Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but life-threatening syndrome initially described as a complication of adenoviral (Ad) vector anti-COVID-19 vaccines and characterized by thrombosis in unusual sites (most frequently cerebral venous sinus - CVST- and splanchnic vein thrombosis - SVT), often associated with multiple venous and/or arterial thromboses, thrombocytopenia, strongly increased circulating D-dimer levels and positivity for anti PF4/heparin antibodies, occurring 5-30 days after vaccination.¹, ², ³ Besides platelet activation, a strong stimulation of neutrophils has also been reported in VITT patients with the formation of neutrophil extracellular traps (NETs).⁴ More recently, new VITT-like disorders sharing with VITT thrombocytopenia, markedly raised D-dimer levels, platelet activating anti-PF4 antibodies and unusual site thrombosis, have been described following the administration of other vaccines, like Gardasil 9 for human papillomavirus (HPV) and mRNA-based Covid-19 vaccines, during bacterial or viral respiratory infections, and in patients with a monoclonal gammopathy of undetermined significance.⁵, ⁶

Thrombotic events in VITT are due to the generation of anti-PF4 antibodies activating platelets and neutrophils through FcγRIIA.¹ Anti-PF4 antibodies persist for some time after an acute VITT episode with differences depending on the assay used.⁷, ⁸

In fact, anti-PF4 antibodies were reported to persist in 94.3% of VITT patients at a median follow-up of 3 months in one study (range: 4 to 19 weeks),⁹ in 72% of VITT patients at a median follow-up of 3.5 months in another (range: 5 to 23 weeks),⁸ in 78.5% and in 77.7% of VITT patients at a median 6 month follow-up in a third (range: 3 to 36 weeks) and a fourth (range: 24 to 29 weeks) study,¹⁰, ¹¹ in 100% of VITT patients at a median 7 month follow-up in a fifth (range: 28 to 40 weeks),¹² and in 55% of VITT patients at a median follow-up of 20 months (range: 13 to 99 weeks) in a sixth study,¹³ using different assays. However, the ability of the residual antibodies to activate platelets was reported only in few VITT patients, for instance in 34% of VITT patients at a median follow-up of 3 months,⁹ in 42.8% at a median 5 month follow-up,¹² in 26% and 11% at a median 6 month follow-up (^10,11), and in 8.5% at a median follow-up of 20 months,¹³ using different platelet-activating assays. To the best of our knowledge, no studies with longer follow-up have been reported to date.

Besides anti-PF4 antibodies, some studies showed that the autoimmune response triggered by Ad-vector vaccines involves the development of autoantibodies against platelet surface glycoproteins,¹⁴, ¹⁵, ¹⁶, ¹⁷, ¹⁸ but no studies have assessed the persistence of these antibodies after an acute VITT episode.

Finally, acute VITT is associated with a strong neutrophil activation with NET formation. NET biomarkers were shown to persist for at least 6 months in patients previously hospitalized for COVID-19, and to be linked to pulmonary fibrosis, cardiovascular abnormalities, and neurological dysfunction in long COVID.¹⁹ Also MMP-9 was significantly elevated in the serum of long COVID patients compared to healthy controls.²⁰ To the best of our knowledge, no data have been reported on the possible persistence of neutrophil activation in patients with a previous VITT. Anti-PF4 persistence and their platelet and neutrophil activating activity may be important in the decision about anticoagulant treatment duration and in the estimate of the risk of relapse, thus a more precise definition of this issue may facilitate the identification and management of potential sequelae.

We carried out a multicenter study in 16 patients who survived an acute VITT episode to evaluate the persistence of anti-PF4/heparin antibodies and their platelet-activating activity, of anti-platelet autoimmunity, and of neutrophil activation.

Our data show that in patients with a previous acute VITT episode anti-PF4/heparin antibodies persist long: in 75% of cases at 6 months and still in 11% at 2.4 years, when assessed by an immunological assay (ELISA), but that a progressive and striking decrease in the intensity of antibody positivity occurs. Notwithstanding, at 6-month follow-up anti-PF4 antibodies still induced platelet activation in 56% of cases, whereas at 2.4 years no VITT patients exhibited a positive response in the functional platelet activation assay. Our findings corroborate previous evidence indicating a decline in the platelet-activating capacity of anti-PF4 antibodies, despite their persistence, although previously reported in shorter follow-ups.⁹, ¹⁰, ¹¹, ¹², ¹³ The exact causes of anti-PF4 antibody persistence in some subjects are still largely unknown and may be due to a large extent to differences in immune responses. In fact, the available evidence suggests the existence of two antibody profiles in VITT patients: those whose platelet-activating antibody levels decrease over time and those with persistent platelet-activating antibodies.²⁵ However, even in patients with persistence of platelet-activating antibodies, neither thrombotic nor thrombocytopenic relapses were observed either in our cohort, or in previous reports,⁸, ¹⁰, ¹², ¹³ suggesting their clinical irrelevance. Thus, our study, that to the best of our knowledge is the first evaluating the persistence of anti-PF4 antibodies at >2 years follow-up, shows that anti-PF4 antibodies persist long after an acute VITT episode, but that they lose their platelet activating ability, as well as their clinical significance.

We also observed that the persistence of anti-PF4 antibody depends on the immunological assay used, confirming the need to carry out more than one immunological test for anti-PF4 detection in VITT.⁷, ⁸

Besides anti-PF4 antibodies, at T0 28% of patients were also positive for anti-platelet autoantibodies, confirming previous results,¹⁴ and, similar to anti-PF4/heparin antibodies, they decreased over time. However, anti-platelet autoantibodies did not seem to be of clinical significance, given that they were reported also in 34% of patients who underwent adenoviral-vector anti SARS-CoV-2 vaccination without suffering from VITT,¹⁵ and that in our case series they did not associate with thrombocytopenia in the follow-up.

A novel aspect of our study is the analysis of plasmatic markers of neutrophil activation²⁴ in the long-term follow-up of VITT patients. Both MMP-9 and MMP-9/NGAL were strikingly elevated at diagnosis compared to healthy subjects. Moreover, their plasmatic levels correlated with anti-PF4 O.D. values, in accordance with previous data reporting the synergistic role of platelets and neutrophils in VITT immunopathogenesis.²⁶, ²⁷ However, they normalized already at the 6 months follow-up.

We recognize that our study has some limitations. First, the small number of VITT patients enrolled; however VITT is a rare event with an estimated incidence ranging from 3.2 to 16.1 cases every 100,000 vaccinated subjects for ChAdOx1 nCoV-19, and 1.7 to 3.7 cases for Ad26.COV2.S) and, similarly, previous studies on anti-PF4 antibody-persistence in VITT were performed in small cohorts.⁹, ¹¹, ²⁸ Second, the relatively high fraction of patients lost at the second follow-up, however, at least at 6 months the case series was still reasonably consistent. Finally, the fact that anti-PF4/heparin antibodies and the assessment of their platelet-activating activity were conducted using a variety of assays, since laboratory investigations during the acute VITT episode were performed independently at each participating center without central coordination. However, all the assays at T1 and T2 were performed by one centralized laboratory, enhancing the comparability of results.

However, all the assays at T1 and T2 were performed by one centralized laboratory, enhancing the comparability of results. In conclusion, our results suggest that the immunological abnormalities underlying VITT although persistent seems to be clinically irrelevant, however long-term monitoring could be beneficial for identifying and managing eventual sequelae. ²⁹ Elucidating the mechanisms behind this persistence is crucial for the targeted long-term monitoring and effective management strategies of VITT patients.