Glutathione S‐Transferase Omega 1 Promotes Macrophage Inflammation‐Associated Coagulopathy in Sepsis by S‐Glutathionylating ANXA5 to Enhance Its Ubiquitination

Abstract

ABSTRACT Sepsis‐associated coagulopathy contributes to organ dysfunction and mortality. Macrophage‐mediated inflammation disrupts anticoagulant homeostasis, but the role of glutathione S‐transferase omega 1 (GSTO1) in this process remains unclear. Clinical samples from sepsis patients were analyzed for correlations among GSTO1, ANXA5, and coagulation parameters were evaluated using Spearman rank correlation analysis. In vitro, loss‐ and gain‐of‐function approaches were employed in macrophages to examine the regulatory axis among GSTO1, ANXA5, and HUWE1. Protein interactions, post‐translational modifications (S‐glutathionylation and ubiquitination), and protein stability were assessed using co‐immunoprecipitation, Western blotting, and cycloheximide chase assays. Macrophage polarization and tissue factor activity were detected with a commercial assay kit and flow cytometry. Coagulation function was assessed by incubating macrophage‐conditioned media with human plasma and measuring APTT, PT, and TT. In vivo, myeloid‐specific GSTO1 knockout mice and pharmacological GSTO1 inhibition were utilized to dissect the function of GSTO1 in a cecal ligation and puncture (CLP)‐induced septic mouse model. The results indicated that upregulation of GSTO1 was associated with coagulation abnormalities in sepsis, where it promoted macrophage‐mediated coagulopathy. GSTO1 reduced ANXA5 protein stability by facilitating its S‐glutathionylation at Cys314 in a catalytic activity‐dependent manner. Additionally, GSTO1 promoted HUWE1‐dependent ubiquitination and subsequent degradation of ANXA5. ANXA5 overexpression rescued the loss of anticoagulant function caused by GSTO1‐induced inflammation in macrophages. In vivo, knockout or inhibition of GSTO1 reduced the incidence of coagulopathy in a CLP‐induced septic mouse model. GSTO1 facilitated ANXA5 degradation via S‐glutathionylation and HUWE1‐mediated ubiquitination, promoting macrophage inflammation and anticoagulation impairment, identifying GSTO1 as a candidate for translational exploration in sepsis‐associated coagulopathy.