Col003 attenuates sepsis-associated coagulation dysfunction in mice in association with reduced platelet activation and NET formation

Background: Coagulation dysfunction is a major contributor to the increased mortality associated with sepsis (Levi et al., 2013; Gando et al., 2019 [1,2]), in which platelet activation, neutrophil activation, and neutrophil extracellular trap (NET) formation play important roles. Recent studies have suggested that HSP47 is involved in venous thromboembolism and platelet/neutrophil activation (Thienel et al., 2023a [3]); however, whether HSP47 contributes to platelet-mediated NET formation and coagulation dysfunction during sepsis remains unclear.

Objectives: This study aimed to investigate the involvement of Heat Shock Protein 47 (HSP47) in platelet activation, NET formation, and coagulation dysfunction during sepsis, and to evaluate the potential effect of pharmacological modulation of HSP47-associated responses.

Methods: A murine model of sepsis was established using cecal ligation and puncture (CLP), and mice were treated with Col003, a functional inhibitor of HSP47-associated responses. Platelet HSP47 signal, histopathological injury, coagulation parameters, platelet activation, granule release, platelet-leukocyte aggregate formation, and NET formation were assessed. Seven-day survival was monitored in the main CLP intervention experiment, and 48 h short-term survival was assessed in the Col003 timing experiment. Additional in vitro experiments using bone marrow-derived neutrophils were performed to explore the involvement of the TLR2-MyD88 pathway in HSP47-associated NET formation.

Results: CLP induced an increase in platelet HSP47 signal, coagulation abnormalities, inflammatory cytokine production, platelet activation, platelet granule release, platelet-leukocyte aggregate formation, and NET formation. Col003 treatment attenuated these CLP-associated changes and improved 7-day survival. In the timing experiment, early administration 3 h before CLP showed the most pronounced effects on platelet HSP47 signal, CitH3 expression, and 48 h survival. In platelet-neutrophil co-culture experiments, platelets from CLP mice promoted NET formation, whereas platelets from Col003-treated CLP mice showed a reduced NET-inducing capacity. In vitro pathway-validation experiments further showed that recombinant mouse HSP47 increased TLR2, MyD88, and CitH3 expression in bone marrow-derived neutrophils, while inhibition of TLR2 or MyD88 reduced HSP47-induced CitH3 expression.

Conclusions: These findings suggest that HSP47-associated responses are involved in sepsis-associated coagulation dysfunction and are linked to platelet activation and NET formation. Early Col003 intervention attenuated CLP-induced pathological changes, supporting HSP47-associated pathways as potential targets for further investigation in sepsis-associated coagulopathy. Further studies are needed to determine whether delayed Col003 administration remains effective after sepsis has been established.