 7.4 for 30 min at 37˚C. These mixtures were transferred to the VWF coated plate and incubated for 2 hours at 37˚C. Then, the plate was washed three times with 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl2, 0.1% Tween 20 after which FVIII bound to VWF was detected with an HRP-labeled monoclonal antibody (CAg 12).26
Surface plasmon resonance analysis
Surface plasmon resonance (SPR) analysis was carried out using a Biacore T-200 biosensor system (GE Healthcare) as previously described.22 A monoclonal antibody to FVIII (EL14) was coupled to a CM5 sensor chip (GE Healthcare) to 5,000 response units (RU) density using the amino coupling activation method according to manufacturer’s suggestions (GE Healthcare). Subsequently, 3,000 RU of FVIII were immobilized to the chip via EL14 antibody. Next, increasing concentrations of D’-D3 fragments were passed over the chip at a flow rate of 30 μL/min in a buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl2 and 0.05% Tween 20 at 25°C. An empty channel was utilized to correct for non-specific binding to the dextran matrix.
Results
HDX-MS on the isolated D’-D3 fragment of VWF
To facilitate identification of the FVIII binding residues within the D’ domain (TIL’-E’ subdomains), we made use of the D’-D3 monomer in which the cysteine residues involved in dimerization of the D3 domains (VWD3- C8_3-TIL3-E3 subdomains) were replaced by serine residues.29 The D’-D3 fragment was transferred from H2O to D2O-containing buffer to assess time-dependent deu- terium incorporation into the protein backbone. 178 pep- tides were identified covering 92% of the D’-D3 sequence (Figure 1A and Online Supplementary Table S1). For each peptide, we plotted the percentage of deuterium incorpo- ration of the identified peptides at three different time points (Figure 1B). The overall result showed that almost all peptides from the N-terminal TIL’ subdomain of D’-D3 exhibit limited to no change in deuterium incorporation at these time points. Only the peptide that includes the N- terminus of the TIL’ subdomain showed increased deuteri- um incorporation. Apart from several peptides in the C8_3 subdomain of the D3 domain, most of the peptides in the other subdomains showed incorporation of deuterium in time. Peptides with the most marked change in deuterium incorporation correspond to unstructured regions in the recently published crystal structure of D’-D3.30 This find- ing confirms that amino acid backbone hydrogens in unstructured regions exhibit an enhanced rate of hydro- gen-deuterium exchange compared to structured regions. It further implies that these regions are unstructured in solution as can be predicted by the crystal structure.
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