Abstract
Thrombocytopenia remains a challenging clinical condition with limited treatment options. Here, we demonstrated that miltefosine stimulated megakaryocyte (MK) differentiation in vitro. Miltefosine significantly accelerated platelet recovery, enhanced platelet function, and boosted MK production and differentiation in irradiated mice. RNA sequencing revealed association of CCR5, MAPK, and JAK2/STAT3 signaling pathways in miltefosine-mediated MK differentiation. Molecular docking, drug affinity responsive target stability (DARTS), and surface plasmon resonance (SPR) assays confirmed direct binding of miltefosine to CCR5. Inhibition of CCR5 disrupted miltefosine's effects on MK differentiation and activation of MAPK and JAK2/STAT3 signaling pathways, as well as key transcription factors GATA1, EGR1, and TAL1. Similarly, blockade of the MAPK or JAK2/STAT3 signaling pathways hindered miltefosine-induced MK differentiation and transcription factor activation. Our findings establish CCR5 as a therapeutic target for thrombocytopenia and identify miltefosine as a CCR5 agonist that promotes MK differentiation and platelet production via MAPK and JAK2/STAT3 signaling.