Plasma from patients with VITT sensitizes healthy donor platelets to become procoagulant. (A) Schematic diagram and representative flow cytometry plots depicting platelet P-selectin expression (CD62P) and GSAO uptake on (i) unstimulated donor platelets and upon stimulation with (ii) 5 µM SFLLRN alone or (iii) a synergistic combination of SFLLRN and plasma containing VITT anti-PF4 antibodies or (iv) plasma from a patient with bacterial sepsis. PPs are defined as GSAO⁺/CD62P⁺ platelet events (red quadrant). (B) Confocal imaging of healthy donor platelet-rich plasma exposed to 5 µM SFLLRN alone or SFLLRN and VITT plasma. Platelets are identified by CD41a antibody (cyan, top panel), whereas P-selectin (CD62P in yellow, middle panel) marks activated platelets. GSAO uptake is shown in yellow (bottom panel). PPs are characterized by ballooning morphology and GSAO uptake (bottom right panel). (C) PP flow cytometry was performed by using healthy donor whole blood treated with platelet agonist 5 µM SFLLRN (n = 43) and incubated with plasma from healthy individuals (n = 32), ChAdOx1 nCoV-19–vaccinated patients with thrombocytopenia and thrombosis but without detectable anti-PF4 antibodies (VITT neg, n = 20), vaccinated patients who were not thrombocytopenic with detectable anti-PF4 antibodies (ELISA false pos, n = 4), or clinically confirmed SRA-positive VITT patients with thrombocytopenia, thrombosis, and detectable anti-PF4 antibodies (VITT pos, n = 23). PP percentages were defined by the proportion of GSAO⁺/CD62P⁺ platelet events. Kruskal-Wallis test with Dunn’s correction for multiple comparisons was performed. Error bars indicate mean ± standard deviation. *P < .05, ****P < .0001.