title: Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry.
abstract: Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA;
authors.forename: Po-Lin
authors.initials: PL
authors.lastname: Chiu
authors.affiliation: Biogen, Cambridge, MA; and.
authors.forename: George M
authors.initials: GM
authors.lastname: Bou-Assaf
authors.affiliation: Biogen, Cambridge, MA; and.
authors.forename: Ekta Seth
authors.initials: ES
authors.lastname: Chhabra
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA;
authors.forename: Melissa G
authors.initials: MG
authors.lastname: Chambers
authors.affiliation: Biogen, Cambridge, MA; and.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Peters
authors.affiliation: Biogen, Cambridge, MA; and.
authors.forename: John D
authors.initials: JD
authors.lastname: Kulman
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Walz
mesh_terms: D001665:Binding Sites; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D014841:von Willebrand Factor; D005169:Factor VIII