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Blood, 1 January 2001, Vol. 97, No. 1, pp. 169-174
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Antigenicity of putative phospholipid membrane-binding residues
in factor VIII
Rachel T. Barrow,
John F. Healey,
Marc G. Jacquemin,
Jean-Marie R. Saint-Remy, and
Pete Lollar
From the Winship Cancer Institute, Emory University,
Atlanta, GA; and the Center for Molecular and Vascular Biology,
Katholieke Universiteit, Leuven, Belgium.
Most inhibitory antibodies to human factor VIII (fVIII) bind to
epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding
of fVIII to phospholipid. The x-ray structure of the human fVIII C2
domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and
Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of
a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged
phosphatidylserine. In this study, 8 active mutants of human fVIII
(Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu,
Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and
Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on
the basis of differences between human, porcine, murine, and canine
fVIII at proposed phospholipid binding sites, were expressed. The
antigenicity of the mutants toward 5 C2-specific polyclonal human
antibodies was measured by using the Bethesda assay. A human monoclonal
anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody,
NMC VIII-5, were also included in the analysis. In comparison with
wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252
single mutants had lower antigenicity toward most of the inhibitors. In
contrast, the Val2223Ala and Lys2227Glu mutants usually showed
increased antigenicity. These results suggest that C2 inhibitors
frequently target the Met2199/Phe2200 and Leu2251/Leu2252  -hairpins
and are consistent with the hypothesis that these residues participate
in binding to phospholipid membranes. In contrast, Val2223 and Lys2227
may oppose antibody binding sterically or through stabilization of a
low-affinity membrane-binding conformation of the C2 domain.
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