Abstract
A platelet glycoprotein (GP) IIIa epitope library was constructed by insertion of randomly cleaved GPIIIa cDNA fragments in the prokaryotic expression vector lambda gt22 and screened with purified anti-PlA1 antibodies for clones expressing a PlA1 epitope. Five independent clones were isolated and characterized by nucleotide sequencing. The smallest anti-PlA1 reactive clone obtained encoded the amino terminal 66 residues of mature GPIIIa. Substitution of leucine33 (PlA1) with a proline33 (PlA2) by in vitro mutagenesis resulted in the loss of anti- PlA1 reactivity; however, this clone still reacted with anti-GPIIIa polyclonal antibodies. These data indicate that a PlA1 alloantigenic epitope is located within a small, unglycosylated fragment of GPIIIa containing the polymorphism responsible for the PIA phenotype. Furthermore, these results prove that small recombinant mimics of a PlA1 epitope may be synthesized and used for detection of these alloantibodies.
