Platelets have a complex impact on the outcome of viral infections, with both favorable and unfavorable effects. Being anucleate cells, platelets contribute to the host defense against viruses by engulfing RNA viruses and recruiting immune cells to eliminate viruses (Negrotto et al., 2015; Zhao et al., 2023), which helps prevent the spread of the viruses. For example, platelets containing SARS-CoV-2 mRNA were detected in COVID-19 patients (Manne et al., 2020; Zaid et al., 2020). Platelets can also assist in regulating immune-cell responses to pathogens by binding and interacting to various leukocytes, such as monocytes, neutrophils, eosinophils, and lymphocytes (Koupenova et al., 2018). In terms of adaptive immune defense, platelets stimulate B cells to produce immunoglobulins (IgG1, IgG2, and IgG3) (Negrotto et al., 2015; Koupenova et al., 2018), thus enhancing the antibody-mediated immune response against the virus. However, platelets can also exacerbate inflammation by increasing the recruitment of neutrophils, promoting the formation of neutrophil extracellular traps (Koupenova et al., 2018), and contributing to an elevated risk of thrombosis (Koupenova et al., 2014; Sciaudone et al., 2023), and tissue injury (Antoniak and Mackman, 2014) during viral infections.

There is evidence to suggest a correlation between platelet abnormalities and COVID-19. Several studies have indicated that patients with COVID-19 demonstrate hyperactive platelets, which are believed to play a role in the pathophysiology of the disease (Chan and Rout, 2020; Manne et al., 2020; Qu et al., 2020; Zaid et al., 2020). In addition, SARS-CoV-2 vaccination can, albeit rarely, induce platelet related vaccine-induced immune thrombotic thrombocytopenia or thrombosis with thrombocytopenia syndrome (Marcucci and Marietta, 2021), highlighting the fundamental pathological condition/disease of platelet disorders in COVID-19.

Epidemiological studies on post-COVID-19 sequelae (also known as long COVID, LC) face considerable challenges in interpretation and integration due to significant disparities in sampling methods, inclusion criteria, participant demographics, diagnostic standards, and overall study designs. The clinical presentations of long COVID are varied and can impact multiple organ systems, with symptoms that may change over time. Current estimates indicate a conservative incidence rate of 10% among infected individuals worldwide; however, the actual number is likely considerably higher due to many unreported cases. Incidence rates for long COVID are estimated to range from 10% to 30% among non-hospitalized patients, 50% to 70% among those who were hospitalized, and 10% to 12% among vaccinated individuals (Davis et al., 2023; Greenhalgh et al., 2024). These symptoms of LC may potentially be linked, at least partially, to immune cell dysregulation (Altmann et al., 2023). The underlying mechanisms of LC are still under investigation. Intriguingly, platelet dysfunction and hypercoagulability have been consistently linked with LC (Pretorius et al., 2021). Platelet-secreted serotonin (5-HT) is known to modulate the function and proliferation of T cells and B cells (Aune et al., 1993; Iken et al., 1995; Abdouh et al., 2001). Recently, several mechanisms have been reported to contribute to the decrease in circulating 5-HT levels in LC patients. These include impaired absorption of tryptophan, the precursor of 5-HT, in the gastrointestinal tract, decreased storage of 5-HT in platelets due to thrombocytopenia, and increased breakdown of 5-HT by enzymes involved in its metabolism (Wong et al., 2023).

Insufficient research has explored the role of platelets in the alterations observed in immune cell profiles among patients recovering from COVID-19. This study addresses this gap by investigating a subgroup of moderate COVID-19 unvaccinated patients (5.3%) who exhibited elevated platelet counts (> 300×10⁹/L), exceeding the upper threshold (above high limited, AHL) on the 48th day post-symptom onset (DPSO 48). The primary objective was to examine the correlation between platelets and various immune cell populations, including CD3⁻CD19⁺ B-cells, different T-cell subsets (CD3⁺CD4⁺ T-cells, CD3⁺CD8⁺ T-cells, double-negative T-cells (DN T-cells), regulatory T-cells (Tregs)), and CD3⁻CD16⁺CD56⁺ natural killer (NK) cells, within the context of moderate COVID-19 cases exhibiting AHL platelet levels and normal platelet levels during recovery.

