Mimotope/anti-mimotope probing of structural relationships in platelet glycoprotein Ib alpha.
A bacteriophage library displaying random decapeptides was used to characterize the binding preference of C-34, a monoclonal antibody originally raised against platelet-type von Willebrand disease platelets heterozygous for the mutation 23OWKQ (G --> V)233V234 in the alpha chain of glycoprotein Ib (GPIb alpha). Three rounds of biopanning C-34 against the library resulted in striking convergence upon the sequence WNWRYREYV. Since no portion of this sequence corresponds to a recognizable peptide sequence within human platelet GPIb alpha, it may be considered a "mimotope" of the naturally occurring C-34 epitope, presumably bearing similarity to it in three-dimensional structure. Synthetic AWNWRYREYV peptide preincubated with C-34 fully neutralized the ability of C-34 to inhibit platelet aggregation, with an IC50 of approximately 6 microg/ml. When biotinylated AWNWRYREYV was subsequently bioparmed against the original decapeptide library, the sole clone demonstrating inhibitory activity above background level in a functional platelet assay displayed the sequence RHVAWWRQGV, and chemically synthesized peptide fully inhibited ristocetin-induced aggregation, with an IC50 of 200-400 microg/ml. Synthesized RHVAWWKQGV peptide exerted only slight inhibition, whereas RHVAWWKQVV peptide showed potent inhibitory activity. Moreover, whereas synthesized wild-type 228YVWKQGVDVK237 GPIb alpha peptide was virtually without inhibitory activity, the 228YVWKQ(G -->V) 233VDVK237 peptide fully inhibited ristocetin-induced aggregation, with an IC50 of approximately 400 microg/ml. These studies raise the possibility of an intramolecular association of peptide regions within GPIb alpha that may play a role in the regulation of von Willebrand factor-dependent platelet aggregation.
Miller
J L
JL
Department of Pathology, State University of New York Health Science Center, Syracuse 13210, USA.
Lyle
V A
VA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D018604:Epitope Mapping; D015723:Gene Library; D006801:Humans; D051379:Mice; D015335:Molecular Probes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D017354:Point Mutation; D014842:von Willebrand Diseases / Q000097:blood / Q000235:genetics
D000911:Antibodies, Monoclonal; D015335:Molecular Probes; D019038:Platelet Glycoprotein GPIb-IX Complex