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

Sanjeev Raut; Sylvie Villard; Sabrina Grailly; Jean-Guy G. Gilles; Claude Granier; Jean-Marie R. Saint-Remy; Trevor W. Barrowcliffe

doi:10.1160/TH02-09-0086

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 90(03): 385-397
DOI: 10.1160/TH02-09-0086

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

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

Sanjeev Raut

¹Division of Haematology, National Institute for Biological Standards and Control, Hertfordshire, UK

,

Sylvie Villard

²UMR-CNRS, Montpellier, France, University of Leuven, Leuven, Belgium

,

Sabrina Grailly

³Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium

,

Jean-Guy G. Gilles

³Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium

,

Claude Granier

²UMR-CNRS, Montpellier, France, University of Leuven, Leuven, Belgium

,

Jean-Marie R. Saint-Remy

³Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium

,

Trevor W. Barrowcliffe

¹Division of Haematology, National Institute for Biological Standards and Control, Hertfordshire, UK

› Author Affiliations

Abstract

Summary

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 ~ nM range) and recognised sequences V³⁵⁷-F³⁶⁰ on the acidic a1 region and E⁷²⁴-L⁷³¹ 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 (IC₅₀: 77 nM and 40 nM respectively), and (b) VWF (IC₅₀: 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.

Keywords

Factor VIII - monoclonal antibody - epitope - phospholipid - VWF

Full Text

References

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