The platelet antigens, Pl(A2) and Pl(A2), are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the Pl(A1) form of GPIIIa into the expression vector pGEX1. To express the Pl(A2) antigen, nucleotide 196 of the Pl(A1) coding sequence was mutated to the Pl(A2) allelic form. When transformed and induced in Escherichia coli, the vivo constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (Pl(A1)), the other proline (Pl(A2)). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the Pl(A1) fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the Pl(A1) fusion protein competes for all of the anti- Pl(A1) antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional Pl(A1) epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-Pl(A1) antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to Pl(A1) alloantigens.
ASJC Scopus subject areas
- Cell Biology