FcγRIIA-mediated activation of cells and inhibition by PF4 antagonists. (A) Activation of DT40 cells transfected with FcγRIIA and a luciferase reporter. Basal condition is a buffer only control, Hep indicates heparin, and IV.3 is the anti-FcγRIIA monoclonal antibody in the presence of anti-IgG antibody. (B) Dose response of DT40 activation by heparin in the presence of constant amounts of PF4. (C) Dose response of antagonists for inhibition of DT40 activation. Data are the mean ± SEM of at least 3 independent experiments performed in triplicate. Curves in panel C represent fit of data to the indirect Hill equation. (D) Inhibition of DT40 activation by plasma obtained from 3 HIT patients. Both antagonists completely inhibited activation by KKO and patient samples at a concentration of 500μM. Data represent the mean ± SEM of at least 2 independent experiments performed in triplicate. (E) Activation of platelets as measured by release of ¹⁴C-5-hydroxytryptamine creatinine sulfate. Data are representative of at least 2 experiments. Antagonists were present at a concentration of 2.5mM. (F) As in panel E, except that activation was by plasma from patients with HIT, and several antagonist concentrations were measured.