Distinct Binding Characteristics of Pathogenic Anti-platelet Factor 4/polyanion Antibodies to their Antigen Coated on Different Substrates

Scientific Research Question: Anti-PF4/heparin antibodies (anti-PF4/H Abs) are rather frequent in patients treated with heparin, but only a subset of these antibodies can cause heparin-induced thrombocytopenia (HIT). Enzyme immuno-assays using PF4/H complexes coated on the solid phase cannot differentiate between non-pathogenic and pathogenic anti-PF4/H Abs. We question if substrate for immobilization of antigen plays an important role in differencing these anti-PF4/H Abs. To clarify this, we compared binding forces of the same anti-PF4/H Abs when interacting with PF4/H complexes coated either on a solid phase or on platelet membranes.

Methodology: Anti-PF4/H Abs were purified from patients' sera by two steps affinity chromatography. Binding forces between single antibodies and PF4/H complexes were measured by single-molecule force spectroscopy (SMFS), in which antibodies were immobilized on cantilevers while PF4 of different concentrations or PF4/H complexes were immobilized either on a solid phase or on platelets.

Findings: Only pathogenic, platelet activating- but not non-pathogenic anti-PF4/H Abs showed significantly stronger binding forces to PF4/H complexes immobilized on platelets compared with PF4/H complexes coated on a solid phase. This indicates that pathogenic anti-PF4/H Abs recognize their antigen differently on a cell membrane compared with a solid phase.

Conclusion: Cell membrane-based antigen presentation may allow developing assays for enhancing the specificity of antigen tests for pathogenic anti-PF4/P Abs.