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 Japanes e boy with type 3 von Willebrand disease has been characterized. The antibo dy was non-precipitating IgG and the main subclasses were IgG(2) and IgG(4) . The antibody inhibited completely ristocetin-induced platelet aggregation (RIPA) and high shear stress-induced platelet aggregation (SIPA). Its pred ominant inhibitory role was focused, therefore, on the interaction between vWF and platelet gycoprotein Ib. The antibody reacted with a 52/48 kDa tryp tic 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 fragmen t (residues 465-728). These findings suggested that the essential epitope r esided in the amino-terminal flanking region of the Al domain. We synthesiz ed overlapping peptides corresponding to the region containing D3/A1 bounda ry. 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 n eutralized the inhibitory effect of the alloantibody on SIPA. The data are consistent with the presence of an epitope within residues 458-472 which re acted with the 52/48 kDa fragment. Furthermore, the specific component of the antibody, directed against resid ues 458-472, blocked vWF binding to GPIb, in absence of exogenous agonist. Our results suggest that the region flanking the A1 domain plays an importa nt role in regulating vWF binding to GPIb.