VITT patient anti-PF4 antibodies trigger NETosis. (A-B) Confocal laser scanning microscopy images of in vitro NETs generated by human neutrophils and platelets following incubation with VITT patient serum alone (A) or VITT patient serum in combination with PF4 (B). (C-D) NET formation induced in healthy control serum alone (C) or healthy serum in the presence of PF4 (D). (E-H) NET formation induced by affinity-purified (Affi.pur.) anti-PF4 IgG from VITT patients (E); combination of anti-PF4 IgG from VITT patients and PF4 (F); buffer; (G) or PF4 only (H). (I-J) Neutrophils (without [w/o] platelets) were incubated with VITT patient serum (I) or VITT patient serum and PF4 (J), and NET formation was measured. (K) Quantification of NETs (NETosis [%]) from confocal laser scanning microscopy images obtained from nuclear and extracellular DNA fluorescent channels was performed using at least 12 individual images for each condition, n = 3 experimental replicates. Statistical analysis was performed using the Welch and Brown-Forsythe ANOVA multiple comparisons test with post hoc 2-stage step-up method per Benjamini, Krieger, and Yekutieli, q = 0.05.