AN AMINO-ACID POLYMORPHISM WITHIN THE RGD BINDING DOMAIN OF PLATELET MEMBRANE GLYCOPROTEIN-IIIA IS RESPONSIBLE FOR THE FORMATION OF THE PEN(A) PEN(B) ALLOANTIGEN SYSTEM

The human Pen(a)/Pen(b) alloantigen system represents a naturally occurring polymorphism of human platelet membrane glycoprotein (GP) IIIa, and has previously been implicated in the onset of two important clinical syndromes, neonatal alloimmune thrombocytopenic purpura and posttransfusion purpura. To investigate the molecular basis of the polymorphism underlying the Pen alloantigen system, we used the polymerase chain reaction to amplify platelet-derived GPIIIa mRNA transcripts. DNA sequence analysis of amplified GPIIIa cDNAs from nucleotides 161 to 1341 (encompassing amino acid residues 22414) revealed a G526 <-> A526 polymorphism that segregated precisely with Pen phenotype in twelve other individuals examined. This nucleotide substitution results in an Arg (CGA) to Gln (CAA) polymorphism at amino acid 143 of GPIIIa. Interestingly, this polymorphic residue is located within the putative RGD binding site (residues 109-171) of GPIIIa. Platelet aggregation patterns of a Pen(b/b) individual, however, were nearly normal in response to all physiological agonists tested, indicating that this polymorphism does not grossly affect integrin function. Short synthetic peptides encompassing residue 143 were unable to mimic either the Pen(a) or Pen(b) antigenic determinants, suggesting that the Pen epitopes are dependent upon proper folding of the polypeptide chain. Finally, we constructed allele-specific recombinant forms of GPIIIa that differed only at amino acid residues 143. Whereas anti-Pen(a) alloantibodies were able to recognize the Arg143 recombinant form of GPIIIa, anti-Pen(b) antibodies were not. Conversely, anti-Pen(b) alloantibodies were reactive only with the Gln143 isoform of the GPIIIa molecule. It thus appears that amino acid 143 of GPIIIa not only associated with Pen phenotype, but specifically controls the formation and expression of the Pen alloantigenic determinants.