Platelet Toll-like Receptor 4 Research Articles

Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection.

SARS-CoV-2 is associated with an increased risk of venous and arterial thrombosis, but the underlying mechanism is still unclear. We performed a cross-sectional analysis of platelet function in 25 SARS-CoV-2 and 10 healthy subjects by measuring Nox2 (NADPH oxidase 2)-derived oxidative stress and thromboxane B2, and investigated if administration of monoclonal antibodies against the S protein (Spike protein) of SARS-CoV-2 affects platelet activation. Furthermore, we investigated in vitro if the S protein of SARS-CoV-2 or plasma from SARS-CoV-2 enhanced platelet activation. Ex vivo studies showed enhanced platelet Nox2-derived oxidative stress and thromboxane B2 biosynthesis and under laminar flow platelet-dependent thrombus growth in SARS-CoV-2 compared with controls; both effects were lowered by Nox2 and TLR4 (Toll-like receptor 4) inhibitors. Two hours after administration of monoclonal antibodies, a significant inhibition of platelet activation was observed in patients with SARS-CoV-2 compared with untreated ones. In vitro study showed that S protein per se did not elicit platelet activation but amplified the platelet response to subthreshold concentrations of agonists and functionally interacted with platelet TLR4. A docking simulation analysis suggested that TLR4 binds to S protein via three receptor-binding domains; furthermore, immunoprecipitation and immunofluorescence showed S protein-TLR4 colocalization in platelets from SARS-CoV-2. Plasma from patients with SARS-CoV-2 enhanced platelet activation and Nox2-related oxidative stress, an effect blunted by TNF (tumor necrosis factor) α inhibitor; this effect was recapitulated by an in vitro study documenting that TNFα alone promoted platelet activation and amplified the platelet response to S protein via p47phox (phagocyte oxidase) upregulation. The study identifies 2 TLR4-dependent and independent pathways promoting platelet-dependent thrombus growth and suggests inhibition of TLR4. or p47phox as a tool to counteract thrombosis in SARS-CoV-2.

Heme stimulates platelet mitochondrial oxidant production to induce targeted granule secretion

Hemolysis, a pathological component of many diseases, is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether these mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules, including the glycoprotein thrombospondin-1 (TSP-1), from the α-granule of platelets. We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet toll-like receptor 4 (TLR4) signaling and leads to the downstream phosphorylation and inhibition of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging of mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.

Toll-like receptor 4 activation in platelets from myocardial infarction patients

IntroductionPlatelet toll-like receptor 4 (TLR4) is overexpressed in patients with myocardial infarction (MI) but it remains to elucidate if it is activated and the potential trigger. MethodsSerum levels of lipopolysaccharides (LPS) and platelet aggregation (PA) by collagen alone or in combination with a TLR4 inhibitor (TLR4i) were studied ex vivo in platelets from 40 MI patients and 40 controls matched for age, sex and atherosclerotic risk factors; platelet TIR domain-containing adaptor protein (TIRAP) and TIRAP-MyD88 interaction were also investigated by western blot and co-immunoprecipitation, respectively. In vitro experiments were conducted to see if LPS triggers platelet TIRAP phosphorylation. ResultsSerum LPS was significantly higher in patients compared to controls (29.5±7.1 vs 16.2±3.8 pg/mL; p<0.001). Collagen-stimulated platelets from MI pre-treated with TLR4i showed a significant decrease of PA compared to platelets stimulated with collagen. Ex vivo study showed that TIRAP phosphorylation as well as TIRAP-MyD88 co-immunoprecipitation were higher in patients compared to controls. In vitro study showed that LPS, at concentrations like those found in MI, dose-dependently activated TIRAP and amplified the platelet response to the agonist, an effect blunted by TLR4i. ConclusionThe study provides evidence that in MI patients platelet TLR4 is activated and suggests circulating LPS as potential trigger.

Lipopolysaccharide-Activated Canine Platelets Upregulate High Mobility Group Box-1 via Toll-Like Receptor 4.

