Figure 2. Plasma from patients with COVID-19 promotes activation of coagulation on endothelial cells.

HUVECs were cultured overnight in the presence of 10% pooled plasma from patients with severe (S, ICU), moderate (Mod, non-ICU), or mild (outpatient) COVID-19 or healthy controls (HC). (A–D) Cells were analyzed for their ability to generate factor Xa or thrombin. Where indicated, cells were pretreated with Angpt-1 (300 ng/mL) or AKB-9778 (5 μM) for 30 minutes prior to incubation with plasma. (A and C) Representative experiments are depicted as mean absorbance (405 nm) or the first derivative of arbitrary fluorescence units as a function of time. (B and D) The rate of reaction for factor Xa and thrombin were converted to nM/min and U/mL, respectively, by comparison to standard curve. For factor Xa and thrombin generation assays, each data point represents the mean of 3 technical replicates, with 3–5 biologic replicates performed in total. (E and F) Cells were stained with annexin V to assess for phosphatidylserine externalization (n = 3 per group). Total fluorescent area was quantified and normalized for number of nuclei. (F) Graph represents the mean total fluorescence per 3 × 3 tile scan image ± SD. Scale bar: 50 μm. Significance was determined by 1-way ANOVA using Dunnett’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.