MIMOTOPE ANTI-MIMOTOPE PROBING OF STRUCTURAL RELATIONSHIPS IN PLATELET GLYCOPROTEIN IB-ALPHA

A bacteriophage library displaying random decapeptides was used to cha racterize the binding preference of C-34, a monoclonal antibody origin ally raised against platelet-type von Willebrand disease platelets het erozygous for the mutation (230)WKQ(G --> V)V-233(234) in the alpha ch ain of glycoprotein Ib (GPIb alpha). Three rounds of biopanning C-34 a gainst the library resulted in striking convergence upon the sequence WNWRYREYV, Since no portion of this sequence corresponds to a recogniz able peptide sequence within human platelet GPIb alpha, it may be cons idered a ''mimotope'' of the naturally occurring C-34 epitope, presuma bly bearing similarity to it in three-dimensional structure, Synthetic AWNWRYREYV peptide preincubated with C-34 fully neutralized the abili ty of C-34 to inhibit platelet aggregation, with an IC50 of approximat e to 6 mu g/ml. When biotinylated AWNWRYREYV was subsequently biopanne d against the original decapeptide library, the sole clone demonstrati ng inhibitory activity above background level in a functional platelet assay displayed the sequence RHVAWWRQGV, and chemically synthesized p eptide fully inhibited ristocetin-induced aggregation, with an IC50 of 200-400 mu g/ml. Synthesized RHVAWWKQGV peptide exerted only slight i nhibition, whereas RHVAWWKQVV peptide showed potent inhibitory activit y. Moreover, whereas synthesized wild-type (Z28)YVWKQGVDVK(237) GPIb a lpha peptide was virtually without inhibitory activity, the (228)YVWKQ (G --> V)(VDVK237)-V-233 peptide fully inhibited ristocetin-induced ag gregation, with an IC50 of approximate to 400 mu g/ml. These studies r aise the possibility of an intramolecular association of peptide regio ns within GPlb alpha that may play a role in the regulation of von Wil lebrand factor-dependent platelet aggregation.