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 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.
Highlights
Factor VIII binds to phospholipid membranes and to von Willebrand factor via its second C domain, which has lectin homology
We have tested the parallel model of factor VIII binding to phospholipid membranes by performing functional studies on factor VIII in which many of the proposed membrane-binding residues have been altered by site-directed mutagenesis
Data were analyzed according to Equation 1 except that ⌬Pro von Willebrand factor (vWf) was substituted for PL, KD referred to the dissociation constant for binding to ⌬Pro vWf, and kf referred to the fluorescence related to free ⌬Pro vWf plus nonspecific binding of ⌬Pro vWf to F-8-Superose microspheres lacking factor VIII
Summary
Vol 277, No 8, Issue of February 22, pp. 6374 –6381, 2002 Printed in U.S.A. Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs*. 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 -hairpin turn, and 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. Recent publication of two crystal structures for the C2 domain of factor V [22] and a single structure for the C2 domain of factor VIII [23] has provided the basis for a model membrane-binding mechanism Both C2 domains display two hydrophobic spikes at the tips of protruding -hairpin turns. It is logical to test any factor VIII C2 mutants with decreased phospholipid affinity to determine whether the vWf binding affinity is decreased
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