Evaluation of Anti-PF4 Positivity and Platelet Activation after COVID-19 Vaccination with Ad26.COV2.S in Brazil

Introduction: Mass vaccination emerged as the key global measure to reduce morbidity and mortality associated with the severe acute respiratory syndrome coronavirus 2 (SARS-COV2). The advance of vaccine coverage worldwide enabled us to document its efficacy and safety. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but life-threatening clinical event that occurs 5 to 30 days after vaccination, characterized by arterial or venous thrombosis, thrombocytopenia, markedly elevated D-dimer, and strongly positive platelet factor 4 antibodies (anti-PF4). Its pathophysiology is not yet fully understood, but studies have demonstrated similarity to heparin-induced thrombocytopenia and the pivotal role in platelet activation. The Janssen COVID-19 vaccine (Ad26.COV2.S), based on an adenoviral vector, has been associated with VITT in 1 case per 263,000 doses administered. Given the safety concerns, its use is restricted in the USA since May 2022, however, it is still in use in Brazil and other countries. In this study, we sought to investigate the prevalence of positive anti-PF4, hematologic parameters and to describe platelet activation profiles in a cohort of individuals who received the Ad26.COV2.S vaccine in Rio Janeiro, Brazil.

Methods: We conducted an observational longitudinal study at Instituto Nacional de Infectologia Evandro Chagas (INI/FIOCRUZ), from July 1^st, 2021 to August 4^th, 2021 to evaluate clinical and laboratory parameters following Ad26.COV2.S COVID-19 vaccination. Eligible individuals were ≥18 years old, with no personal history of vaccination against COVID-19, who sought Ad26.COV2.S COVID-19 vaccination at our institution. Volunteers attended 3 clinical visits; the first visit occurred before vaccination (week 0) and the other 2 visits occurred at 1 (week 1) and 3 weeks (week 3) after vaccination, respectively. Signs and symptoms of thrombosis, suspected vaccine side effects or COVID-19 infection were monitored during all visits. Platelet count (reference range: 150- 450 x 10³/µL) and d-dimer (elevated if ≥ 0.55mg/L) were measured at each visit. Anti-PF4 using Lifecodes PF4 IgG ELISA assay (positivity defined as optical density (OD) ≥ 0.40, and strong reaction defined as OD >1) were obtained in week 3. Samples from individuals who had positive anti-PF4 on week 3 were tested for anti-PF4 on week 0. Individuals with positive anti-PF4 on week 3, new onset thrombocytopenia, or elevated D-dimer were submitted to functional platelet activating property tests.

Results: A total of 631 individuals were included. Of those, 324 (51.34%) self-identified as males and 307 (48.65%) as females. The median age was 28 years (IQR 23 - 33). Of all included participants, 522 (82.73%) attended the second visit (week 1) and 573 (90.81%) attended the third visit (week 3). A total of 5 (0.79%) individuals had thrombocytopenia, of those 2 had thrombocytopenia at baseline (week 0) and additional 3 developed thrombocytopenia by week 3. All of those were classified as mild thrombocytopenia, ranging from 109-149 x 10³/µL. Compared to baseline, 31 (4.91%) individuals had d-dimer elevation. Of the 573 volunteers who attended visit 3, 9 (1.57%) had a positive anti-PF4, of those 5 (0.87%) since week 0 and the remaining 4 (0.70%) became positive by week 3. Eight (88.88%) showed weak reaction and only 1 presented strong reaction, OD range 0.453-1.440 (Figure 1). None of the participants with positive anti-PF4 exhibited symptoms or concomitant thrombocytopenia, however 2 had associated d-dimer elevation. Samples with abnormal results were submitted to further investigation and revealed slightly increased plasmatic concentrations of tissue factor (coagulation factor III), CD62p (P-selectin) and IL-1β. These and other hematologic alterations are summarized in Table 1. No thrombotic events or clinical findings suggestive of VITT were observed during the study period.

Conclusions: Our study demonstrated a low prevalence of thrombocytopenia, d-dimer elevation and positive anti-PF4, hallmark features of VITT, after Ad26.COV2.S COVID-19 vaccination. Moreover, in our cohort they did not correlate with the clinical picture of VITT. These results suggest that routine screening of these laboratory parameters may not be recommended for predicting risk of developing VITT in individuals receiving the Ad26.COV2.S COVID-19 vaccination.

No relevant conflicts of interest to declare.