Four hydrophobic amino acids of the factor VIII C2 domain are constituents of both the membrane-binding and von Willebrand factor-binding motifs

Abstract

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 β-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 Arg2215, Arg2220, Lys2227, Lys2249, Gln2213, Asn2217, and Phe2196/Thr2197 had specific activities that were > 70% of the wild type. Mutants at Arg2209, Lys2227, Trp2313, and Arg2320 were degraded within the cell. Hydrophobic spike mutants at Met2199/Phe2200, Leu2251/Leu2252, and Met2199/Phe2200/Leu2251/Leu2252 (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 ∼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 ∼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.