| Abstract | BACKGROUND: The single-nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen-1 (HPA-1) system encoding a Leu (HPA-1a) or Pro (HPA-1b) at position 33. HPA-1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires alpha(IIb)beta3 integrin from the platelets of HPA-genotyped donors. OBJECTIVES: We set out to define the beta3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble beta3 peptides for HPA-1 antibody detection. METHODS: We designed two sets (1a and 1b) of four soluble beta3 domain-deletion peptides (deltaSDL, deltabetaA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA-1a-specific phage antibodies were defined by analyzing binding patterns to the beta3 peptides and canine platelets, and models of antibody-antigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). RESULTS: Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight beta3 peptides. Testing of FMAIT samples indicated that deltabetaA-Leu33 is the superior peptide for HPA-1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA-1a antibodies was confirmed by the study of HPA-1a phage antibody footprints and the reactivity pattern of clinical samples with the four beta3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. CONCLUSIONS: Soluble recombinant beta3 peptides can be used for detection of clinical HPA-1a antibodies. |
| Authors | P Stafford, C Ghevaert, K Campbell, C Proulx, G Smith, L M Williamson, E Ranasinghe, N A Watkins, J A Huntington, W H Ouwehand
(Affiliation: Department of Haematology, University of Cambridge, Cambridge, UK.)
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| Journal | Journal of thrombosis and haemostasis : JTH
(J Thromb Haemost)
Vol. 6
Issue 2
Pg. 366-75
(Feb 2008)
ISSN: 1538-7836 England |
| PMID | 18045240
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
|
| Chemical References |
- 1a alloantigen, human
- Antigens, Human Platelet
- Epitopes
- Integrin beta3
- Isoantibodies
- Platelet Glycoprotein GPIIb-IIIa Complex
- Recombinant Fusion Proteins
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| Topics |
- Animals
- Antigen-Antibody Reactions
- Antigens, Human Platelet
(chemistry, genetics, immunology)
- Blood Platelets
(metabolism)
- Dogs
- Enzyme-Linked Immunosorbent Assay
- Epitopes
(chemistry, immunology)
- Female
- Humans
- Infant, Newborn
- Integrin beta3
(chemistry, genetics, immunology)
- Intracranial Hemorrhages
(etiology, immunology)
- Isoantibodies
(blood, chemistry, immunology)
- Models, Molecular
- Platelet Glycoprotein GPIIb-IIIa Complex
(chemistry, immunology)
- Polymorphism, Single Nucleotide
- Pregnancy
- Protein Binding
- Protein Conformation
- Protein Interaction Mapping
- Protein Structure, Tertiary
- Recombinant Fusion Proteins
(immunology)
- Sequence Deletion
- Thrombocytopenia, Neonatal Alloimmune
(diagnosis, immunology)
|
