Deletion analysis of the 66–amino acid segment from the N-terminus of human GPIIIa that is recognized by anti–HPA-1a antibodies. (A) Amino acid sequence of the immunoreactive 66–amino acid segment is shown above. *Site of the human polymorphism. Proposed loops of the cloverleaf structure are indicated, loop 1 (dashed line), loop 2 (solid line), and loop 3 (dashed and dotted line) with the proposed disulfide bonds between Cys₁₃-Cys₂₃, Cys₂₆-Cys₃₈, and Cys₁₆-Cys₄₉ shown with solid lines above the sequence. The deletion mutations and their sequences are shown below. (B) Results of an ELISA are graphically displayed. Letters (a-g) refer to deletion mutations illustrated in A. In this assay, microtiter wells were coated in triplicate with the deletion mutations (expressed as GST fusion proteins). An anti–HPA-1a antiserum was tested for its ability to bind the various human mutations. Antibodies bound were detected using Protein A/G conjugated with HRP, and TMB (tetramethylbenzidine) was used as the colorometric substrate. The average of the absorption at 450 nm of the triplicate was divided by the average of duplicate wells reacted with anti-GST. (C) Immunoblot (top box) of the deletion mutations incubated with the same anti–HPA-1a antiserum tested in ELISA, and corresponding Coomassie blue–stained SDS-polyacrylamide gel electrophoresis (PAGE) (bottom box) of deletion mutations. Molecular weight of the intact fusion protein, h3a 1-66, is indicated on the left of the blot and gel.