Novel PF4/heparin complexes drive platelet activation in heparin-induced thrombocytopenia Free

Understanding the interaction between platelet factor 4 (PF4) and heparin is essential for elucidating the pathogenesis of heparin-induced thrombocytopenia (HIT). Heparin, a highly sulfated polyanion, consists of a heterogeneous mixture of polysaccharide chains of varying lengths. Its interaction with the positively charged PF4 tetramer is primarily driven by non-specific electrostatic forces, resulting in the formation of PF4/heparin (PF4/H) complexes. These complexes can elicit an immune response, leading to the generation of anti-PF4/H antibodies. When these antibodies bind to the PF4/H complexes, they form immunocomplexes that engage the FcγRIIa receptor on platelets, triggering activation and leading to thrombocytopenia and thrombosis. The formation of PF4/H complexes is dependent on the stoichiometric PF4:heparin ratio (PHR), which governs their size, structure, antigenicity, and immunogenicity. Ultralarge PF4/H complexes (ULCs), which form optimally at a PHR near 1:1, have been shown to play a crucial role in the pathogenesis of HIT, as they are the complexes that drive platelet activation through HIT antibody binding. However, recent structural insights suggest that PF4/H complexes can also form at PHRs higher than previously recognized, indicating that multiple structural variants may contribute to HIT. Based on these findings, we hypothesized that high-PHR complexes are also involved in the pathogenesis of HIT.

By testing plasma samples from 16 HIT patients across a wide range of stoichiometric ratios between PF4 and heparin, we found that while ELISA-based binding and complex formation peak at a PHR of 1.5:1, this ratio completely inhibits platelet activation. In contrast, maximal platelet activation occurs at a much higher PHR of 15:1. Using nanoparticle tracking analysis, we observed that PF4/heparin complexes formed at a 15:1 ratio exhibit a broad size distribution ranging from approximately 200 to 3,000 nm. This is significantly larger than the size range of 1.5:1 complexes, which are mostly confined between 200 and 500 nm. The increased size of the 15:1 complexes likely explains the enhanced platelet activation, as larger complexes can accommodate more HIT antibodies and form higher-valency immunocomplexes. Since ELISA failed to detect HIT antibody binding to 15:1 ULCs, we developed an alternative flow cytometry–based assay. The large size and high light-scattering properties of the 15:1 complexes allow them to be easily visualized and distinguished from background noise by flow cytometry. To test for antibody binding, PF4/heparin complexes were incubated with purified IgG from HIT plasma samples, alongside IgG from healthy donors as controls. Complex-bound IgG was detected using a fluorescent secondary nanobody. Pathogenic HIT IgG showed significantly greater binding to the 15:1 complexes compared to healthy donor IgG, confirming that HIT antibodies can indeed recognize and bind to these larger complexes in solution.

In summary, our findings reveal that PF4 and heparin can assemble into two distinct types of ULCs in a PHR-dependent manner. In addition, we developed a flow cytometry–based assay to detect HIT antibody binding to PF4/H complexes in solution. Using this approach, we identified the presence of a previously unrecognized type of high-PHR ULCs capable of binding HIT antibodies and inducing platelet activation.