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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-06-1818.
Submitted June 19, 2002
Accepted September 24, 2002
Shielding the front strand ß3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ib
Arnaud Bonnefoy, Hiroshi Yamamoto, Chantal Thys, Morikazu Kito, Jos Vermylen, and Marc F Hoylaerts*
Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
Pharmaceutical Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan
* Corresponding author; email: marc.hoylaerts{at}med.kuleuven.ac.be.
Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand Factor (vWf) A1 domain to platelet GPIb . Blocking this interaction represents a promising approach to the treatment of arterial thrombosis. Comparison of amino acid sequences of the vWf A1 domain in several species, expressing vWf recognized by the blocking monoclonal antibody AJvW-2, suggested nine residues (H563, I566, D570, A581, V584, A587, R616, A618 and M622) to contribute to the epitope for AJvW-2 and/or to be part of the GPIb binding site. GST/human vWf A1 fusion proteins, in which these amino acids were mutated to their murine counterpart, were tested for their capacity to bind AJvW-2 or heparin, to interfere with botrocetin or ristocetin mediated vWf binding to GPIb, or to induce flow-dependent platelet tethering in a perfusion chamber. Thus, mutations H563R, I566L, D570A, and A587T, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The H563R, I566L and D570A mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that H563, I566, D570, R616 and A618 take part in GPIb binding, their mutation impairing platelet recruitment. In agreement with the surface distribution of vWf type 2M mutations, this study demonstrates overlapping of the epitope for AJvW-2 and the GPIb binding site, located around the front pocket of the A1 domain and defined by strands ß3 and ß4 and helix 3, and provides a mechanistic basis for vWf neutralization by this antibody.
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