Construction, expression and antigenic characterisation of recombinant human platelet antigen-1 (HPA-1)

• Main Author: Anani Sarab, Gholamreza
• Published: University of Aberdeen 2010
• Subjects: 616.079; Blood Platelets
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.531846

Previously it has been shown that sequences containing both Trp²⁵ and Leu³³ are the most effective at inducing Th cell proliferation in HLA-DRB3*0101 positive women, alloimmunised with anti-HPA-1a.  The Leu³³/Pro³³ polymorphism is embedded in the N-terminal plexin/semaphorin/integrin (PSI) domain of GPIIIa.  In the present study, amino acids 1-62 of the GPIIIa (Leu³³ or Pro³³) PSI domain were cloned into the vector pGEX-6p-1.  The recombinant proteins were expressed and tested by ELISA, Luminex and Absorption Assays.  The presence of the HPA-1a/-1b epitope was confirmed by the ability of PSI-Leu³³/-Pro³³ recombinant fragments to specifically capture its corresponding HPA-1 antibody.  Cells from a human B cell line (HHKB), homozygous for HLA-DRB3*0101, were pulsed with the recombinant PSI domain fragment of GPIIIa expressing the HPA-1a antigen.  MHC class II/peptide complexes were isolated from the pulsed cells using an immunoaffinity column.  A nested set of naturally processed and presented HPA-1a derived peptides, each containing the residues Trp²⁵ – Leu³³ core epitope was identified.  For the first time a naturally processed and presented HPA-1a peptide that spans the HPA-1a polymorphism has been identified, bound to the class II molecule encoded by HLA-DRB3*0101.  The efficient processing and presentation of this peptide, which includes the putative dominant Th epitope, is likely to be an important contributory factor in the immunogenicity of HPA-1a.  Such peptides may also provide the basis for novel treatments to tolerise the corresponding Th response in HPA-1b1b women at risk of NAIT with an HPA-1a-positive fetus.