Four hydrophobic amino acids of the factor VIII C2 domain are constituents of both the membrane-binding and von Willebrand factor-binding motifs.
Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a beta-hairpin turn, and also by net positive charge and specific interactions with phospho-l-serine. To test this model, we prepared 16 factor VIII mutants in which single or multiple amino acids were changed to alanine. Mutants at Arg(2215), Arg(2220), Lys(2227), Lys(2249), Gln(2213), Asn(2217), and Phe(2196)/Thr(2197) had specific activities that were >70% of the wild type. Mutants at Arg(2209), Lys(2227), Trp(2313), and Arg(2320) were degraded within the cell. Hydrophobic spike mutants at Met(2199)/Phe(2200), Leu(2251)/Leu(2252), and Met(2199)/Phe(2200)/Leu(2251)/Leu(2252) (4-Ala) exhibited 43, 59, and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipid-limiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced approximately 20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced approximately 5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipid-binding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.
Gilbert
Gary E
GE
Veterans Affairs Boston Healthcare System, Brigham and Women's Hospital, and Harvard Medical School, West Roxbury, Massachusetts 02132, USA. ggilbert@rics.bwh.harvard.edu
Kaufman
Randal J
RJ
Arena
Andrew A
AA
Miao
Hongzhi
H
Pipe
Steven W
SW
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014841:von Willebrand Factor / Q000378:metabolism*
D014841:von Willebrand Factor; D005169:Factor VIII; D005170:Factor X