title: Anti-heavy-chain monoclonal antibodies directed to the acidic regions of the factor VIII molecule inhibit the binding of factor VIII to phospholipids and von Willebrand factor.
abstract: Recent studies have shown that inhibitors develop against acidic regions of the FVIII molecule, which contain important functional sites. However, their mechanisms of inhibition are not well understood. In this study, two anti-human FVIII mouse monoclonal antibodies (MAbs), directed towards the exposed acidic regions of the FVIII molecule, were developed, characterised and their mechanisms of inhibition investigated. The two MAbs, F7B4 and F26F6, had inhibitory titres of 32 and 944 BU/mg respectively, had high affinities for the FVIII molecule (K(D) approximately nM range) and recognised sequences V(357)-F(360) on the acidic a1 region and E(724)-L(731) on the acidic a2 region of the FVIII heavy-chain (HC), respectively. F7B4 inhibited the rate of FXa generation by activated FVIII, whilst both antibodies inhibited FVIII activation by thrombin and blocked thrombin cleavage of FVIII. Furthermore, F7B4 and F26F6 inhibited FVIII binding to (a) phospholipids (IC(50): 77 nM and 40 nM respectively), and (b) VWF (IC(50): 93 nM and 267 nM respectively), despite both having HC specificity. Experiments with F(ab')(2) fragments confirmed the above findings. Taken together these data represent novel findings in that anti-acidic HC antibodies can inhibit FVIII function by a variety of mechanisms, in particular by interfering with the binding of FVIII to phospholipids & VWF.
authors.affiliation: Division of Haematology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK. sraut@nibsc.ac.uk
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Raut
authors.forename: Sylvie
authors.initials: S
authors.lastname: Villard
authors.forename: Sabrina
authors.initials: S
authors.lastname: Grailly
authors.forename: Jean-Guy G
authors.initials: JG
authors.lastname: Gilles
authors.forename: Claude
authors.initials: C
authors.lastname: Granier
authors.forename: Jean-Marie R
authors.initials: JM
authors.lastname: Saint-Remy
authors.forename: Trevor W
authors.initials: TW
authors.lastname: Barrowcliffe
mesh_terms: D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D018604:Epitope Mapping; D000939:Epitopes; D005169:Factor VIII / Q000276:immunology* / Q000378:metabolism; D015951:Factor Xa / Q000096:biosynthesis; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D024342:Amino Acids, Acidic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010743:Phospholipids; D014841:von Willebrand Factor; D005169:Factor VIII; D013917:Thrombin; D015951:Factor Xa