Figure 6.
Treatment with VITT IgG and PF4 induces endothelial activation. (A) Relative mRNA expression of endothelial cells to glyceraldehyde-3-phosphate dehydrogenase in endothelial cells incubated with media (untreated), treated with VITT IgG and 25 μg/mL PF4, or with vax control IgG and 25 μg/mL PF4. Expression was determined for E-selectin (SELE), P-selectin (SELP), VCAM-1 (VCAM1), ICAM-1 (ICAM1), and VWF. There were n = 3 repeats of n = 3 VITT ELISA positive IgG (patients 3, 4, 11) and n = 3 vax control IgG (patients 1, 2, 5). One-way ANOVA with Dunn post hoc analysis was used for comparisons. (B) Left: release of VWF after stimulation of endothelial cells with thrombin, VITT IgG and 25 μg/mL PF4, vax control IgG and 25 μg/mL PF4, or untreated. Representative images. Right: quantification of the average number of VWF strings per field after stimulation with thrombin, VITT IgG + PF4, vax control IgG + PF4, and untreated. A total of 2 to 3 fields of view were used for n = 3 VITT ELISA positive IgG (patients 3, 4, 11) and n = 3 vax control IgG (patients 1, 2, 5). One-way ANOVA with Tukey post hoc test was used for comparisons. (C) Left: representative immunofluorescent images of TF staining (green) of HUVECs after incubation with IgG isolated from VITT or vax control serum. Nuclei are stained with Hoechst (blue). Right: TF fluorescence intensity units after incubation of HUVECs with VITT IgG or vax control IgG; average of n = 3 fields of view from 4 VITT ELISA positive (patients 2, 4, 6, 13) and 3 vax controls (patients 1, 2, 5). Unpaired t tests were used for comparisons. (D) Left: kinetic curve of Xa generation after treatment of endothelial cells with media (untreated), VITT IgG + 25 μg/mL PF4, vax control IgG + 25 μg/mL PF4, and 10 ng/mL TNF-α (left panel). Right: factor Xa generation after treatment of endothelial cells with VITT IgG + PF4 vs vax control IgG + PF4. There were 3 repeats for 3 VITT ELISA positive IgG samples (patients 4, 6, 13) and 3 vax control IgG samples (patients 1, 2, 5). Unpaired t tests were used for comparison.

Treatment with VITT IgG and PF4 induces endothelial activation. (A) Relative mRNA expression of endothelial cells to glyceraldehyde-3-phosphate dehydrogenase in endothelial cells incubated with media (untreated), treated with VITT IgG and 25 μg/mL PF4, or with vax control IgG and 25 μg/mL PF4. Expression was determined for E-selectin (SELE), P-selectin (SELP), VCAM-1 (VCAM1), ICAM-1 (ICAM1), and VWF. There were n = 3 repeats of n = 3 VITT ELISA positive IgG (patients 3, 4, 11) and n = 3 vax control IgG (patients 1, 2, 5). One-way ANOVA with Dunn post hoc analysis was used for comparisons. (B) Left: release of VWF after stimulation of endothelial cells with thrombin, VITT IgG and 25 μg/mL PF4, vax control IgG and 25 μg/mL PF4, or untreated. Representative images. Right: quantification of the average number of VWF strings per field after stimulation with thrombin, VITT IgG + PF4, vax control IgG + PF4, and untreated. A total of 2 to 3 fields of view were used for n = 3 VITT ELISA positive IgG (patients 3, 4, 11) and n = 3 vax control IgG (patients 1, 2, 5). One-way ANOVA with Tukey post hoc test was used for comparisons. (C) Left: representative immunofluorescent images of TF staining (green) of HUVECs after incubation with IgG isolated from VITT or vax control serum. Nuclei are stained with Hoechst (blue). Right: TF fluorescence intensity units after incubation of HUVECs with VITT IgG or vax control IgG; average of n = 3 fields of view from 4 VITT ELISA positive (patients 2, 4, 6, 13) and 3 vax controls (patients 1, 2, 5). Unpaired t tests were used for comparisons. (D) Left: kinetic curve of Xa generation after treatment of endothelial cells with media (untreated), VITT IgG + 25 μg/mL PF4, vax control IgG + 25 μg/mL PF4, and 10 ng/mL TNF-α (left panel). Right: factor Xa generation after treatment of endothelial cells with VITT IgG + PF4 vs vax control IgG + PF4. There were 3 repeats for 3 VITT ELISA positive IgG samples (patients 4, 6, 13) and 3 vax control IgG samples (patients 1, 2, 5). Unpaired t tests were used for comparison.

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