Circulating Activated Platelets in Children With Long Covid: A Case-Controlled Preliminary Observation, 2024, Buonsenso, Danilo MD, MSc, PhD et al

Abstract
We investigated if children with Long Covid (n=14) have activated platelets compared with healthy controls (n=14). Platelet activation and secretion markers were investigated by flow cytometry using MoAbs directed against P-selectin, CD63, and PAC-1 in quiescent platelets and in platelets stimulated with 10-µM adenosine diphosphate and 25-µM protease activated receptor 1-activating peptide. Circulating platelets of patients with Long Covid had significantly increased expression of the activation marker cytometry using MoAbs directed against P-selectin (P = 0.019).

BACKGROUND
Long Covid is a condition characterized by persisting symptoms several weeks after COVID-19. In adults with this condition, hypercoagulability, endotheliopathy, and thromboinflammation with complement dysregulation have been demonstrated and considered pathogenetic mechanisms.1,2 In addition, persistent activation of circulating platelets was demonstrated in adults 1 year after COVID,3 possibly expression of immune dysregulation and persistent inflammation triggered by the initial infection.1–3

About the pediatric population, observational studies have demonstrated that children also can develop Long Covid. A recent state-of-the-art narrative review showed how children can present persistent symptoms from acute SARS-CoV-2 infection (eg, cough, headaches, fatigue, and loss of taste and smell), new symptoms such as dizziness, or exacerbation of underlying conditions, or develop conditions de novo, including postural orthostatic tachycardia syndrome, myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune conditions, and multisystem inflammatory syndrome in children.4 Although these symptoms tend usually to improve over time, studies have found that children can suffer from Long Covid for ≥18 months,4 therefore having a significant impact on daily life.

Although Long Covid has been documented in children as well, no studies have investigated the pathogenetic mechanisms of the disease in this population. Given the growing evidence of ongoing thromboinflammation in adults with Long Covid,4 we performed this study aiming to explore if activated platelets are more expressed in children with Long Covid compared with controls, as a possible indirect sign of endothelial inflammation.



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METHODS
This study is part of a prospective follow-up of children after the first microbiologically confirmed acute SARS-CoV-2 infection (based on polymerase chain reaction on nasopharyngeal swabs), as previously described.5 Among these cohorts, Long Covid was defined as the persistence of otherwise unexplained symptoms negatively impacting daily life for at least three months after initial infection.6 Children seen in our follow-up study after initial infection and without complaining about ongoing symptoms and returned to usual pre-COVID activities were classified as recovered. Among the cohort of patients with Long Covid, the most severe group of children having persisting symptoms for 6 to 12 months affecting at least 3 different systems was invited to participate in this study, aiming to explore whether platelet activation is present in pediatric Long Covid. Patients who were already taking medications were excluded from the study. Also, children with signs and symptoms suggesting an acute infection (eg, new acute onset of fever) were excluded.

For every patient, an age- and sex-matched healthy control that fully recovered from a previous SARS-CoV-2 infection was invited to participate. Each couple of patients and control was sampled and analyzed the same day. Platelet activation and secretion markers were investigated by flow cytometry using MoAbs directed against P-selectin, CD63, and PAC-1 in quiescent platelets and in platelets stimulated with 10-µM adenosine diphosphate and 25-µM thrombin receptor activating peptide. Mean fluorescence intensity was measured and expressed as a fold increase of the patient samples relative to paired control samples. Results were expressed as median and ranges.

Statistical analysis was performed by GraphPad Prism 6 using Student’s t test, and P values were calculated based on the 2-tailed test. P < 0.05 was considered statistically significant.

The Long Covid cohort was enrolled as part of a larger, prospective, multidisciplinary follow-up study of children with SARS-CoV-2 infection, approved by the local ethics committee (Ethic approval ID4518, Prot0040139/21), and informed consent was provided. Written and informed consent was obtained from parents/caregivers and from children older than 5 years of age according to local guidance of the ethics committees.

 

RESULTS
Fourteen children with ongoing Long Covid and 14 healthy controls were included. Details about demographic and clinical data are provided in Table 1. Fatigue and postexertional malaise were the commonest persisting symptoms. The most frequent combination of persisting symptoms was fatigue plus neurocognitive problems (including headache) and musculoskeletal pains. Circulating platelets of patients with Long Covid had significantly increased expression of cytometry using MoAbs directed against P-selectin compared to controls (Fig. 1A), whereas CD63 and PAC-1 expression in patients with Long Covid were not significantly different compared to controls (Fig. 1D and 1G). After in vitro platelet stimulation by ADP and thrombin receptor activating peptide, all activation and secretion markers were similarly expressed in Long Covid children and controls.

 

We acknowledge that the small number of children represents the main limitation of our study, due to the late start of the study, in 2023, and to choose to recruit only the more severe cases that had not received previous treatments. Also, markers of activated platelets were not elevated in all patients, suggesting that these events may explain only partially why some patients develop Long Covid. More extensive studies investigating further mechanisms involved in the pathogenesis of the disease are needed for children with Long Covid.