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



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.



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).



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.



Soluble recombinant beta3 peptides can be used for detection of clinical HPA-1a antibodies.
Stafford
P
P
Department of Haematology, University of Cambridge, Cambridge, UK.
Ghevaert
C
C
Campbell
K
K
Proulx
C
C
Smith
G
G
Williamson
L M
LM
Ranasinghe
E
E
Watkins
N A
NA
Huntington
J A
JA
Ouwehand
W H
WH
D000818:Animals; D000937:Antigen-Antibody Reactions; D016824:Antigens, Human Platelet / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001792:Blood Platelets / Q000378:metabolism; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D039661:Integrin beta3 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D020300:Intracranial Hemorrhages / Q000209:etiology / Q000276:immunology; D007518:Isoantibodies / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D008958:Models, Molecular; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000276:immunology; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017384:Sequence Deletion; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000175:diagnosis / Q000276:immunology*
D016824:Antigens, Human Platelet; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007518:Isoantibodies; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins