1815: EFFECTS OF BILIRUBIN ON EARLY MULTIPLE ORGAN FAILURE IN THE SECOND-HIT SEPSIS ANIMAL MODEL

Abstract

Learning Objectives: Platelets are thrombotic cells that are increasingly recognized as mediators of the immune response. Platelet activation is significantly increased in sepsis patients compared to ICU control patients. However, the role of activated platelets in contributing to sepsis pathophysiology remains unclear. We previously demonstrated NLRP3 inflammasome activation in sepsis-induced activated platelets from cecal-ligation puncture (CLP) rats. Platelet activation was associated with increased pulmonary edema and glomerular injury in CLP vs Sham controls. In this study, we investigated whether inhibition of platelet activation would attenuate NLRP3 activation and renal and pulmonary injury in response to CLP. Methods: CLP was performed in male and female Sprague Dawley rats (n=4/group) to induce abdominal sepsis and Sham rats served as controls. A subset of CLP animals were treated with clopidogrel (10mg/kg/day, CLP+Clop) to inhibit platelet activation. At 72 hours post-CLP, platelet activation was assessed via flow cytometry, NLRP3 assembly was evaluated using confocal microscopy, IL-1β and IL-18 were measured in plasma and tissues, lung wet/ dry ratio and plasma creatinine were measured to assess pulmonary injury and renal function. Results: Activated platelets were 9 ± 2% in Sham, 25 ± 2% in CLP and significantly decreased to 14 ± 0.1% in CLP+Clop (p<0.05). Importantly, co-localization of NLRP3 inflammasome assembly was inhibited in platelets of CLP+Clop vs CLP. Increases in plasma IL-1β and IL-18 in response to CLP were inhibited with clopidogrel treatment. Plasma IL-1β: Sham42 ± 1 pg/mL, CLP374 ± 108 pg/mL, CLP+Clop-85 ± 26 pg/mL (p<0.05); Plasma IL-18: Shamnot detected, CLP17 ± 4 pg/mL, CLP+Clop4 ± 2 pg/mL (p<0.05). Similar results were observed in lung and kidney homogenates. Lung wet/dry ratio was 4 ± 0.4 in Sham, 5 ± 0.4 in CLP, and 3 ± 0.3 in CLP+Clop (p<0.05). Plasma creatinine rose from 0.67 ± 0.03 mg/dL in Sham to 1.46 ± 0.03 mg/dL in CLP, and decreased to 1.01 ± 0.09 mg/dL in CLP+Clop (p<0.05) indicating improved renal function with clopidogrel treatment. Conclusions: These data provide evidence that platelet activation mediates NLRP3 inflammasome activation in response to CLP, has a role in contributing to multi-organ injury in sepsis, and thus may be a novel therapeutic target to improve sepsis outcomes.