Immunologic and structural analysis of eight novel domain-deletion β₃integrin peptides designed for detection of HPA-1 antibodies

Abstract

WH. Immunologic and structural analysis of eight novel domain-deletion b 3 integrin peptides designed for detection of HPA-1 antibodies. J Thromb Haemost 2008; 6: 366-75.See also Stafford P, Garner SF, Huiskes E, Kaplan C, Kekomaki R, Santoso S, Tsuno NH, Watkins NA, Ouwehand WH. Three novel b3 domaindeletion peptides for the sensitive and specific detection of HPA-4 and six low frequency b3-HPA antibodies. This issue, pp 376-83.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 a IIb b 3 integrin from the platelets of HPAgenotyped donors. Objectives: We set out to define the b 3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble b 3 peptides for HPA-1 antibody detection. Methods: We designed two sets (1a and 1b) of four soluble b 3 domain-deletion peptides (DSDL, DbA, 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 b 3 peptides and canine platelets, and models of antibodyantigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAI-T). Results: Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight b 3 peptides. Testing of FMAIT samples indicated that DbA-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 b 3 -Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant b 3 peptides can be used for detection of clinical HPA-1a antibodies.

Three novel β3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency β3-HPA antibodies

“…4A, B), as expected, as the epitope recognized by this antibody was previously localized to the plexin-semaphorinintegrin (PSI) domain of b3 (residues 1-54) [22] which remained unaltered in three new b3 fragments. Similarly, SZ21, a pseudo anti-HPA-1a [30], bound equally well to all three b3 fragments, while as expected the negative control antibody RFGP56 against aIIbb3 did not bind to any of the three peptides (Fig.…”

“…MoAb SZ21 which has pseudo anti-HPA-1a specificity [30], and the human recombinant anti-HPA-1a CamTran007 [22,36] both require residues outside the PSI domain, and showed normal binding to the three new DSDL constructs, further corroborating their structural integrity. The expression of b3-DSDL-8bw demonstrated that the introduction of Cys636 in the bTD domain did not affect the secretion of monomeric protein by S2 cells.…”

“…We believe that this reflects alteration of the specific Y2/51 epitope in the DSDL-SuperRare fragment, rather than an overall lack of structural integrity. Previous studies from our laboratory with four b3 domain deletion fragments indicate that residues from the hybrid domain are required for an intact Y2/51 epitope [22]. Data from the current study indicates that either residue 62 (HPA-10) or 407 (HPA-7) are critical to antibody binding, and as Cos-7 cells transfected with the ITGB3*003 (Gln62, HPA10bw) allele have already been shown to have normal reactivity with Y2/51 [37], it appears that the replacement at residue 407 is critical to the loss of reactivity.…”

“…In an attempt to overcome this problem, we have recently made a new generation of CaM-tagged b3-Leu33 and b3-Pro33 domain deletion fragments and demonstrated that the bAdeletion mutants are the best targets for the sensitive and specific detection of HPA-1 antibodies [22]. We also observed that the near full-length extracellular domain of b3, consisting of residues 1-690, can be successfully expressed in Drosophila S2 cells as a monomer by deleting the specificity determining loop (residues 160-188) and by fusion to CaM.…”

“…Generation of recombinant b3 peptides: b3-DSDL-4b, DSDL8bw, and DSDL-SuperRare Recently, we have reported the successful expression of two recombinant b3 peptides, from which the bA domain was deleted, for the detection of HPA-1 antibodies [22]. However, two out of the nine immunologically relevant sequence variants in the ITGB3 gene (ITGB3*004 and ITGB3*005) reside in the segment coding the bA domain (Fig.…”