Fig 2: Scheme of proposed procoagulant mechanisms in VITT. Early reactions: Initial VITT reactions are triggered by complexes formed by PF4 and vaccine constituents and are accompanied by an inflammation-induced “danger signal.” Both events occur early following vaccination (days 1-2). Late reactions: In some vaccine recipients, PF4/vaccine-induced activation of B cells produces high-titer anti-PF4 autoantibodies that bind to platelets and induce platelet activation. Anti-PF4 antibodies together with platelets activate granulocytes (neutrophils) to release procoagulant NETs (NETosis), culminating in VITT-associated thrombosis.

Fig 3: 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.

Fig 4: Complex formation of PF4, vaccine components, and anti-PF4 antibodies on platelet surfaces. (A) 3D super-resolution microscopy of PF4, AV and affinity-purified anti-PF4 antibodies (obtained from VITT patients) reveals complex formation. Upper left panel: PF4 (green) and AV hexon protein (purple) accumulation on platelet surfaces. Scale bar, 1 µm. Lower left panel: Localization microscopy (dSTORM) of PF4 (green) at ChAdOx1 vaccine aggregates (AV, purple). Scale bar, 200 nm. Right panel: colocalization of ChAdOx1 AV hexon protein (AV; purple), PF4 (green) and purified anti-PF4 IgG from VITT patient sera (blue). Scale bar, 200 nm. (B) Relative composition of 192 complexes analyzed. Approximately 44.5% of complexes investigated showed VITT anti-PF4 IgG bound to particles containing both PF4 and AV hexon proteins. (C) Analyses of ChAdOx1 nCoV-19 vaccine by DLS. Diameter of ChAdOx1 nCoV-19 vaccine aggregates before (left) and after addition of PF4 (+PF4, 10 µg/ml). Incubation of mouse anti-P F4 recombinant antibody (clone RTO) or purified anti-PF4 IgG from VITT patients increased the size of vaccine aggregates in the presence of PF4. Addition of DNA further increased the size of PF4/vaccine aggregates (+DNA). In contrast, addition of heparin (100 IU/mL) dissociated previously formed vaccine/PF4/anti-PF4 IgG complexes. Each data point represents 12 runs of n =3 individual measurements. Statistical assessment by ordinary 1-way analysis of variance (ANOVA) with Sidak's multiple comparisons test. (D) Negative charge indicated by surface ? potential (?,13 mV) of ChAdOx1 nCoV-19 vaccine particles in the presence of buffer (control), UFH (1 IU/mL), PF4 (reduced the negative charge; -5 mV), and coapplication of PF4 and heparin. Statistical assessment by Brown-Forsythe and Welch ANOVA test followed by Dunnett's T3 multiple comparisons test. Transmission electron microscopy images of aggregates formed in the vaccine upon addition of PF4. (E) Vaccine without added PF4 shows the virion particles and multiple small amorphous structures. (F) Aggregate (arrowhead) formation in the vaccine following addition of PF4. Biotinylated PF4 (arrow) is labeled with 10 nm gold particles. (G) Aggregate (arrowhead) as in panel F. Here, AV capsid protein (arrow) is labeled with 10 nm gold particles. Scale bars, 100 nm.