HPA-1a phenotype–genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet β3 integrin that disrupts the HPA-1a epitope

Abstract

IntroductionThe ␤3 integrin subunit (glycoprotein [GP] IIIa, CD41) forms a heterodimeric complex with the ␣IIb integrin subunit (GPIIb) on the surface of platelets (␣IIb␤3, GPIIb/IIIa, CD61/41) and functions as a major fibrinogen receptor. Activation of ␣IIb␤3 occurs through so-called inside-out signaling that follows the binding of platelet receptors to components of the subendothelial cell matrix (eg, the binding of ␣ 2 ␤ 1 and GPVI to collagen) or soluble ligands (eg, adenosine diphosphate and thrombin). The activated conformation of ␣IIb␤3 binds fibrinogen, fibronectin, and vitronectin and has a pivotal role in clot formation after blood vessel damage. 1,2 The gene encoding ␤3 integrin has several single nucleotide polymorphisms (SNPs) that result in single amino acid substitutions of immunologic, and possibly functional, consequence. 3 Platelet alloantigen systems encoded by SNPs in the ␤3 integrin gene are of clinical relevance. The C196T SNP, encoding for a Leu33Pro substitution, is the most immunogenic human platelet alloantigen (HPA) system. 4 In ␤3Leu33-negative (ie, ␤3Pro33 homozygous), HLA-DRB3*0101-positive persons, exposure to the ␤3Leu33 form is highly immunogenic and alloimmunization causes neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet refractoriness. 3,[5][6][7] Alloimmunization against ␤3Leu33 occurs in 1 in 365 pregnant women, and ␤3Leu33-specific maternal alloantibodies (anti-HPA-1a) cause severe thrombocytopenia in 1 in 1100 neonates. 8,9 In such cases, the treatment of choice is ␤3Leu33-negative donor platelets, the provision of which requires the phenotyping of large numbers of donors. 5,10 We have previously reported on a recombinant human immunoglobulin (Ig)G1 specific for ␤3Leu33, which can be used for large-scale donor phenotyping. [11][12][13] In the process of phenotyping more than 6000 donors using this assay, we identified one donor with a ␤3Leu33 weak phenotype but a heterozygous genotype. Here we describe the molecular basis of this unique phenotype, suggesting that Arg93 of the ␤3 integrin contributes to the formation of the HPA-1a B-cell epitope. Patients, materials, and methods Donor samplesMore than 6000 EDTA-anticoagulated whole blood donor samples were ␤3Leu33 phenotyped by enzyme-linked immunosorbent assay (ELISA) with our recombinant antibody CAMTRAN-007 as described previously. 11 A single donor (Donor A) with a ␤3Leu33 weak phenotype and a heterozygous genotype was identified. Genomic DNA samples from healthy apheresis donors were from the National Blood Service donor DNA repository. Informed consent was obtained for all samples. Monoclonal antibody immobilization of platelet antigensThe binding of human polyclonal anti-␤3Leu33 and anti-␤3Pro33 was studied using monoclonal antibody immobilization of platelet antigens (MAIPA) with platelets from healthy donors and Donor A. 18,19 MAIPA was performed using platelet-rich plasma obtained from citrate-anticoagulated donor blood samples and the mAb NIBSC-85/661 to specifically capture ␣IIb␤3 fro...

Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics

“…Polymorphic substitution of the distally located residue Arg 93 at the hybrid/PSI interface (Fig. 5f) disrupts the HPA-1a epitope 40 , demonstrating the importance of the interface for structural integrity of the PSI domain. The I-EGF1 domain also participates in the HPA-1 epitope 41 , further emphasizing the tight linkage between the hybrid, PSI and I-EGF1 domains.…”

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

“…The binding of HPA-1a phage antibodies to human (donor from NHSBT Cambridge, UK) and dog (from M. Coetezee, University of Cambridge, UK) platelets was detected by the platelet immunofluorescence test [24], and fluorescence was recorded on a Beckman-Coulter XL-MCL, running SYSTEM II software (Beckman Coulter, High Wycombe, UK), as previously described [11].…”

“…Truncated and soluble b 3 integrin peptides, either on their own or complexed with a IIb , have previously been expressed, and it has been suggested that the 66 N-terminal residue plexin/semaphorin/integrin (PSI) domain, which contains the HPA-1 polymorphism at position 33, may suffice for HPA-1 antibody detection [9]. However, studies from Valentin and colleagues and more recently from our laboratory have unequivocally shown that certain categories of HPA-1a antibodies, so-called type II, require residues in the hybrid and epidermal growth factor (EGF) domains for binding [10,11].…”

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

“…Previous studies highlighted the potential of this approach by demonstrating the detection of HPA-1 antibodies using Escherichia coli expressed mini-b3 (residues 1-66) and HPA-2 antibodies using a truncated version (residues 1-289) of insect cell expressed GPIba [33,34]. However, we and others have shown that the previously proposed mini-b3 recombinant protein for HPA-1 antibody is unsuitable because residues from domains distant of the PSI domain are critical to the integrity of the HPA-1 epitope [35,36].…”