Toll-like receptor 4 regulates platelet function and contributes to coagulation abnormality and organ injury in hemorrhagic shock and resuscitation

Abstract

Background: Growing evidence indicates that the presence of toll-like receptor 4 (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 (HS), however, remains unexplored.

Methods and results: Using thromboelastography and platelet aggregometry, we demonstrate that platelet function is impaired in a mouse model of HS with resuscitation. Further analysis using cellular-specific TLR4 deletion in mice revealed that platelet TLR4 is essential for platelet activation and function in HS with resuscitation 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 knockout mice were protected from lung and liver injury and exhibited a marked reduction in systemic inflammation as measured by circulating interleukin-6 after HS with resuscitation.

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 HS and resuscitation.

Keywords: blood platelets; inflammation; shock, hemorrhagic; toll-like receptor 4.

Conflict of interest statement

Conflict of Interest Disclosures: None.

© 2014 American Heart Association, Inc.

Figures

Figure 1

Changes of platelet function in hemorrhagic shock mouse models. Thromboelastography (TEG) was used to investigate platelet function via maximal amplitude (MA) after HS or HS-R. A: MA value significantly decreased in HS-R mice compared with control and HS alone. Data shown are means ± SEM, n = 8–10 mice/group. *p

Figure 2

TLR4 contributes to platelet function impairment in HS-R. A: TEG from wide type (WT) mice and TLR4−/− subjected to HS-R or unmanipulated control. A: MA values showing reduction WT but not TLR4−/− mice after HS-R. Data shown are means ± SEM, n= 8–10 mice/group. *p

Figure 3

Platelet TLR4 contributes to platelet function impairment in HS-R. A: Whole blood of cell specific TLR4−/− mice were subjected to HS-R or unmanipulated control and analyzed by TEG. Data shown are means ± SEM, n= 4–6 mice/group. *ploxP/loxP and TLR4loxP/Pf4-cre mice following HS-R or control. Data are means ± SEM, n= 4–6 mice/group. *p<0.05 vs. control.

Figure 4

Assessment of platelet activation by flow cytometry. Platelet rich plasma from TLR4loxP/Pf4-cre and TLR4loxP/loxP mice was isolated and stained for CD41 and CD62p (100uM ADP). A: Percentage of CD62p positive. Data are means ± SEM, n = 8–10 mice/group. *p<0.05 vs. control. B: Representative dual parameter dot-plot figures obtained by flow cytometry for CD41 and CD62p. C: Treatment of PRP from both TLR4loxP/Pf4-cre and TLR4loxP/loxP mice with 0.1U/ml thrombin confirms equal CD62p upregulation. Data are means ± SEM, n = 5 mice/group. p=NS between thrombin groups.

Figure 5

TLR4 regulates platelet aggregation following HS-R. Aggregometry was performed on wild-type (TLR4 +) or mice lacking TLR4 on their platelets (TLR4 −). Results are expressed as the area under the aggregometry curve, measured as arbitrary units/minute (AU*min). A: Ex vivo collagen (5ug/ml) treatment of platelets demonstrates no significant difference in aggregation between groups. N=8 mice/group. p=NS. B: Aggregometry from either control (pre-shock blood draw) vs HS-R (post-shock). Collagen (5ug/ml) was present as the agonist in all samples. N=5 mice/group. C: Representative aggregometry tracings from control and HS-R. Each y axis block represents 10% change and each x axis block is 1 minute.

Figure 6

Effect of HS-R on platelet sequestration into lung and liver. TLR4loxP/Pf4-cre mice and TLR4loxP/loxP mice were subjected to HS-R or unmanipulated control. Platelet sequestration into lung and liver of the mice were assessed by measuring the expression of CD41 using western blotting and immunofluorescence. A: The expression of CD41 in liver (A) and lung (B), analyzed by Western blotting. Data are means ± SEM, n = 8–10 mice/group. *p<0.05 vs. control. Livers (C) and lungs (D) were stained with CD41 (green), F-actin (white) antibody and counterstained with Hoechst to detect nuclei (blue) and imaged by confocal microscopy (Olympus FV1000 confocal, magnification ×200). Representative images of five individual experiments.

Figure 7

Effect of HS-R on lung and liver injury and cytokine release. TLR4loxP/Pf4-cre mice and TLR4loxP/loxP mice were subjected to HS-R or unmanipulated control and liver and lung injury was assessed by histology, circulating AST and ALT concentrations, lung MPO activity and circulating IL-6. Liver (A) and lung (B) were sectioned and stained with H&E and tissue damage induced by HS-R was assessed by light microscopy (Nikon FX series). Black arrows show the regions of necrosis. (Original magnification ×100). Serum AST (C) and ALT (D) levels were assayed and are presented as means ± SEM (n = 8–10 mice/group). Aliquots of the mouse lung tissues were homogenized to prepare total protein samples and MPO levels were assayed by ELISA (E). The levels are presented as means ± SEM (n = 8–10 mice/group). Serum samples from mice treated as described above were used to assay IL-6 concentration by ELISA (F) and the levels are presented as means ± SEM (n = 8–10 mice/group). *p<0.05 vs. control. #p<0.05 vs. HS-R group of TLR4loxP/loxP mice.

Figure 8

Platelet TLR4 is necessary and sufficient for the induction of platelet dysfunction and organ injury in HS-R. Wild-type (LOXP) and mice lacking TLR4 on platelets (PF4-cre) were depleted of their platelets using anti-CD41 antibody and subsequently transfused donor platelets isolated from either their littermates (LOXPtransLOXP, PF4-cretransPF4-cre) as controls or from donors with opposite TLR4 status with respect to their platelets (LOXPtransPF4-cre, PF4-cretransLOXP) to test the specific role of TLR4 on platelets in HS-R. (A) TEGs pre and post HS-R are shown and the MA are quantified in (B) as means ± SEM (n = 5 mice/group). (C) rSerum AST levels including both sham and HS-R (means ± SEM (n = 5 mice/group)). *p<0.01 vs. control. #p<0.05 vs. HS-R group of LOXPtransLOXP mice.