Lymphopenia and thrombocytopenia have been recognized as detrimental factors linked to unfavorable outcomes in the acute phase of SARS-COV-2 infection (Henry et al., 2020; Huang and Pranata, 2020; Lippi et al., 2020). Nevertheless, the exact mechanism connecting the abnormal correlation between lymphopenia and thrombocytopenia and their consequences to the disease necessitates further investigation.

During the recovery phase, a majority of COVID-19 patients generally experience a normalization of lymphocyte and platelet counts, as demonstrated by previous research (Mandal et al., 2021; Alfadda et al., 2022; Lechuga et al., 2023). In our investigation, we found that 5.3% of 281 moderate COVID-19 patients displayed elevated platelet counts (>300×10⁹/L) on DPSO 48, which aligns with the results reported in a previous longitudinal study spanning 7 months. In the aforementioned study, asymptomatic patients had a platelet count of 266.16 ± 80.6 (×10⁹/L, mean ± SD) after six months of follow-up, while symptomatic patients had a platelet count of 253.68 ± 52.2 (×10⁹/L, mean ± SD). By examining the platelet mean values and standard deviations reported in their findings (Alfadda et al., 2022), it suggests that there is a subgroup within their cohort whose platelet counts remained above 300×10⁹ cells/L even at the six-month follow-up.

Interestingly, our study found that convalescent COVID-19 patients with elevated platelet counts had higher cell counts of total T cells, CD4 and CD8 T cells, and Tregs compared to convalescent patients with normal platelet counts. Previous reports have not specifically examined the relationship between platelets and T cell subsets in convalescent COVID-19 patients. Therefore, our study provides initial evidence showing an association between higher platelet counts and increased cell counts of total T cells and its subsets in convalescent COVID-19 patients.

Numerous pieces of evidence indicate that platelets have the ability to influence the functions of CD4 and CD8 T cells (Morrell et al., 2014; Yu et al., 2020). However, our understanding of their role in the expansion or proliferation of these T cells remains limited. A study conducted by Elzey et al. revealed that a reduction in platelet levels led to a decrease in the production of CD8 T cells (Mandal et al., 2021). Conversely, when platelets were reintroduced in platelet-depleted mice, the number of CD8 T cells in the spleen and liver increased after an infection with lymphocytic choriomeningitis virus (Mandal et al., 2021). These findings emphasize the significance of platelets in the expansion of antigen-specific CD8 T cells. As for CD4 T cells, mitochondria derived from platelets were found to directly and differentially up-regulate Naïve and central memory CD4 T cells, while down-regulating effector memory CD4 T cells (Morrell et al., 2014).

Recently, there has been a suggestion that platelet-derived 5-HT may be involved in the underlying mechanism of LC (Wong et al., 2023). Previous studies have demonstrated that 5-HT promotes the proliferation of both B cells and T cells (Aune et al., 1993; Iken et al., 1995; Abdouh et al., 2001). In our study, although there was no significant difference in B cell counts between HMo and NMo cases, we observed a positive correlation between platelet counts and B cell counts only in HMo cases (r = 0.636, p = 0.03). This suggests that higher platelet counts are associated with higher B cell counts in a subset convalescent COVID-19 patients.

Platelets exert harmful effects primarily through the inflammatory response during viral infection (Morrell et al., 2014). Inflammatory-activated platelets enhance the adhesion of lymphocytes to the endothelium, directing them towards endothelial veins in various inflammatory sites and causing damage (Koupenova et al., 2018). In the convalescent phase of COVID-19, the inflammatory responses subside, as indicated by low levels of CRP, an inflammatory marker, in both HMo and NMo cases in this study. Therefore, during the convalescence of COVID-19, platelets mainly play a role in modulating immune homeostasis in patients. Persistent SARS-CoV-2 shedding is strongly linked to reduced T cell and B cell counts, which can contribute to chronic immune activation in LC (Hu et al., 2020).

