VITT patient antibodies activate platelets in a PF4, FcγRIIA, and polyanion dependent manner. (A) Platelet activation by VITT sera in the presence of buffer (n = 60 independent experiments), PF4 (n = 78), DNA (n = 18), and short-chain polyphosphate (polyP70; n = 29). Numbers refer to total experiments with 14 VITT sera and healthy donor platelets. A shorter reaction time indicates stronger platelet activation. Patient sera and platelet donors were selected for these experiments by assessing those VITT sera that did not induce strong platelet activation in the presence of buffer to enable cross-reactivity testing. (B) Sera of VITT patients were incubated with washed platelets from healthy donors in the presence or absence of PF4 (10 µg/mL) and/or monoclonal antibody IV.3, which blocks the platelet FcγRIIA receptor (n = 31). Datasets were compared using Wilcoxon matched-pairs signed rank test.