ROCK2 Inhibition Suppresses Cytotoxic T Lymphocyte-Mediated Platelet Destruction in Primary Immune Thrombocytopenia Running title: ROCK2 inhibition reduces CTL platelet injury

Cytotoxic T lymphocyte (CTL)-mediated platelet destruction represents an important pathogenic mechanism in ITP patients. Rho-associated coiled-coil kinase 2 (ROCK2) is an emerging regulator of immune balance, but its role in pathogenic CTL activation in ITP remains undefined. Here, we demonstrated that selective ROCK2 inhibition with KD025 potently suppressed CTL-mediated platelet destruction. In vitro , KD025 treatment of CTLs from ITP patients suppressed key effector functions, reducing degranulation as measured by CD107a expression, diminishing the secretion of cytotoxic molecules such as granzyme B and perforin, and decreasing CTL-platelet conjugate formation, resulting in reduced platelet apoptosis and activation. RNA-sequencing revealed downregulation of cytotoxic and glycolytic programs, with enrichment of JAK-STAT signaling. Mechanistically, KD025 reversed the pathogenic metabolic shift in ITP CTLs by lowering glycolytic flux and restoring mitochondrial respiration, accompanied by decreased STAT3 phosphorylation. IL-6-mediated STAT3 activation largely reversed these effects, indicating a ROCK2-STAT3-dependent mechanism. In vivo , both daily KD025 administration to an active ITP mouse model and transplantation of KD025-pretreated CD8 ⁺ T cells into irradiated Rag1 ^−/− mice alleviated CTL-mediated platelet apoptosis and increased platelet counts. Collectively, these findings identified a ROCK2-STAT3 regulatory axis integrating transcriptional and metabolic control of CTL pathogenicity and support ROCK2 inhibition as a promising therapeutic strategy for ITP.