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The platelet antigens, PlA1 and PlA2, 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 PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). 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 PlA1 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 PlA1 fusion protein competes for all of the anti-PlA1 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 PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

Neonatal alloimmune thrombocytopenia (NATP) and post-transfusion purpura (PTP) are acquired bleeding disorders caused by alloimmune thrombocytopenia. In most cases, the thrombocytopenia is due to an alloantibody directed against the platelet glycoprotein IIb-IIIa (GPIIb-IIIa) complex. During the course of routine studies on the role of GPIIb-IIIa in inherited and acquired bleeding and thrombotic disorders, we unexpectedly identified an individual whose platelets reacted by non-reduced Western blot analysis with anti-GPIIIa polyclonal antisera, but did not react with a commercially available monoclonal antibody (SZ21) specific for GPIIIa. We screened all 14 GPIIIa exons for possible nucleotide changes which might alter amino acids and found variations in only exons 3 and 10. Nucleotide sequencing revealed that only the exon 3 alteration changed the predicted amino acid sequence. This variation was caused by homozygosity for the uncommon P1A2 allele of the GPIIIa gene. Platelets from two additional unrelated normal individuals known to be homozygous for P1A2 also lacked reactivity with SZ21 by Western blot. Using flow cytometry with intact platelets, we observed a markedly reduced binding of SZ21 to platelets with the P1A2 genotype. Scatchard analyses indicated that SZ21 bound to P1A1/A1 platelets with a Kd of approximately 8.26 x 10(-10) M, and to P1A2/A2 platelets with a Kd of approximately 5.58 x 10(-9) M. Thus, we have characterized a readily available monoclonal antibody able to distinguish between the two P1A alleles of the GPIIIa gene. Because incompatibility for this platelet polymorphism is the most common cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura, and because platelet immunophenotyping reagents lack specificity and are not easily available, this monoclonal antibody could facilitate the management of patients with these disorders.

Baka is a platelet alloantigen whose putative allele, Bakb, has not been identified previously. By using a serum, “Har,” obtained from a patient with posttransfusion purpura, we describe the platelet alloantigen Bakb. The Har serum reacted with an NP-40-extractable platelet membrane protein of 142 kd with mobility similar to platelet glycoprotein IIb alpha. We found that the antigen recognized by the Har serum is inherited in an autosomal dominant mode with an apparent gene frequency of .39. Chi-square analysis of observed and expected phenotype frequencies indicated that serum Har recognizes Bakb, the anticipated allele of Baka. Our findings provide new evidence for polymorphism of glycoprotein IIb and for the association of posttransfusion purpura with alloimmunization to determinants on this glycoprotein.

Posttransfusion purpura typically occurs in PLA1 negative blood recipients who have been previously immunized to the PLA1 antigen. Following transfusion, severe thrombocytopenia develops with the formation of anti-PLA1. Since the patients' platelets lack the PLA1 antigen, one would not expect this antibody to destroy autologous platelets. In this study we show that PLA1 antigen exists in stored blood and can absorb to PLA1 negative platelets making them PLA1 reactive. Incubating PLA1 (-) platelets with ultracentrifuged plasma from PLA1 (+) blood donors allowed anti-PLA1 to bind to PLA1 (-) platelets. Control plasma from PLA1 (-) blood donors did not lead to anti-PLA1 binding. Using an inhibition assay, we showed that stored blood contains PLA1 material that was not removed by ultracentrifugation. The material absorbing to PLA1 (-) platelets represented the PLA1 antigen, which was confirmed by Western blotting. After incubating plasma containing PLA1 antigen with PLA1 (-) platelets, reactivity at 95,000 D was observed. Native PLA1 (+) platelets showed a similar band. When PLA1 (-) platelets were incubated with plasma from a PLA1 (-) donor, this band was not present. These studies show that a soluble form of PLA1 antigen exists in stored blood that can absorb to PLA1 (-) platelets. Consequently, anti-PLA1 can bind to these platelets leading to thrombocytopenia. These observations may explain the autologous destruction of platelets in posttransfusion purpura.