Involvement of the EGF Domain in the Formation of the Human Platelet Alloantigen, HPA-1a

The human platelet alloantigen, HPA-1a (also known as Pl^A1), is most frequently responsible for several clinically important alloimmune platelet disorders, including neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura, and less frequently, platelet transfusion refractoriness. A single C29523T nucleotide substitution, resulting in a Leu33Pro amino acid polymorphism within PSI domain of the integrin β3 subunit, controls the expression of the HPA-1a/HPA-1b alloantigenic epitopes. Small linear or cyclic synthetic peptides encompassing amino acid 33, however, fail to bind human anti-HPA-1a alloantibodies, leading to the notion that the HPA-1a antigenic determinant is conformationally dependent, and may require noncontiguous regions of the polypeptide chain that are linearly distal to polymorphic residue 33. This concept is supported by the finding that antibodies from some alloimmunized women fail to recognize a Cys₄₃₅Ala mutant form of β3, and by the observation that the PSI domain of β3 is immediately adjacent in the crystal structure of β3 to the first epidermal growth factor (EGF1 - residues 436-472) domain of the molecule. The purpose of the present investigation, therefore, was to localize the amino acid(s) within EGF1 that contribute to the HPA-1a alloantibody binding interface. Two complementary approaches were employed. In the first, site-directed mutagenesis was used to introduce four amino acid substitutions (T30A, S32P, Q33L, and N39D) into mouse β3 cDNA that have previously been shown to be necessary for reconstituting the human anti-HPA-1a epitope into the murine protein. The resulting mouse β3 integrin subunit containing this “humanized” PSI domain (termed APLDmβ3) was able to bind both the murine HPA-1a-selective monoclonal antibody, SZ21, as well as human maternal anti-HPA-1a antisera. Structural analysis indicates that S₄₆₉ and Q₄₇₀ at the C-terminus of EGF1 are immediately adjacent to Leu₃₃ in human β3. Ser469 is conserved in the mouse, however residue 470 is a methionine in the murine protein. Based on these observations, we introduced an additional M₄₇₀Q amino acid substitution within APLDmβ3 to produce APLDQmβ3. As predicted, binding of mAb SZ21 and human anti-HPA-1a alloantisera to HEK293 cells expressing an aIIb/APLDQmβ3 complex was increased ~30% over that which bound to cells expressing similar levels of aIIb/APLDmβ3. Conversely, substitution of M for Q at position 470 within human β3 resulted in a marked reduction in the ability of SZ21 and human anti-HPA-1a alloantisera to bind. Taken together, our results demonstrate that this region of the conformationally close EGF1 domain contributes importantly to the expression of the HPA-1a alloantigenic determinant. These findings may have important implications for diagnostic and therapeutic use of synthetic or recombinant HPA-1a mimetics.