The biallelic human platelet alloantigen (HPA) 1 system encodes a leucine to proline substitution at position 33 in the beta3 integrin. Homozygous individuals can be immunized by the non-self allele-encoded protein following transfusion or during pregnancy. In post-transfusion purpura (PTP), a subsequent recall alloantibody response against the non-self form of beta3 is paralleled by the destruction of autologous platelets, leading to profound thrombocytopenia. Although serological evidence suggests platelet autoantibodies are responsible, such autoantibodies are poorly defined. We used variable gene phage display to isolate alphaIIbbeta3 autoantibodies formed in the acute phase of PTP and determined the epitopes recognized. An immunoglobulin G (IgG)-encoded variable heavy-chain domain (VH) gene repertoire containing 4.7 x 10(7) single-chain Fv fragments was cloned and three alphaIIbbeta3 antibodies were isolated (clones 2F2, E3 and B12). All three used different VH genes with a low level of somatic mutation for genes derived from gamma-encoding mRNA. Two (2F2 and E3) recognized an overlapping epitope and their binding was inhibited by sera from patients with PTP; all three recognized Ca2+-dependent compound epitopes on alphaIIbbeta3. Our results support the theory that a transient loss of tolerance for alphaIIbbeta3 with autoantibody formation occurs in PTP.