The use of 1e12, a monoclonal anti-platelet factor 4 antibody to improve the diagnosis of vaccine-induced immune thrombotic thrombocytopenia

Background Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-COV-2 due to prothrombotic IgG antibodies to platelet factor 4 (PF4), and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin. Objectives We assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIA) in this context. Methods The ability of F(ab')2 fragments of 1E12, 1C12 and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine scanning mutagenesis was performed to define the amino acids (AA) involved in the interactions between the monoclonal antibodies and PF4. Results A strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition 93%, n=8), whereas it had no effect on the binding of HIT antibodies (median: 6%, n=8). In contrast, 1C12 and 2E1 inhibited VITT (median: 74 and 76%, respectively) and HIT antibodies (median: 68 and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for one patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key AAs on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition. Conclusions A simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.