The mechanisms linking elevated platelet counts to increased T-cell numbers remain unclear. In addition to the previously mentioned role of platelet-derived 5-HT in promoting T-cell proliferation, the convalescent phase of COVID-19 may involve diminished inflammatory cytokines that also connect elevated platelets and T-cell counts. For instance, high mobility group box 1 (HMGB1), which is actively secreted by necrotic cells in response to inflammatory signaling, may contribute to the hyper-inflammatory response seen in COVID-19 (Al-Kuraishy et al., 2022), leading to complications such as acute respiratory distress syndrome (ARDS) and thrombosis. Notably, plasma levels of HMGB1 show a negative correlation with platelet counts in immune thrombocytopenia (ITP). The natural HMGB1 inhibitor, 18β-glycyrrhetinic acid (18β-GA), exhibits anti-inflammatory and immune-modulatory effects. It enhances the production of Tregs, restores the balance of CD4 T-cell subsets, and increases the suppressive functions of Tregs by inhibiting HMGB1 activity in ITP patients. These findings suggest that inhibiting HMGB1 signaling may effectively restore immune balance in ITP (Wang et al., 2023). Moreover, our study indicates a correlation between elevated platelet counts and Tregs, CD4, and CD8 T cells, without altering the CD4/CD8 T-cell ratio, implying a restoration of immune homeostasis. This is further supported by our observation that the frequencies of all T-cell subsets remained consistent among healthy controls, mildly ill patients, and those with mild COVID-19 on day 48 post-symptom onset (DPSO). In contrast, individuals with long COVID showed increased frequencies of CD8 T cells, Tregs, and natural killer (NK) cells compared to recovered patients, highlighting disruptions in immune homeostasis and a persistent cytotoxic memory immune response (Galan et al., 2022).

Tregs play a crucial role in autoimmune diseases. Clinical studies have revealed that COVID-19 patients exhibit lower levels of Tregs in comparison to the general population. This reduction in Tregs can result in compromised control over inflammation, an imbalance in the Treg/Th17 ratio, and an increased susceptibility to respiratory failure (Alsalman et al., 2022). Additionally, decreased Tregs contribute to the development of LC and exacerbate the prognosis of the disease (Dhawan et al., 2023). Emerging evidence has substantiated the crucial role of Tregs in maintaining immune balance and preventing the onset of autoimmune disorders. Furthermore, defects or deficiencies in Tregs have been correlated with a wide array of autoimmune conditions, allergies, and chronic inflammatory diseases (Cheru et al., 2023). Our study reveals a positive correlation between elevated platelet counts and increased Tregs levels, suggesting that platelets and/or their mediators may stimulate the expansion of Tregs. Given the significant role of Tregs in the immune response, further investigation is warranted to explore the connections between platelets and Tregs in the context of LC.

CD8 T cells have been shown to be a crucial type of immune cell induced by vaccination for protection against SARS-CoV-2. Activation and proliferation of CD8 T cells targeting the spike protein of SARS-CoV-2 can be observed following breakthrough infections. Moreover, this response is only observed in individuals who were previously vaccinated, while it is absent in unvaccinated individuals (Painter et al., 2023). These findings are consistent with the results of Zhang et al.’s study, which identified CD8 T cells specific to two subgroups of the vaccine and potentially serving as long-term memory cells (Zhang et al., 2023). In our study, we found that individuals with higher platelet counts have higher CD8 T cell counts (by 36.9%) on DPSO 48, suggesting that platelets may play a key role in enabling T cells to provide effective long-term protection against future SARS-CoV-2 infections.

In this study, we acknowledge several limitations that may affect the interpretation of our findings. The generalizability of our results is constrained by the study population and context, and we did not assess SARS-CoV-2-specific immune responses, which could have offered deeper insights into the immune dynamics involved. Additionally, potential confounding factors may influence our results, necessitating careful consideration in future analyses. The absence of a healthy control group is another notable limitation that we plan to address in upcoming studies to enhance the robustness of our conclusions. Furthermore, we encountered significant limitations related to the short duration of follow-up, particularly for patients affected by long COVID (LC). To fully examine variations attributed to disease severity, treatment, and other potential factors, large-scale studies with extended follow-up periods are essential. While our study provides valuable insights into lymphocyte subtype cell profiles in post-COVID-19 individuals, employing additional analytical methodologies focused on immune cell subtypes will yield a more detailed assessment of immune dysregulation in recovering patients and the specific cellular characteristics associated with LC.

In summary, 5.3% of moderate COVID-19 unvaccinated patients show high platelet counts (> 300x10⁹/L), which is linked to increased levels of total T-cells, CD4 and CD8 T-cells, and Tregs compared to patients with normal platelet counts. This does not affect the CD4/CD8 T-cell ratio. Furthermore, the significant positive correlation observed between platelet counts and B cell counts, particularly in cases of hematological malignancies, provides compelling evidence of a strong association and suggests underlying pathways linking platelets and B cells. These findings are crucial for advancing our understanding of platelet-related immunopathology and may help identify reliable biomarkers for recognizing patients with immune disorders who have achieved immunological balance during their recovery.