Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare thromboembolic complication associated with two severe acute respiratory coronavirus 2 (SARS-CoV-2) adenoviral vaccine vectors, ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen/Johnson & Johnson). Pathological immunoglobulin G (IgG) antibodies that bind platelet factor 4 (PF4) are the major driver of VITT pathogenesis. These anti-PF4 antibodies bind PF4 to form immune complexes that activate platelets in a Fcγ receptor IIa (FcγRIIa) dependent manner, promoting a prothrombotic environment. Endothelial cells can also promote thrombus formation in response to vessel injury. Whether aberrant endothelial cell activation also contributes to VITT pathogenesis is unknown. Given the fundamental role of ECs in the coagulation cascade, we hypothesised that anti-PF4 IgG antibodies can activate ECs, further amplifying the coagulation cascade and VITT pathogenesis. We incubated human umbilical vein endothelial cells (HUVECs) with sera from VITT patients and anti-PF4 monoclonal antibodies (mAbs) derived from serum antibody sequences for 24 hours, in the presence of PF4 and assessed prothrombotic phenotypes by immunofluorescence. Antibody-PF4 immune complexes from VITT serum or anti-PF4 mAbs upregulated and clustered the adhesion molecule P-selectin on HUVECs. These immune complexes also increased platelet adherence to endothelial cells, but did not increase endothelial ICAM-1 expression, indicating a selective mechanism of endothelial cell activation. Only anti-PF4 immune complexes induced P-selectin clustering, indicating that this is PF4-dependent. These results suggest P-selectin augments thrombosis by modulating platelet, monocyte and neutrophil activation, and targeting endothelial cell P-selectin may have benefit in treating VITT.