The Pla Alloantigen System Is a Sensitive Indicator of the Structural Integrity of the Amino-terminal Domain of the Human Integrin β3 Subunit

Within the native integrin αIIbβ3, the conformation of the amino-terminal domain of the β3 subunit has a significant influence on the availability of the Leu33/Pro33 polymorphism that defines the A1 and A2 alleles, respectively, of the PlA alloantigen system. The majority of anti-PlA1 IgG antibodies, affinity-purified by adsorption to either (A1/A1)-platelets or purified αIIb(A1)β3, fail to bind to the Leu33 polymorphic loop within Cys26-Cys38 in native β3 unless this sequence is maintained in a proper orientation by two noncontiguous human β3 sequences. By comparing IgG binding to recombinant β3 molecules composed of segments of human, Xenopus and avian sequences expressed by Spodoptera frugiperda cell lines, we have found that neither region alone is sufficient for full expression of the epitope. One sequence, that most proximal to Leu33/Pro33, lies within β3[54-133], and a second distal sequence is located within β3[435-490]. Proximity of the distal sequence to the Cys26-Cys38 loop in native β3 is confirmed by the fact that both anti-PlA1 IgG and the murine monoclonal antibody SZ21, specific for β3[28-35], completely inhibit the binding of rabbit polyclonal IgG specific for the sequence β3 [479-485]. Anti-PlA1 IgG antibodies from all of 7 donors selectively bind to the epitope within the native conformation of β3. However, in one of the seven donors, roughly 60% of the anti-PlA1 IgG binds exclusively to (A1)β3, and not to either denatured (A2)β3 or nondentaured (native) (A1)β3. This IgG subpopulation cannot be purified by adsorption to intact (A1/A1)-platelets or to αIIb(A1)β3 integrin. The physiologic relevance of this exceptional type of anti-PlA1 antibody is debatable, but the presence of IgG with this specificity only in an immunized subject suggests that the epitope may well be presented on a "denatured" form of β3 in vivo. Regardless, our results indicate that the amino-terminal domain of the native β3 molecule associates with noncontiguous β3 sequences, and the epitope recognized by a majority of anti-PlA1 antibodies is a sensitive indicator of correct tertiary structure within this region of β3.