Elevated levels of pro-thrombotic eNOS-negative platelets in COVID-19 patients

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.