Immunologic and structural analysis of eight novel domain-deletion beta3 integrin peptides designed for detection of HPA-1 antibodies.

STAFFORD, P., GHEVAERT, C., CAMPBELL, K., PROULX, C., SMITH, G., WILLIAMSON, L. M., RANASINGHE, E., WATKINS, N. A., HUNTINGTON, J. A. and OUWEHAND, W. H. (2008). Immunologic and structural analysis of eight novel domain-deletion beta3 integrin peptides designed for detection of HPA-1 antibodies. Journal Of Thrombosis And Haemostasis, 6 (2), 366-375.

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Link to published version:: 10.1111/j.1538-7836.2008.02858.x

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.

Item Type: Article
Research Institute, Centre or Group: Biomolecular Sciences Research Centre
Identification Number: 10.1111/j.1538-7836.2008.02858.x
Depositing User: Jamie Young
Date Deposited: 03 Jun 2015 13:29
Last Modified: 03 Jun 2015 13:29
URI: http://shura.shu.ac.uk/id/eprint/9999

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