Background: Nitric oxide (NO) regulates both thrombosis and vascular leakage, two of the major complications of severe Covid19. Platelet-derived NO inhibits adhesion/aggregation and within microvascular endothelial cells NO-signalling mediates vascular endothelial growth factor (VEGF/VPF)-induced permeability. Recently, we identified novel platelet subpopulations with a differential ability to produce NO and the presence/absence of endothelial nitric oxide synthase (eNOS). We showed that eNOS-negative (eNOSneg) platelets initiate adhesion and aggregation, while eNOS-positive (eNOSpos) platelets limit aggregate growth. As inflammatory cytokines are known to counter-regulate eNOS and VEGF expression, we hypothesized that the ratio of eNOS-negative to –positive platelets may be increased in COVID-19 patients along with their VEGF content.

Aims: To determine the eNOS-positive to –negative platelet ratio and platelet VEGF levels within the blood of hospitalized COVID-19 patients.

Methods: Platelets were isolated from age- and sex-matched COVID-19 patients (ICU and non-ICU) and COVID-negative controls. Platelets were immunostained for eNOS, VEGF, and surface CD62P then analyzed using flow cytometry. A multiplex assay was used to measure plasma cytokines.

Results: The percentage of eNOSneg platelets within the blood of COVID-19 patients (ICU and non-ICU) was higher than that within healthy controls and their levels correlated with disease severity (81.2±2.8% ICU vs. 66.0 ±3.1% non-ICU vs. 6.1±1.3% controls, P-value < 0.001; Fig. 1). Accordingly, the percentage of eNOSpos platelets in COVID-19 patients was lower compared to controls, while the platelet VEGF content and surface CD62P of COVID-19 patients in ICU was higher than that of healthy controls (Fig. 2). Further, COVID-19 patients demonstrated higher TNFα, IL-6, and IL-1β plasma concentrations than controls.

Conclusion(s): Preliminary data suggests that eNOS-negative to -positive platelet ratios and VEGF content increase with COVID-19 severity, potentially predisposing patients to thrombosis and enhanced vascular permeability upon platelet activation. These platelet changes may be due to the actions of inflammatory cytokines on megakaryocytes.

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Figure 1. characterization of isolated platelet based on eNOS in COVID-19 patients’ -ICU & Non-ICU- vs Healthy controls. Statistical test: Tukey’s multiple comparisons test.

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Figure 2. comparison of the VEGF level content-A- within platelet, and surface CD62P-B- on platelets in COVID-19 ICU patients’ vs healthy controls. Statistical test: unpaired T-test.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/a-comparison-of-enos-based-platelet-subpopulation-ratios-and-platelet-vegf-content-within-covid-19/