Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada.
Thromb Res. 2024 Dec;244:109178. doi: 10.1016/j.thromres.2024.109178. Epub 2024 Oct 3.
Platelet-rich microvascular thrombi are common in severe COVID-19. Endogenous nitric oxide (NO)-signaling limits thrombus formation and previously we identified platelet subpopulations with a differential ability to produce NO based on the presence or absence of endothelial nitric oxide synthase (eNOS). eNOS expression is counter-regulated by cytokines, and COVID-19-associated immune/inflammatory responses may affect the transcriptome profile of megakaryocytes and their platelet progeny.
We investigated whether the percentage of eNOS-negative to eNOS-positive platelets increases in COVID-19 patients and whether this change may be due to the actions of pro-inflammatory cytokines on megakaryocytes.
Platelets were isolated from hospitalized COVID-19 patients and COVID-19-negative controls. Platelet eNOS was measured by flow cytometry and plasma inflammatory cytokines by ELISA. Megakaryocytes from eNOS-GFP transgenic mice and the Meg-01 cell line were characterized to identify an appropriate model to study eNOS-based platelet subpopulation formation in response to inflammatory cytokines.
COVID-19 patients demonstrated a significant increase in eNOS-negative and a concomitant decrease in eNOS-positive platelets compared to controls, and this change was associated with disease severity as assessed by ICU admission. A higher eNOS-negative to -positive platelet percentage was associated with enhanced platelet activation as measured by surface CD62P. Accordingly, COVID-19 patients demonstrated higher TNF-α, IL-6, and IL-1β plasma concentrations than controls. Inflammatory cytokines associated with COVID-19 promoted eNOS-negative Meg-01 formation and enhanced subsequent eNOS-negative platelet-like particle formation.
COVID-19 patients have a higher percentage of eNOS-negative to -positive platelets, likely as a result of inflammatory response reducing megakaryocyte eNOS expression, which predisposes to thrombosis.
富含血小板的微血管血栓在严重的 COVID-19 中很常见。内源性一氧化氮（NO）信号限制血栓形成，我们之前根据内皮型一氧化氮合酶（eNOS）的存在与否，确定了具有不同产生 NO 能力的血小板亚群。eNOS 的表达受到细胞因子的反向调节，COVID-19 相关的免疫/炎症反应可能会影响巨核细胞及其血小板前体的转录组谱。
我们研究了 COVID-19 患者中 eNOS 阴性血小板与 eNOS 阳性血小板的比例是否增加，以及这种变化是否可能是由于促炎细胞因子对巨核细胞的作用。
从住院的 COVID-19 患者和 COVID-19 阴性对照中分离血小板。通过流式细胞术测量血小板 eNOS，通过 ELISA 测量血浆炎症细胞因子。对 eNOS-GFP 转基因小鼠和 Meg-01 细胞系中的巨核细胞进行了表征，以确定一种合适的模型，用于研究炎症细胞因子对基于 eNOS 的血小板亚群形成的影响。
与对照组相比，COVID-19 患者的 eNOS 阴性血小板显著增加，而 eNOS 阳性血小板相应减少，这种变化与 ICU 入院评估的疾病严重程度有关。eNOS 阴性血小板与 eNOS 阳性血小板的比例较高与表面 CD62P 测量的血小板活化增强有关。相应地，COVID-19 患者的 TNF-α、IL-6 和 IL-1β 血浆浓度高于对照组。与 COVID-19 相关的炎症细胞因子促进 Meg-01 中 eNOS 阴性的形成，并增强随后的 eNOS 阴性血小板样颗粒的形成。
COVID-19 患者的 eNOS 阴性血小板与 eNOS 阳性血小板的比例较高，可能是由于炎症反应降低了巨核细胞 eNOS 的表达，从而导致血栓形成。
