Specific high-affinity interaction between TPO and IFN-γ. (A) Evaluation of the binding affinity between NT647-labeled IFN-γ and TPO, SCF, or Flt3L using MST. Specific IFN-γ:TPO heteromer formation was detected. (B) Evaluation of the binding affinity between NT647-labeled TPO and IFN-γ, SCF, or Flt3L using MST. Specific TPO:IFN-γ heteromer formation was detected. (C) Evaluation of the binding affinity between eltrombopag and IFN-γ in the presence of 1000-fold molar excess concentration of eltrombopag using MST. IFN-γ did not interact with eltrombopag. (D) Fluorescence far-red emission of eltrombopag (E; 665-720 nm) upon excitation at 470 nm showed no changes in intensity, even at saturating concentrations of IFN-γ, indicating no interaction between IFN-γ and eltrombopag. Background fluorescence of buffer alone (B) is shown. (E) Model depicting TPO:IFN-γ heteromers impairing TPO:c-MPL interaction, downstream cell signaling, and survival of human HSPCs (left panel). Eltrombopag does not complex with IFN-γ and can overcome the IFN-γ–mediated obstruction of TPO cell signaling pathways, resulting in improved HSPC survival (right panel). (A-B) K_D values are listed in Table 2. (A-D) Data are mean ± SEM; curve fit by Langmuir isotherm nonlinear regression.