Human platelet antigen 1a epitopes are dependent on the cation-regulated conformation of integrin α(IIb)β(3) (GPIIb/IIIa).
BACKGROUND
The HPA-1a (Leu(33)) polymorphism of platelet integrin αIIbβ3 is the target of alloantibodies in 70-80% cases of neonatal alloimmune thrombocytopenia (NAIT) in Caucasians and reliable detection of these antibodies is essential for appropriate clinical management. However, the ability to detect such antibodies is highly variable between laboratories and, in a number of clinical cases where there is a HPA-1 genotype mismatch between mother and neonate, HPA-1a antibodies are undetectable. Furthermore, some studies have not shown a consistent relationship between maternal anti-HPA-1a level and neonatal platelet count. Since the integrity and conformation of the αIIbβ3 complex are dependent on divalent cations, we investigated whether HPA-1a epitope integrity and/or conformation might be affected by the presence of the cation chelator EDTA in patient samples or in assay buffers, thus providing a possible explanation for the variable sensitivity of current assays.


RESULTS
Exposure of the αIIbβ3 complex to EDTA resulted in reduced reactivity of three anti-HPA-1a mAbs (B2, 19-7 and 23-15). More significantly, cation chelation adversely affected detection of polyclonal anti-HPA-1a, not only in the platelet immunofluorescence assay, where alloantibody binding was reduced compared to control platelets (mean MFI reduction 44.5%, range 17.3-69.7%, n=4), but also in the commonly used monoclonal antibody specific immobilisation of platelet antigens assay (MAIPA) where both alloantibody and monoclonal capture antibody binding were reduced (mean OD reduction 82.8%, range 68.3-96.6%, n=9).


CONCLUSIONS
These data show that HPA-1a antibodies recognise epitopes on αIIbβ3 that are sensitive to EDTA treatment and that cation chelation grossly reduces the sensitivity of the MAIPA assay by diminishing not only HPA-1a alloantibody binding but also 'capture' monoclonal antibody binding. These findings may, in part, explain the current variability in antibody measurement and will guide the development of more sensitive tests for anti-integrin antibodies in NAIT and other conditions.
NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK. dave.allen@ndcls.ox.ac.uk
David L
DL
Allen
Sofia
S
Abrahamsson
Michael F
MF
Murphy
David J
DJ
Roberts
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016824:Antigens, Human Platelet / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001792:Blood Platelets / Q000276:immunology / Q000378:metabolism*; D004492:Edetic Acid / Q000378:metabolism; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D039661:Integrin beta3; D007502:Iron Chelating Agents / Q000378:metabolism*; D007518:Isoantibodies / Q000276:immunology / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D016824:Antigens, Human Platelet; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007502:Iron Chelating Agents; D007518:Isoantibodies; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D004492:Edetic Acid