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.
Shibata
M
M
Department of Pediatrics, Nara Medical University, Kashihara Japan.
Shima
M
M
Fujimura
Y
Y
Takahashi
Y
Y
Nakai
H
H
Sakurai
Y
Y
Asatani
M
M
Nomura
A
A
Take
H
H
Giddings
J C
JC
Yoshioka
A
A
D000918:Antibody Specificity; D001665:Binding Sites / Q000276:immunology; D001777:Blood Coagulation / Q000276:immunology; D002648:Child; D018604:Epitope Mapping; D006801:Humans; D007518:Isoantibodies / Q000276:immunology* / Q000494:pharmacology; D008297:Male; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D014842:von Willebrand Diseases / Q000097:blood / Q000276:immunology*; D014841:von Willebrand Factor / Q000276:immunology* / Q000378:metabolism
D007518:Isoantibodies; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor