Diverse epitopes in anti-PF4/heparin antibodies predicted by in silico docking 2310336

Abstract

Abstract Introduction Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder caused by antibodies that recognize platelet factor 4 (PF4) when it forms complexes with heparin (PF4/H). These antibodies engage the Fc receptor on platelets, resulting in aberrant platelet activation and thrombus formation. The epitope on PF4 recognized by HIT antibodies has been partially characterized using polyclonal IgG as well as KKO, a mouse-derived “HIT-like” antibody. However, HIT is a polyspecific disorder, and the epitopes involved may not be fully elucidated. Methods Anti-PF4/H antibodies were cloned from HIT patients previously. The structures of these antibodies were modeled from amino acid sequence using AlphaFold3. These models were then docked to PF4 using the ClusPro webserver, and the top clusters were loaded into PyMol and PDBePISA for further analysis of interacting residues. Fab versions of cloned antibodies were used to assess competitive binding, using inhibition of platelet activation measured by flow cytometry as a readout. Results Residues on PF4 within 4Å of the antibody were recorded, and the percentage overlap of these residues with those involved in KKO binding was calculated. Percentage overlap of residues ranged from 0% to 48%. However, KKO fab was able to completely inhibit P-Selectin expression for all tested clones, including the clone with 0% overlapping residues. Conclusion In silico docking methods suggest that anti-PF4/H antibodies in HIT patients bind to diverse epitopes on PF4. Many of the tested clones bound to overlapping regions on the PF4 surface, but one clone was predicted to bind to a completely distinct epitope from that bound by KKO. However, validation of this finding using an in vitro platelet activation assay found that KKO still effectively inhibited the ability of all antibody clones to activate platelets, suggesting that despite the diversity in predicted epitopes, the tested HIT clones share a method of platelet activation susceptible to inhibition by KKO fab. Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)