Longitudinal Characteristics of Anti-Platelet Factor 4 Immunoglobulin G in Vaccine-Induced Immune Thrombotic Thrombocytopenia

Introduction: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenoviral vector-based vaccines against the SARS-CoV-2 virus. VITT typically presents as moderate to severe thrombocytopenia often accompanied by arterial and/or venous thrombi occurring in unusual locations. The thrombocytopenia and thrombosis seen in VITT are triggered by antibodies against platelet factor 4 (PF4, CXCL4), leading to the activation of platelets and inducing a hypercoagulable state, similar to what is observed in heparin-induced thrombocytopenia (HIT). In a previous study, we identified a unique VITT antibody binding site. involving amino acids located at the heparin binding region of PF4. In this study, we describe the change in VITT anti-PF4 IgG binding regions and binding response, as well as the ability of longitudinal patient samples to activate platelets at initial presentation and up to 109 days after vaccination with Vaxzevria AZD1222 (AstraZeneca ChAdOx1 nCoV-19).

Methods: We longitudinally studied sera from VITT patients (n = 7) sent for confirmatory testing to the McMaster Platelet Immunology Laboratory. These VITT patient samples were received at time of diagnosis and follow up samples up to 109 days after vaccination (range 8 – 109 days). Confirmed VITT patients presented with clinical features including thrombocytopenia (platelet count < 150 x 10⁹/L) with thrombotic manifestations and tested positive in both the anti-PF4 IgG/A/M immunoassay (positive optical density (OD) > 0.4) and in the PF4-serotonin release assay (PF4-SRA, positive release > 20%). Alanine scanning mutagenesis was used to identify the changes in antibody binding sites on PF4 from VITT patients at diagnosis and one follow-up time point. We also determined the change in binding responses and dissociation rates at time of diagnosis and follow-up anti-PF4 antibodies using biolayer interferometry (BLI).

Results: Anti-PF4 immunoglobulin G antibody levels are positive and high at diagnosis (OD median 2.35, range 1.895 – 3.347) and decreased but remain relatively high and positive (OD >1.0, median 2.09, range 1.178 – 2.631) in all 7 VITT patients, longitudinally. In addition, most follow-up samples were weaker at activating platelets with or without exogenous PF4 compared to their initial sample. However, follow-up sera beyond 45 days still strongly activated platelets with PF4 in 2/7 (28.5%) VITT patients in the PF4-SRA. In 4/7 (57.1%) VITT patients, the number of amino acids on PF4 that continued to be important for VITT antibody binding decreased from 7 on average at time of diagnosis to 2 amino acids in the defined VITT binding site at follow-up. Binding responses to PF4 and PF4/heparin measured by BLI also decreased from initial diagnosis (PF4, median 1.872 nm, range 0.587 – 2.443 nm; PF4/heparin, median 1.362 nm, range 0.450 – 2.072 nm) when compared to follow-up (PF4, median 0.356 nm, range 0.052 – 1.770 nm; PF4/heparin, median 0.159 nm, range 0.006 – 1.206 nm) in all 7 VITT patient samples. Binding responses are a measure of abundance of antigen-specific antibodies present in a given sample. Lastly, we also measured dissociation rates of the antibodies with BLI, which reflects the avidity of an antibody for a given antigen. In similar trend as the binding responses, the dissociation rates of the anti-PF4 and anti-PF4/heparin antibodies decreased in 4/7 (57.1%) VITT patients at follow-up when compared to initial diagnosis.

Conclusion: Despite persistent anti-PF4 antibody levels in VITT patients over time, there is reduced platelet activation possibly due to the loss of antibodies to the VITT binding region necessary for platelet activation, or due to a decrease in binding response and avidity. This study suggests that VITT is similar to HIT because over time, there is a decline in platelet activating anti-PF4 antibodies. Clinical implications of persistent anti-PF4 antibodies in VITT require further study.