Signaling pathways involved in immune complex–mediated procoagulant platelet activation. Platelet activation depends on extracellular calcium and is partially mediated through Syk signaling. (A) Platelets from n = 4 healthy donors were exposed to TTS sera after preincubation with the indicated inhibitors and blocking agents. (B) Platelets from n = 4 healthy donors were exposed to 2 μg/mL histone octamer and a mix of the corresponding mouse antihistone antibodies (0.1 μg/mL each) after preincubation with the indicated inhibitors and blocking agents. Statistics were performed using an ordinary 1-way analysis of variance followed by an uncorrected Fisher least significant difference test. Controls are the uninhibited conditions with TTS sera (A) and generated histone/antihistone complexes (B) ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (C) Quantification of cleaved caspase 3/7 activity after treatment with platelet activation–inducing or noninducing plasma samples (n = 4 per group) as determined by flow cytometry. Viable platelets were assessed without paraformaldehyde fixation after incubation with TTS sera inducing PS exposure (PS+) compared with sera from patients with TTS and/or immune thrombocytopenia that do not induce PS exposure (PS−). Gating and analyses of MFIs were performed using FlowJo version 10 (BD Biosciences). AB, antibody; MFI, mean fluorescence intensity.