Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

• Published in: The Journal of biological chemistry Vol. 288; no. 1; pp. 393 - 400
• Main Authors: Castro-Núñez, Lydia, Bloem, Esther, Boon-Spijker, Mariëtte G., van der Zwaan, Carmen, van den Biggelaar, Maartje, Mertens, Koen, Meijer, Alexander B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.01.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Binding Sites; Blood Coagulation Factors; Chemical Footprinting; Dose-Response Relationship, Drug; Factor VIII; Factor VIII - chemistry; Humans; Kinetics; Lysine - chemistry; Mass Spectrometry (MS); Mass Spectrometry - methods; Molecular Conformation; Molecular Sequence Data; Protein Binding; Protein Structure and Folding; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Serine - chemistry; Surface Plasmon Resonance; Surface Plasmon Resonance (SPR); von Willebrand Factor; von Willebrand Factor - chemistry; Mass Spectrometry (MS); Chemical Footprinting; Surface Plasmon Resonance (SPR); von Willebrand Factor; Blood Coagulation Factors; Factor VIII
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.400572

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. Background: von Willebrand factor (VWF) protects factor VIII (FVIII) from rapid clearance and degradation. Results: Mass spectrometric footprinting revealed that FVIII protects Lys-773 and the N-terminal Ser-764 of VWF from chemical modification. VWF(S764A) showed increased and VWF(K773A) showed decreased FVIII binding. Conclusion: The N terminus of VWF is critical for FVIII binding. Significance: This study sheds new light on the mechanism of FVIII-VWF complex assembly.