Abstract
Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.
Highlights
Platelet adhesion to subendothelial structures, to the thrombogenic compound collagen, is one of the first steps in a sequence of reactions that can lead to arterial thrombosis
We raised a monoclonal antibody, 82D6A3, against human von Willebrand factor (VWF) that prevents the binding of VWF to collagen [24] and that is antithrombotic in a baboon arterial thrombosis model [33]
Characterization of 82D6A3 and Its Fab Fragments under Static and Flow Conditions—monoclonal antibody (mAb) 82D6A3, an anti-human VWF antibody, and its Fab fragments inhibit binding of purified VWF to human collagen type I in a specific and dose-dependent manner with an IC50 of 20 ng/ml for the IgG and 1 g/ml for the Fab fragments. mAb 82D6A3 recognizes the A3-domain of VWF as it binds to the recombinant VWF A3domain but not to ⌬A3-VWF (Fig. 1) [24, 35]
Summary
Materials—Human placental collagen type I and III were purchased from Sigma. The collagens were solubilized in 50 mmol/liter acetic acid and subsequently dialyzed against phosphate-buffered saline PBS (48 h, 4 °C) to obtain fibrillar collagen. Inhibition of VWF Binding to Collagen by 82D6A3 and Its Fab Fragments—A 96-well plate (Greiner, Frickenhausen, Germany) was coated overnight with human collagen type I (25 g/ml in PBS) and blocked with 3% milk powder solution. Bound phages were detected after a 1-h incubation with a polyclonal anti-M13-HRP-conjugated antibody (Amersham Biosciences), and visualization was performed as described above. Binding of 82D6A3 to VWF Mutants—Microtiter plate wells (Costar, Cambridge, MA) were coated with a 2.5 g/ml concentration of a polyclonal antibody directed against the DЈ- and D3-domains of VWF [37] in 50 mM carbonate buffer, pH 9.6, and blocked with 3% bovine serum albumin in PBS, 0.1% Tween 20. Wells were incubated with expression medium diluted with PBS, 0.1% Tween 20, 3% bovine serum albumin to a final VWF concentration of 100 ng/ml for 60 min at 37 °C. The main and side chain atoms of the Ser and Arg residues were used as anchors to superimpose the structures
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
