Cooperative effect between anti-PF4/H and anti-PF4 antibodies increases cell activation and thrombotic risk in HIT

• Anti-PF4/heparin and anti-PF4 antibodies can cooperate to induce platelet activation, thrombocytopenia and thrombus formation

• The synergistic effect between PF4-specific antibodies is FcγRIIA-dependent and occurs in the absence of heparin

Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy, frequently associated with thrombosis. IgG antibodies to heparin-platelet factor 4 complexes (PF4/H) play a central role in HIT by activating platelets and leukocytes via FcγRIIA receptors. However, some patients also develop IgG against unmodified PF4 (anti-PF4), but their implication in the pathophysiology of HIT is unclear. We therefore assessed the impact of the joint presence of anti-PF4/H and anti-PF4 antibodies on cellular activation, platelet count and thrombus formation, using chimeric monoclonal IgG1 antibodies specific for either PF4/H complexes (5B9) or PF4 alone (1E12). As expected, 5B9 or plasma samples from HIT patients co-incubated with washed platelets without heparin did not induce platelet activation. But when a non-activating concentration of 1E12 was present with 5B9, significant platelet activation was observed. This functional cooperation was Fc-dependent and involved FcγRIIA receptors, as it was no longer detectable with F(ab')2 fragments of 1E12 or 5B9 or with ibrutinib, which inhibits the FcγRIIA pathway. 5B9 at a non-activating concentration of 1E12 also induced thrombus formation without heparin under flow conditions. Furthermore, when the two antibodies were injected together into hFcγRIIA/hPF4 transgenic mice, thrombocytopenia always occurred, with pulmonary thrombi in one third of the injected mice, similar to that observed after injection of 5B9 and heparin. These results support that functional cooperation may exist between anti-PF4 antibodies of different specificity, and promote cell activation, thrombocytopenia and thrombosis. This process may also increase the risk of thrombosis in HIT even after heparin treatment has been discontinued.