Shielding the front-strand beta 3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibalpha.
Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand factor (VWF) A1 domain to platelet GPIbalpha. 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 9 residues (His563, Ile566, Asp570, Ala581, Val584, Ala587, Arg616, Ala618, and Met622) to contribute to the epitope for AJvW-2 or to be part of the GPIbalpha-binding site. Glutathione-S-transferase (GST)-human VWF A1 fusion proteins, in which these amino acids were mutated to their murine counterparts, 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 His563Arg, Ile566Leu, Asp570Ala, and Ala587Thr, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The His563Arg, Ile566Leu, and Asp570Ala mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that His563, Ile566, Asp570, Arg616, and Ala618 take part in GPIbalpha 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 GPIbalpha-binding site, located around the front pocket of the A1 domain and defined by strands beta3, beta4, and helix alpha3, and it provides a mechanistic basis for VWF neutralization by this antibody.
Bonnefoy
Arnaud
A
Center for Molecular and Vascular Biology, K.U. Leuven, Belgium.
Yamamoto
Hiroshi
H
Thys
Chantal
C
Kito
Morikazu
M
Vermylen
Jos
J
Hoylaerts
Marc F
MF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites / Q000276:immunology; D001792:Blood Platelets / Q000378:metabolism; D003435:Crotalid Venoms / Q000494:pharmacology; D018604:Epitope Mapping; D005982:Glutathione Transferase / Q000235:genetics; D006493:Heparin / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010973:Platelet Adhesiveness; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D016376:Oligonucleotides, Antisense; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins; C023592:glycocalicin; D014841:von Willebrand Factor; C030961:botrocetin; D006493:Heparin; D005982:Glutathione Transferase