Dupuy, AlexanderLiu, XiaomingKong, YvonneQi, MiaoPerdomo, JoseFenwick, JemmaTieng, JessicaJohnston, BedeShi, Qiyu SaraLarance, MarkZhang, YingqiJu, Lining ArnoldColeman, PaulGamble, JenniferGardiner, Elizabeth E.Poncz, MortimerTran, HuyenChen, VivienPassam, Freda H.2025-12-162025-12-162473-9529PubMed:40085945ORCID:/0000-0001-9453-9688/work/187725259https://hdl.handle.net/1885/733795449Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious complication of the ChAdOx1 nCOV-19 vaccine. In Australia, the diagnosis of VITT required the detection of antibodies against platelet factor 4 (PF4) in plasma using a PF4/polyanion enzyme-linked immunosorbent assay (ELISA). Half of the patients who fulfilled the clinical criteria for VITT tested positive when using this ELISA and another third tested positive when using platelet activation assays, highlighting limitations in the assays used for VITT. Using a microfluidic device coated with endothelial cells, the Endo-chip, we measured the effects of serum and immunoglobulin G (IgG) from patients with clinical VITT on endothelial thromboinflammation. Our cohort comprised 40 patients (21 ELISA-positive and 19 ELISA-negative patients as measured by PF4/polyanion ELISA), 12 vaccinated patients with venous thromboembolism without VITT, and 17 individuals who received the ChAdOx1 vaccine without adverse events (vax controls). Treatment with VITT serum, plasma, or IgG increased endothelial tissue factor (TF) expression and activity. Perfusion of blood from healthy donors labelled with fluorescent antibodies against platelets, neutrophils, and fibrin through Endo-chips treated with VITT serum or IgG induced a twofold to threefold increase in platelet, neutrophil, and fibrin deposition. Thromboinflammation was enhanced with addition of PF4 and reduced with an inhibitory antibody against TF. We conclude that endothelial activation contributes to thromboinflammation in patients with clinical features of VITT. The Endo-chip offers a platform for the study of endothelial responses in immune thrombosis.The authors thank the vaccine-induced immune thrombotic thrombocytopenia (VITT) Advisory Group of the Thrombosis and Hemostasis Society of Australia and New Zealand for clinical VITT samples. The authors also thank Hester Stuart and Betty Loi from the Heart Research Institute, Sydney, Australia, for help in the recruitment of vaccinated healthy donors (vax controls). The P-selectin G1 hybridoma, developed by R.P. McEver, Oklahoma Medical Research Foundation, was obtained from the Developmental Studies Hybridoma Bank, created by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This research was funded by a Medical Research Future Fund (MRFF) 2021 COVID-19 Vaccine-Associated Thrombosis with Thrombocytopenia Syndrome Grant Opportunity (APP2015305; H.T. [lead], V.C., and F.H.P.), MRFF Cardiovascular grant APP2017914 (F.H.P. and V.C.), a Sydney Nano Research Scheme (Grand Challenge; L.A.J. and F.H.P.), and MRFF Cardiovascular Health Mission grant MRF2023977 (L.A.J. and F.H.P.). L.A.J. is a National Heart Foundation Future Leader Fellow Level 2 (105863).16en© 2025 American Society of Hematology.202510.1182/bloodadvances.2024014165105008133548