Characterization of autoantigenic epitopes on platelet glycoprotein IIb/IIIa using random peptide libraries

Most patients with chronic immune thrombocytopenic purpura (ITP) have autoantibodies directed against the glycoprotein (GP) IIb/IIIa complex. We have used a filamentous phage library that displays random linear hexapeptides to identify peptide sequences recognized by these autoantibodies. Plasma antibody eluates from two patients were used to select for phage displaying autoantibody-reactive peptides. From patient ITP-1 (known to have two distinct autoantibodies), we identified anti-GPIIb/IIIa antibody-specific phage encoding the peptide sequences Arg-Glu-Lys-Ala-Lys-Trp (REKAKW) and Pro-Val-Val-Trp-Lys-Asn (PVVWKN). Patient ITP-2 bound phage encoding the hexapeptide sequence Arg-Glu-Leu-Leu-Lys-Met. Each phage showed saturable dose-dependent binding to immobilized autoantibody, and binding could be blocked with purified GPIIb/IIIa. Patient ITP-1 autoantibody recognition of phage encoding REKAKW could be blocked with a synthetic peptide derived from the GPIIIa cytoplasmic tail; however, the PVVWKN was not. Using sequential overlapping peptides from the GPIIIa cytoplasmic region, an epitope for ITP-1 was localized to the sequence Arg-Ala-Arg-Ala-Lys-Trp (GPIIIa 734-739). Inhibition studies using synthetic peptides showed that phage REKAKW and PVVWKN were recognized by distinct autoantibodies from patient ITP-1. To determine whether individual patients with ITP possessed autoantibodies that recognize similar antigenic determinants on GPIIb/IIIa, the three phage were tested for binding to five other ITP patient autoantibodies. The phage encoding the peptide PVVWKN was found to bind ITP-1 and one other patient autoantibody. This result suggests that ITP patients recognize a limited number of shared epitopes.