Background Growing evidence indicates that the presence of TLR4 on platelets is a key regulator of platelet number and function. Platelets exposed to TLR4 agonists may serve to activate other cells such as neutrophils and endothelial cells in sepsis and other inflammatory conditions. The functional significance of platelet TLR4 in hemorrhagic shock, however, remains unexplored. Methods and Results Using thromboelastography and platelet aggregometry, we demonstrate that platelet function is impaired in a mouse model of hemorrhagic shock with resuscitation (HS-R). Further analysis using cellular specific TLR4 deletion in mice revealed that platelet TLR4 is essential for platelet activation and function in HS-R and that platelet TLR4 regulates the development of coagulopathy after hemorrhage and resuscitation. Transfusion of TLR4 negative platelets into mice resulted in protection from coagulopathy and restored platelet function. Additionally, platelet-specific TLR4 knock-out mice were protected from lung and liver injury and exhibited a marked reduction in systemic inflammation as measured by circulating IL-6 following HS-R. Conclusions We demonstrate for the first time that platelet TLR4 is an essential mediator of the inflammatory response as well as platelet activation and function in hemorrhagic shock and resuscitation.

BackgroundMechanical ventilation (MV) can augment inflammatory response in lipopolysaccharide (LPS) challenged lungs. High mobility group box 1 protein (HMGB1) is a pro-inflammatory mediator in ventilator-induced lung injury, but its mechanisms are not well defined. This study investigated the role of HMGB1 in lung inflammation in response to the combination of MV and LPS treatment.MethodsForty-eight male Sprague-Dawley rats were randomized to one of four groups: sham control; LPS treatment; mechanical ventilation; mechanical ventilation with LPS treatment. Mechanically ventilated animals received 10 ml/kg tidal volumes at a rate of 40 breaths/min for 4 h. In the HMGB1-blockade study, sixteen rats were randomly assigned to HMGB1 antibody group or control antibody group and animals were subjected to MV+LPS as described above. A549 cells were pre-incubated with different signal inhibitors before subjected to 4 h of cyclic stretch. Lung wet/dry weight (W/D) ratio, total protein and IgG concentration, number of neutrophils in bronchoalveolar lavage fluid (BALF), and lung histological changes were examined. The levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2) and HMGB1 in BALF were measured using ELISA. Real-time quantitative PCR and Western blot were used to analyze mRNA and protein expression of HMGB1. Western blot were employed to analyze the activation of IκB-α, NF-κB, JNK, ERK, and p38.ResultsMV significantly augmented LPS-induced lung injury and HMGB1 expression, which was correlated with the increase in IL-1β, IL-6 and MIP-2 levels in BALF. In vivo, intratracheally administration of HMGB1 antibody significantly attenuated pulmonary inflammatory injury. In vitro experiments showed cyclic stretch induced HMGB1 expression through signaling pathways including p38 and NF-κB.ConclusionsThe findings indicated that moderate tidal volume MV augmented LPS induced lung injury by up-regulating HMGB1. The mechanism of HMGB1-mediated lung injury is likely to be signaling through p38 and NF-κB pathways.