HPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA-1a epitope
| Autor: | Graham A. Smith, David L. Allen, Elisabeth Schaffner-Reckinger, Michael F. Murphy, Willem H. Ouwehand, Nicolaas H. C. Brons, Nelly Kieffer, Graham J. Howkins, Paul Metcalfe, Nicholas A. Watkins |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
endocrine system
Genotype medicine.drug_class Immunology CHO Cells Platelet Glycoprotein GPIIb-IIIa Complex Platelet Membrane Glycoproteins Monoclonal antibody Platelet membrane glycoprotein Transfection Biochemistry Polymorphism Single Nucleotide Epitope Antigen-Antibody Reactions Antigens CD Cricetinae medicine Animals Humans Mass Screening Antigens Human Platelet Linear epitope biology Integrin beta3 Antibodies Monoclonal Cell Biology Hematology Sequence Analysis DNA Molecular biology Human platelet antigen Phenotype Amino Acid Substitution Polyclonal antibodies biology.protein Epitopes B-Lymphocyte Antibody |
| Popis: | A single nucleotide polymorphism (SNP) at position 196 in the beta 3 integrin causes a Leu33Pro substitution in the mature protein. Alloimmunization against the beta 3Leu33 form (human platelet antigen [HPA]-1a, Pl(A1), Zw(a)) in patients who are beta 3Pro33 homozygous (HPA-1b1b, Pl(A2A2), Zw(bb)) causes neonatal alloimmune thrombocytopenia, posttransfusion purpura, or refractoriness to platelet transfusion. Studies with recombinant proteins have demonstrated that amino acids 1 to 66 and 288 to 490 of the beta 3 integrin contribute to HPA-1a epitope formation. In determining the HPA-1a status of more than 6000 donors, we identified a donor with an HPA-1a(weak) phenotype and an HPA-1a1b genotype. The platelets from this donor had normal levels of surface alpha IIb beta 3 but reacted only weakly with monoclonal and polyclonal anti-HPA-1a by whole blood enzyme-linked immunosorbent assay (ELISA), flow cytometry, and sandwich ELISA. We reasoned that an alteration in the primary nucleotide sequence of the beta 3Leu33 allele of this donor was disrupting the HPA-1a epitope. In agreement with this hypothesis, sequencing platelet RNA-derived alpha IIb and beta 3 cDNA identified a novel G/A SNP at position 376 of the beta 3 integrin that encodes for an Arg93Gln replacement in the beta 3Leu33 allele. Coexpression of the beta 3Leu33Gln93 encoding cDNA in Chinese hamster ovary cells with human alpha IIb cDNA showed that the surface-expressed alpha IIb beta 3 reacted normally with beta 3 integrin-specific monoclonal antibodies but only weakly with monoclonal anti-HPA-1a. Our results show that an Arg93Gln mutation in the beta 3Leu33 encoding allele disrupts the HPA-1a epitope, suggesting that Arg93 contributes to the formation of the HPA-1a B-cell epitope. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|