Identification of Platelet-Activating Antibodies That Do Not Bind to PF4/Heparin Complexes in Heparin-Induced Thrombocytopenia
Authors: Zhou Lu , Villalobos Daniel , Aster Richard H. , Pabmanabhan Anand , Wang Demin , Wen Renren 
Published: December 2024

Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy, driven by IgG antibodies (Abs) that target platelet factor 4 (PF4) bound to heparin, resulting in platelet activation and a pro-thrombotic state. The PF4/Heparin (PF4/H) ELISA is the most commonly used screening assay for HIT due to its wide accessibility and high sensitivity. Despite its limited specificity, resulting in only a small fraction of positive cases progressing to actual HIT, PF4/H ELISA remains a critical tool in initial diagnostics.
It is generally believed that HIT patients produce two types of Abs - those that bind to PF4/H and may or may not activate platelets. However, there are documented but rare HIT cases with an atypical profile: Abs testing negative in the PF4/H ELISA (ELISA-) but capable of platelet activation, suggesting the presence of a third type of Abs - those that activate platelets but can't recognize PF4/H complex. Since a PF4/H ELISA- result typically excludes an HIT diagnosis, overseeing these cases could lead to the resumption of heparin, potentially resulting in life-threatening thrombosis. Additionally, in HIT cases with a positive ELISA test profile, or classic HIT cases, levels of platelet-activating Abs and PF4/H-reactive Abs are not always correlated, suggesting the presence of ELISA- platelet-activating Abs. We thus investigated whether ELISA- platelet-activating Abs are present in classic HIT.
To identify and characterize ELISA- Abs at the serological level, we firstly purified IgG Abs from the plasma of 5 healthy donors (HD) and 8 HIT patients using protein A beads, followed by the depletion of PF4/H-binding Abs using PF4/H-coated Sepharose beads. The efficiency of IgG purification and PF4/H-specific IgG depletion was confirmed by IgG and PF4/H ELISA. P-selectin expression assay (PEA) was conducted to evaluate the platelet activating ability of purified IgG pre and post depletion of PF4/H-specific Abs. We found that variable, but significant levels of platelet-activating Abs exist in the PF4/H non-reactive IgG portion in all 8 HIT samples, but none of the HD samples. Closely resembling those found in the PF4/H-reactive Abs in HIT patients' plasma, platelet activation induced by post-depletion Abs relied on exogenous PF4 and FcγRIIa and was inhibited by high-dose heparin. These data demonstrate that PF4/H non-reactive IgG Abs coexist with PF4/H-reactive Abs and may contribute to platelet activation in HIT patients.
To verify these findings at the clonal level, we conducted single B-cell culture to induce antibody secretion by the cultured B cells from patients experiencing clinical manifestations of HIT. We screened clonal Abs made by 1506 single IgG1+ B cells isolated from peripheral blood mononuclear cells (PBMCs) of seven HIT patients and identified 18 clones capable of platelet activation. Of these, 13 were unable to bind to PF4/H. The functionality of these Abs was confirmed by recombinant Abs derived from single B-cell clones. This finding suggests that PF4/H non-reactive platelet-activating Abs exist at a higher frequency than those that bind to PF4/H. Interestingly, even though these Abs tested negative in the PF4/H ELISA, they were able to bind to the surface of PF4-treated platelets, and their platelet activation ability correlated with platelet binding affinity, suggesting a potential method for their detection.
In summary, our study identified a novel group of potentially pathogenic Abs in HIT that, while unable to recognize the PF4/H complex, can bind to the PF4-coated platelet surface. These Abs frequently coexist with PF4/H-binding pathogenic Abs and may contribute to HIT pathogenesis. These insights will enrich our understanding of HIT pathogenesis and may pave the way for more advanced diagnostic and therapeutic strategies.