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Shielding the front-strand β3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibα
- Source :
- Blood. 101:1375-1383
- Publication Year :
- 2003
- Publisher :
- American Society of Hematology, 2003.
-
Abstract
- 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.
- Subjects :
- Blood Platelets
Models, Molecular
Von Willebrand factor type A domain
Recombinant Fusion Proteins
Immunology
Sequence Homology
Platelet membrane glycoprotein
Polymerase Chain Reaction
Biochemistry
Mice
Structure-Activity Relationship
Platelet Adhesiveness
Von Willebrand factor
Platelet adhesiveness
Crotalid Venoms
von Willebrand Factor
Animals
Humans
Platelet
Amino Acid Sequence
Binding site
Glutathione Transferase
Binding Sites
Molecular Structure
biology
Heparin
Chemistry
Antibodies, Monoclonal
Platelet Glycoprotein GPIb-IX Complex
Cell Biology
Hematology
Oligonucleotides, Antisense
Molecular biology
Cell biology
Glycoprotein Ib
Mutation
biology.protein
Epitope Mapping
Subjects
Details
- ISSN :
- 15280020 and 00064971
- Volume :
- 101
- Database :
- OpenAIRE
- Journal :
- Blood
- Accession number :
- edsair.doi.dedup.....c14fde5ba112128f248befcb75623dee