High mobility group box-1 (HMGB1) and the toll-like receptor 4 (TLR4) axis is a key mediator of inflammation. Platelet-derived high mobility group box-1 (HMGB1) may also play a critical role in sepsis-mediated thrombosis resulting in complications like disseminated intravascular coagulation and multiple organ failure. While elevated levels of HMGB1 have been documented in humans and dogs with systemic inflammatory response syndrome and sepsis, a better understanding of how platelet agonists and lipopolysaccharide (LPS) mediate platelet HMGB1 expression would open doors to novel therapies for sepsis-mediated thrombosis. Herein, we sought to determine if canine platelets express HMGB1 in the presence or absence of LPS and agonists (ADP or thrombin) and if surface expression of HMGB1 is dependent on platelet TLR4. Canine platelets were unstimulated (resting) or activated with thrombin or adenosine diphosphate (ADP) in the presence or absence of Escherichia coli LPS prior to flow cytometric and western blot analyses for HMGB1 expression. We also treated canine platelets with or without TLR4 function blocking antibody or its isotype control. We discovered that while thrombin upregulated both surface and cellular HMGB1 expression, LPS-mediated activation in the presence of ADP priming led to upregulation of surface HMGB1 expression. This expression was found to be most prominent in platelets that had undergone alpha-granule secretion. Inhibition of TLR4 attenuated LPS-induced HMGB1 expression indicating that exteriorization of HMGB1 may be dependent on the non-genomic pathway of platelet TLR4. Our findings indicate that upregulation of platelet-derived HMGB1 occurs as a result of thrombin or TLR4-mediated activation in dogs. Future studies should explore the translational implication of platelet-derived HMGB1 as novel therapeutic targets in humans and dogs with sepsis.

Pharmacological Modulation of Neutrophil Extracellular Traps Reverses Thrombotic Stroke tPA (Tissue-Type Plasminogen Activator) Resistance.

Background and Purpose- Recanalization of the occluded artery is a primary goal in stroke treatment. Unfortunately, endovascular treatment is not always available, and tPA (tissue-type plasminogen activator) therapy is limited by its narrow therapeutic window; importantly, the rate of early arterial recanalization after tPA administration is low, especially for platelet-rich thrombi. The mechanisms for this tPA resistance are not well known. Since neutrophil extracellular traps (NETs) have been implicated in this setting, our aim was to study whether NET pharmacological modulation can reverse tPA resistance and the role of TLR4 (Toll-like receptor 4), previously related to NET formation, in thrombosis. Methods- To this goal, we have used a mouse photothrombotic stroke model, which produces a fibrin-free thrombus composed primarily of aggregated platelets and thrombi obtained from human stroke patients. Results- Our results demonstrate that (1) administration of DNase-I, which promotes NETs lysis, but not of tPA, recanalizes the occluded vessel improving photothrombotic stroke outcome; (2) a preventive treatment with Cl-amidine, impeding NET formation, completely precludes thrombotic occlusion; (3) platelet TLR4 mediates NET formation after photothrombotic stroke; and (4) ex vivo fresh platelet-rich thrombi from ischemic stroke patients are effectively lysed by DNase-I. Conclusions- Hence, our data open new avenues for recanalization of platelet-rich thrombi after stroke, especially to overcome tPA resistance.

Abstract 369: Muscle Ischemia Induces Nlrp3 Inflammasome Activation in Platelets via Tlr4, Promoting Platelet Aggregation and Interfering With Perfusion Recovery

