Identification of the binding site for an alloantibody to von Willebrand factor which inhibits binding to glycoprotein Ib within the amino-terminal region flanking the A1 domain.

An alloantibody to von Willebrand factor (vWF) which developed in a Japanese boy with type 3 von Willebrand disease has been characterized. The antibody was non-precipitating IgG and the main subclasses were IgG2 and IgG4. The antibody inhibited completely ristocetin-induced platelet aggregation (RIPA) and high shear stress-induced platelet aggregation (SIPA). Its predominant inhibitory role was focused, therefore, on the interaction between vWF and platelet gycoprotein Ib. The antibody reacted with a 52/48 kDa tryptic fragment of vWF (residues 449-728). No reaction was seen, however, with either a 39/34 kDa dispase fragment (480-718) or a recombinant vWF fragment (residues 465-728). These findings suggested that the essential epitope resided in the amino-terminal flanking region of the Al domain. We synthesized overlapping peptides corresponding to the region containing D3/A1 boundary. A peptide, residues 458-472, bound to the antibody and dose-dependently blocked the antibody binding to the 52/48 kDa fragment. The same peptide neutralized the inhibitory effect of the alloantibody on SIPA. The data are consistent with the presence of an epitope within residues 458-472 which reacted with the 52/48 kDa fragment. Furthermore, the specific component of the antibody, directed against residues 458-472, blocked vWF binding to GPIb in absence of exogenous agonist. Our results suggest that the region flanking the Al domain plays an important role in regulating vWF binding to GPIb.