Effect of single amino acid substitutions on the formation of the PIA and bak alloantigenic epitopes

Abstract

The subunits that comprise the platelet-specific integrin αIIbβ3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PIA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PIA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PIA2-associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PIA-specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PIA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PIA1 and PIA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 ↔ Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes. © 1991 by The American Society of Hematology.