Patient-derived monoclonal anti-HPA-1a can induce platetet activation through fcyriia

Abstract Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy-associated disorder caused by maternal alloantibodies targeting paternally inherited human platelet antigens (HPAs). These antibodies traverse the placenta and bind to fetal platelets, causing thrombocytopenia and potentially severe complications including intracranial hemorrhage (ICH). Beyond platelets, antibodies may also target other fetal cells such as endothelial cells and placental trophoblasts thereby exacerbating disease severity. Anti-HPA-1a antibodies, the primary cause of FNAIT, display functional heterogeneity due to differences in epitope specificity, glycosylation, and functional effects, which complicates disease severity prediction. Here, we report the structural and functional characterization of two novel anti-HPA-1a antibodies, D- and M-204, derived from a severe FNAIT patient. In comparison to D-204 and other existing anti-HPA-1a antibodies such as B2G1 and 26.4, M-204 demonstrated a unique binding profile towards HEK-293F cells, expressing αIIbβ3, and platelets, as determined by flow cytometry. Notably, M-204 was capable of inducing platelet aggregation upon binding. To investigate the underlying mechanism behind this aggregation, various antibody formats were generated – Fab, Fab2 and IgG-Fc dead variants. Light transmission aggregometry revealed that M-204 triggered platelet aggregation through an FcɣRIIa-dependent pathway, which could be fully inhibited using the blocking antibody (AT10) targeting this receptor. Structural modelling with AlphaFold and HADDOCK suggests that M-204 binds αIIbβ3 in a distinct orientation compared to D-204. M-204 also contains a Fab glycan, confirmed via LC-MS, which does not interfere with antigen binding or functional activity. Our findings highlight the functional diversity of anti-HPA-1a antibodies and provide new insights into their pathogenic mechanisms.