Figure 4.
VITT serum induces endothelial cell activation, which is enhanced with the addition of PF4. (A) Immunostaining of the HUVEC layer for spike protein (red), nuclei, TF, and P-selectin (lower panel) after incubation with media alone or with media containing ChAdOx1 nCOV-19 adenoviral vaccine. (B) Immunostaining for human IgG (green) in HUVECs after treatment with VITT sera vs vax control sera. Representative images. The fluorescence intensity of IgG per field is shown for VITT and vax controls. Mean ± SD from 3 to 5 fields of view; n = 4 independent experiments, unpaired t test. (C) Representative images of HUVECs stained for TF (green), P-selectin (red), and VCAM-1 (magenta) after exposure to TNF-α (5 ng/mL), VITT serum, VITT serum + PF4 (25 μg/mL), vax control serum, vax control serum + PF4 (25 μg/mL), or media. Nuclear staining using Hoechst is shown in blue. (D) Fluorescence intensity of TF, P-selectin, and VCAM-1 of HUVECS treated with VITT, without or with 25 μg/mL PF4 added, or treated with vax control, without or with 25 μg/mL PF4 added, expressed as fold change in comparison with the fluorescent intensity of media alone. Mean ± SD, from 3 to 5 fields of view; VITT ELISA positive n = 2 (patients 2, 4), VITT ELISA negative n = 2 (patients 3, 9), vax control n = 4 (patients 2, 6, 10, 12). One-way ANOVA with Dunn post hoc test was used for comparison. (E) Fold change in the mRNA expression of F3 (TF) in HUVECs treated with plasma, with or without PF4 (25 μg/mL), in comparison with that of glyceraldehyde-3-phosphate dehydrogenase. Mean ± SD, n = 10 for media, VITT ELISA positive n = 21 (Table 1), VITT ELISA negative n = 17 (Table 1), vax control n = 8 plasma samples. One-way ANOVA with Dunn post hoc test was used for comparisons.

VITT serum induces endothelial cell activation, which is enhanced with the addition of PF4. (A) Immunostaining of the HUVEC layer for spike protein (red), nuclei, TF, and P-selectin (lower panel) after incubation with media alone or with media containing ChAdOx1 nCOV-19 adenoviral vaccine. (B) Immunostaining for human IgG (green) in HUVECs after treatment with VITT sera vs vax control sera. Representative images. The fluorescence intensity of IgG per field is shown for VITT and vax controls. Mean ± SD from 3 to 5 fields of view; n = 4 independent experiments, unpaired t test. (C) Representative images of HUVECs stained for TF (green), P-selectin (red), and VCAM-1 (magenta) after exposure to TNF-α (5 ng/mL), VITT serum, VITT serum + PF4 (25 μg/mL), vax control serum, vax control serum + PF4 (25 μg/mL), or media. Nuclear staining using Hoechst is shown in blue. (D) Fluorescence intensity of TF, P-selectin, and VCAM-1 of HUVECS treated with VITT, without or with 25 μg/mL PF4 added, or treated with vax control, without or with 25 μg/mL PF4 added, expressed as fold change in comparison with the fluorescent intensity of media alone. Mean ± SD, from 3 to 5 fields of view; VITT ELISA positive n = 2 (patients 2, 4), VITT ELISA negative n = 2 (patients 3, 9), vax control n = 4 (patients 2, 6, 10, 12). One-way ANOVA with Dunn post hoc test was used for comparison. (E) Fold change in the mRNA expression of F3 (TF) in HUVECs treated with plasma, with or without PF4 (25 μg/mL), in comparison with that of glyceraldehyde-3-phosphate dehydrogenase. Mean ± SD, n = 10 for media, VITT ELISA positive n = 21 (Table 1), VITT ELISA negative n = 17 (Table 1), vax control n = 8 plasma samples. One-way ANOVA with Dunn post hoc test was used for comparisons.

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