Many α_IIbβ₃ autoepitopes in chronic immune thrombocytopenic purpura are localized to α_IIb between amino acids L1 and Q459

In chronic immune thrombocytopenic purpura (ITP), autoantibodies bind to platelet surface proteins, particularly α_IIb, resulting in platelet destruction by the reticulo-endothelial system. In order to better localize the autoepitopes on α_IIb, we studied the binding of antibodies to Chinese hamster ovary (CHO) cells expressing either α_IIbβ₃ or α_IIb-α_vβ₃ chimaeras in which a segment of α_IIb (either amino acids L1-Q459, L1-F223 or F223-Q459) was substituted for that portion of α_v. We evaluated platelet-associated autoantibodies from 14 ITP patients with α_IIb-dependent antibodies. Ten of 14 bound to α_{IIb (L1-Q459)}-α_vβ₃, showing that autoepitopes were often localized to this region of α_IIb. In addition, each of the autoantibodies binding to α_{IIb (L1-Q459)}-α_vβ₃, also bound to CHO cells expressing either α_IIb(L1-F223)-α_vβ₃ or α_{IIb(F223-Q459)}-α_vβ₃). In two of the three eluates tested, > 95% of the autoantibody binding to α_IIb could be adsorbed using CHO cells expressing any of the three chimaeras, showing that the epitope(s) have contact points on either side of amino acid F223; in the third eluate, only a portion (∼40%) could be adsorbed by the chimaeric cell lines showing that, in this patient, an additional antibody was also present, directed to a site distal to amino acid Q459. The remaining four eluates bound to CHO cells expressing α_IIbβ₃ but to none of the chimaeras, suggesting that these epitopes are also distal to amino acid Q459. We conclude that the binding of many anti-α_IIbβ₃ autoantibodies is dependent on the presence of α_IIb amino acids L1-Q459.