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 immunoglobulin (Ig) G 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 (EIAs) 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 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 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition. CONCLUSION: 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.

Antibodies against platelet factor 4 and the risk of cerebral venous sinus thrombosis in patients with vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenoviral vector-based vaccines against SARS-CoV-2. This syndrome is caused by antibodies against platelet factor 4 (PF4; CXCL4) that lead to platelet activation and is characterized by thrombocytopenia and thrombosis in unusual locations, including cerebral venous sinus thrombosis (CVST). VITT can be classified based on anti-PF4 antibodies properties in vitro: those that require PF4 to activate platelets (PF4-dependent) and those that can activate platelets without additional PF4 (PF4-independent) in the serotonin release assay. OBJECTIVES: We aim to characterize the relationship of VITT platelet-activating profiles with CVST. METHODS: We conducted a retrospective cohort study involving patients with confirmed VITT who were tested between March and June 2021. Data were collected with an anonymized form and cases were identified as VITT with high clinical suspicion according to platelet activation assays. Anti-PF4 antibody binding regions on PF4 were further characterized with alanine scanning mutagenesis. RESULTS: Of the patients with confirmed VITT (n = 39), 17 (43.6%) had PF4-dependent antibodies and 22 (56.4%) had PF4-independent antibodies. CVST occurred almost exclusively in PF4-independent patients (11 of 22 vs 1 of 17; P < .05). Additionally, PF4-independent antibodies bound to 2 distinct epitopes on PF4, the heparin-binding region and a site typical for heparin-induced thrombocytopenia antibodies, whereas PF4-dependent antibodies bound to only the heparin-binding region. CONCLUSION: These findings suggest that VITT antibodies that cause PF4-independent platelet activation represent a unique subset of patients more likely to be associated with CVST, possibly due to the 2 different types of anti-PF4 antibodies.

The clinical and laboratory diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious adverse syndrome occurring 5 to 30 days after adenoviral vector COVID-19 vaccination. Therefore, a practical evaluation of clinical assessments and laboratory testing for VITT is needed to prevent significant adverse outcomes as the global use of adenoviral vector vaccines continues. We received the clinical information and blood samples of 156 patients in Canada with a suspected diagnosis of VITT between April and July 2021. The performance characteristics of various diagnostic laboratory tests were evaluated against the platelet factor 4 (PF4)-14C-serotonin release assay (SRA) including a commercial anti-PF4/heparin immunoglobulin G (IgG)/IgA/IgM enzyme immunoassay (EIA, PF4 Enhanced; Immucor), in-house IgG-specific anti-PF4 and anti-PF4/heparin-EIAs, the standard SRA, and the PF4/heparin-SRA. Of those, 43 (27.6%) had serologically confirmed VITT-positive based on a positive PF4-SRA result and 113 (72.4%) were VITT-negative. The commercial anti-PF4/heparin EIA, the in-house anti-PF4-EIA, and anti-PF4/heparin-EIA were positive for all 43 VITT-confirmed samples (100% sensitivity) with a few false-positive results (mean specificity, 95.6%). These immunoassays had specificities of 95.6% (95% confidence interval [CI], 90.0-98.6), 96.5% (95% CI, 91.2-99.0), and 97.4% (95% CI, 92.4-99.5), respectively. Functional tests, including the standard SRA and PF4/heparin-SRA, had high specificities (100%), but poor sensitivities for VITT (16.7% [95% CI, 7.0-31.4]; and 46.2% [95% CI, 26.6-66.6], respectively). These findings suggest EIA assays that can directly detect antibodies to PF4 or PF4/heparin have excellent performance characteristics and may be useful as a diagnostic test if the F4-SRA is unavailable.

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia.

Vaccine-induced immune thrombotic thrombocytopaenia (VITT) is a rare adverse effect of COVID-19 adenoviral vector vaccines1-3. VITT resembles heparin-induced thrombocytopaenia (HIT) in that it is associated with platelet-activating antibodies against platelet factor 4 (PF4)4; however, patients with VITT develop thrombocytopaenia and thrombosis without exposure to heparin. Here we sought to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine-scanning mutagenesis5, we found that the binding of anti-PF4 antibodies from patients with VITT (n = 5) was restricted to eight surface amino acids on PF4, all of which were located within the heparin-binding site, and that the binding was inhibited by heparin. By contrast, antibodies from patients with HIT (n = 10) bound to amino acids that corresponded to two different sites on PF4. Biolayer interferometry experiments also revealed that VITT anti-PF4 antibodies had a stronger binding response to PF4 and PF4-heparin complexes than did HIT anti-PF4 antibodies, albeit with similar dissociation rates. Our data indicate that VITT antibodies can mimic the effect of heparin by binding to a similar site on PF4; this allows PF4 tetramers to cluster and form immune complexes, which in turn causes Fcγ receptor IIa (FcγRIIa; also known as CD32a)-dependent platelet activation. These results provide an explanation for VITT-antibody-induced platelet activation that could contribute to thrombosis.