These authors should be considered as joint first authors.
Immunologic and structural analysis of eight novel domain-deletion β3 integrin peptides designed for detection of HPA-1 antibodies
Version of Record online: 14 OCT 2009
© 2007 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 6, Issue 2, pages 366–375, February 2008
How to Cite
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 β3 integrin peptides designed for detection of HPA-1 antibodies. Journal of Thrombosis and Haemostasis, 6: 366–375. doi: 10.1111/j.1538-7836.2008.02858.x
- Issue online: 14 OCT 2009
- Version of Record online: 14 OCT 2009
- Received 4 September 2007, accepted 8 November 2007
- β3 integrin deletion peptides;
Summary. 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 αIIbβ3 integrin from the platelets of HPA-genotyped donors.Objectives: We set out to define the β3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble β3 peptides for HPA-1 antibody detection.Methods: We designed two sets (1a and 1b) of four soluble β3 domain-deletion peptides (ΔSDL, ΔβA, 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 β3 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 β3 peptides. Testing of FMAIT samples indicated that ΔβA-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 β3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant β3 peptides can be used for detection of clinical HPA-1a antibodies.