Substitution of murine GPIIIa residues with analogous human amino acids to generate an HPA-1a–like epitope. (A) Comparison of first 66 amino acids from the N-termini of human and murine GPIIIa. Amino acid differences are highlighted and italicized. *Site of human polymorphism for HPA-1a. Underlined sequence refers to the shortest human fragment that maintains the majority of immunoreactivity with clinical anti–HPA-1a antibodies. (B) By site-directed mutagenesis, several amino acids in the N-terminus of the murine GPIIIa were mutated to the human counterpart. The N-terminal regions containing the substitution mutations (left) were expressed as GST fusion proteins (37 kD), electrophoresed on 10% to 15% SDS-PAGE and transferred onto nitrocellulose membranes. Immunoblot (top, right) of murine GPIIIa substitution mutations incubated with an affinity-purified anti–HPA-1a antibody is shown. The same set of proteins (bottom, right) stained with Coomassie blue. Smaller band on gel is contaminating GST protein.9