Characterization of eNOS-based Platelets Subpopulations in COVID-19 and the Impact of Platelet α-Granule Contents on Cancer Cell PD-L1
Creators: Asgari, Amir
Publisher: name: University of Alberta Library
Year: 2024

<p>Thrombosis significantly complicates numerous diseases that have inflammatory components. This condition, characterized by the formation of blood clots within blood vessels, can exacerbate a range of health issues, from cardiovascular diseases to systemic infections like COVID-19, and even cancer. Inflammation plays a critical role in thrombosis by enhancing platelet activation and aggregation, promoting endothelial dysfunction, and altering blood flow, which together increase the risk of clot formation. Earlier research in the Jurasz lab led to identification of two distinct platelet subpopulations distinguished by the presence or absence of endothelial nitric oxide synthase (eNOS). This led to the classification of platelets into eNOS-positive and eNOS-negative groups. It was further demonstrated that while eNOS-negative platelets initiate thrombotic reactions, while eNOS-positive platelets contribute to and ultimately limit thrombus growth. However, little is known about potential changes in the ratios of these platelet subpopulations in diseases.<br>
In the context of severe COVID-19, which is characterized by platelet-rich microvascular thrombi, I investigated whether COVID-19-associated immune and inflammatory responses alter the balance of these platelet subpopulations. Platelets were isolated from age- and sex-matched hospitalized COVID-19 patients and COVID-19-negative controls. Platelet eNOS was measured by flow cytometry and plasma inflammatory cytokines (IFN-, TNF-, IL-6, and IL-1)  by  multiplex ELISA. COVID-19 patients demonstrated significantly elevated ratios of eNOS-negative to -positive platelets than controls and their ratios strongly correlated with disease severity (81.2  2.8%: 19.2  2.8% ICU vs. 66.0  3.1%: 34.7  3.5% non-ICU vs. 6.1  1.3%: 93.5  1.3% controls). Higher eNOS-negative to –positive platelet ratios were associated with enhanced platelet reactivity as measured by surface CD62P. Accordingly, COVID-19 patients demonstrated higher TNF-, IL-6, and IL-1 plasma concentrations than controls. Using the Meg-01 cell line, which demonstrates eNOS-negative and –positive subpopulations of cells, as a megakaryocyte model, demonstrated that inflammatory cytokines associated with COVID-19 promoted eNOS-negative Meg-01 formation and enhanced subsequent eNOS-negative platelet-like particle formation.<br>
Further characterization of eNOS-based platelet subpopulations led to identification of Granule-enriched platelet subpopulations, marked by CD62P, with higher content of VEGF and PDGF in healthy donors. Further research revealed that these platelet subsets (α-granule-enriched platelets with higher content of VEGF and PDGF) were all characterized as eNOS-positive. Strikingly, COVID-19 patients’ platelets demonstrated a higher overall mean in VEGF content and a greater percentage of VEGF-enriched platelet subpopulations compared to COVID-19-negative controls. COVID-19 patients demonstrated higher TNF- plasma concentrations than COVID-19-negative controls. Incubation of Meg-01 cells with TNF-α led to the formation of a distinct Meg-01 subpopulation with higher intracellular VEGF levels (14.4 ± 3.1%).<br>
Building on findings in platelet diversity, particularly the identification of eNOS-positive and eNOS-negative subpopulations with distinct VEGF and PDGF content, I investigated the impact of platelets on cancer cell evasion of the adaptive immune response.<br>
Current understanding of platelets in cancer suggests they facilitate hematogenous metastasis by enabling cancer cells to evade the immune system, although the current knowledge of platelet function in modulating the adaptive immune system in cancer is limited. A major negative regulator of the adaptive response is the immune checkpoint protein Programmed Death Ligand 1 (PD-L1). As platelets secrete factors that may increase PD-L1 expression, we investigated whether they up-regulate cancer cell PD-L1, thus promoting immunoevasion, and whether common anti-platelet drugs inhibit this process. Platelets increased PD-L1 expression by cancer cells, an effect counteracted by the simultaneous neutralization of platelet-derived VEGF and PDGF or the administration of eptifibatide, an anti-platelet agent. Furthermore, A549 lung carcinoma cells incubated with platelets showed diminished capacity to activate T-cells, an immunosuppressive effect reversed by eptifibatide.<br>
In summary, altered ratios of eNOS-negative to eNOS-positive platelets may contribute to the thrombosis observed in COVID-19 and potentially other diseases with inflammatory components. Additionally, platelets may contribute to cancer cell immunoevasion, suggesting that anti-platelet drugs could be beneficial when used in combination with immune checkpoint inhibitor therapy targeting PD-L1. More research is needed to assess the impact of platelet heterogeneity on cancer cell immunoevasion.</p>