Dual Targeting of Net Formation and Coagulation Improves Survival and Organ Protection in Experimental Sepsis

Sepsis is a life-threatening critical illness characterized by dysregulated host responses, in which excessive neutrophil extracellular trap (NET) formation and sepsis-associated coagulopathy play central roles in organ dysfunction and mortality. Targeting a single pathological pathway has shown limited clinical benefit. Cl-amidine, a peptidylarginine deiminase 4 inhibitor, suppresses NET formation, whereas heparin is widely used in critically ill patients to modulate coagulation and neutralize histone-mediated cytotoxicity. Whether simultaneous targeting of NET-driven inflammation and coagulation dysregulation provides enhanced protection in sepsis remains unclear.

Sepsis was induced in C57BL/6J mice using the cecal ligation and puncture model. Mice received postinsult treatment with Cl-amidine (50 mg/kg, intraperitoneally) and/or heparin (1,000 U/kg, intravenously). Seven-day survival was assessed. Circulating NET-related markers (citrullinated histone H3 and myeloperoxidase), inflammatory cytokines, coagulation parameters, and histopathological injury of the lung, liver, and kidney were evaluated.

Combined treatment with Cl-amidine and heparin significantly improved 7-day survival compared with untreated septic mice, whereas Cl-amidine monotherapy did not confer a survival benefit despite significantly reducing NET markers. Cl-amidine significantly reduced NET formation, whereas heparin alone did not show a statistically significant effect on NET markers. Combined treatment further enhanced the reduction of myeloperoxidase levels. Histopathological analysis demonstrated that pulmonary, hepatic, and renal injury were most markedly attenuated in the combination group compared with either monotherapy. Although inflammatory and coagulation parameters were generally improved following treatment, the effects of the combined regimen were comparable to those of the more effective monotherapy for several markers, and no statistically confirmed pharmacological synergy was observed.

Combined administration of Cl-amidine and heparin provides a meaningful survival benefit and attenuates multiorgan injury in experimental sepsis. Although no statistically confirmed pharmacological synergy was observed across all parameters, the combined intervention exerted additive protective effects beyond single-agent treatment. Importantly, the additional histological protection could not be explained by further suppression of systemic cytokines or by changes in global coagulation parameters, and the underlying mechanisms therefore remain incompletely defined. Further preclinical studies addressing bleeding risk, optimal dosing strategies, and mechanistic pathways are warranted before clinical translation.