Introduction: Angiogenesis is an adaptive response to chronic ischemia, but is deficient in peripheral arterial disease (PAD). We have recently shown that the pattern recognition receptors toll-like receptor 4 (TLR4) and nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets control aggregation and thrombosis. The role of platelet TLR4 and NLRP3 in PAD is unexplored. Methods: Unilateral femoral artery ligation (FAL) was performed in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in global NLRP3 knockout (NLRP3 KO) mice. Platelet NLRP3 inflammasome activation was monitored by caspase-1 activation (fluorescent labeled inhibitor of caspase-1, FLICA) and cleavage of IL1β (Western blot). Platelet aggregation was evaluated with impedance aggregometry. Laser Doppler perfusion imaging (LDPI) verified perfusion in the ischemic (right) and non-ischemic (left) limb over time. Angiogenesis and myoblast regeneration were measured histologically. Results: Platelet NLRP3 inflammasome activity was significantly upregulated following FAL (p&lt;0.001), and reversed with TAK242, a TLR4 inhibitor. FAL significantly increased aggregation of circulating platelets, which was significantly suppressed in TLR4 PF4 (p&lt;0.01) and NLRP3 KO mice (p&lt;0.001). Down-regulation of platelet aggregation and caspase-1 activity in TLR4 PF4 mice was nearly completely reversed by nigericin, a NLRP3 activator. Ischemic limb perfusion (Fig 1A) was significantly higher in TLR4 PF4 (Fig1B, p&lt;0.05) and NLRP3 KO mice (Fig1C, p&lt;0.001) than in controls 14d after FAL. Angiogenesis and regeneration were significantly improved in TLR4 PF4 mice (p&lt;0.05). Conclusion: We show that the platelet NLRP3 inflammasome is activated in muscle ischemia via platelet TLR4, which upregulates platelet aggregation and deters recovery from FAL. Thus, platelet TRL4/NLRP3 activation may be a therapeutic target to improve limb salvage in PAD.

Platelets at the crossroads of thrombosis, inflammation and haemolysis.

Platelets play a critical role at the interphase of thrombosis and inflammation, key features in haemolysis-associated disorders. Exercising this role requires expression of pattern recognition receptors by platelets, including toll-like receptor 4 (TLR4) and nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3), the latter forming intraplatelet multiprotein inflammasome complexes. Platelets are a potential target of various damage-associated molecular pattern (DAMP) molecules, such as free haem, a degradation by-product of haemoglobin oxidation during haemolysis, and high-mobility group box 1 (HMGB1), a DNA-binding protein released by dying or stressed cells and activated platelets. We have recently identified platelet TLR4, NLRP3, and Bruton tyrosine kinase (BTK) as critical regulators of platelet aggregation and thrombus formation, suggesting that the BTK inhibitor ibrutinib is a potential therapeutic target. Increasing evidence suggests that these and other DAMP-driven signalling mechanisms employed by platelets might be key in mediating inflammation and thrombosis encountered in haemolytic disorders. However, the precise regulatory triggers and their clinical relevance are poorly understood. We provide new insights into these less-well characterised platelet mechanisms, which are potentially targetable in haemolytic disorders.

Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4.

Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

Platelets Toll-like receptor-4 in Crohns disease.

Platelets are activated in Crohn's disease (CD) and interplay with leukocytes. Engagement of Toll-like receptor-4 (TLR-4), which is expressed in human platelets, may be involved in crosstalks between platelets and leukocytes leading to their mutual activation for host defense. Human neutrophil peptides (HNPs), lipoprotein binding peptides, and sCD14 were determined by enzyme-linked immunosorbent assays in 42 patients with active CD, in 43 patients with CD in remission, and in 30 healthy individuals. Neutrophil-platelet aggregates and binding of the TLR-4 monoclonal antibody to platelets were determined by flow cytometry. Levels of HNPs were higher in patients with CD than in controls (P = 0.0003 vs. active CD and P = 0.01 vs. CD in remission). Likewise, neutrophils with adhering platelets were higher in patients with active CD than in controls (P = 0.004). Binding of the TLR-4 antibody in patients with active CD was similar to that in controls, while patients in remission had significantly higher binding capacities (P = 0.59 and P = 0.003). Incubation of plasma from patients with active disease or patients in remission with platelets from healthy controls confirmed lower binding of the TLR-4 antibody in the presence of plasma from active diseased patients compared to controls (P = 0.039), possibly due to high levels of lipopolysaccharides, as suggested by high levels of sCD14 and lipoprotein binding protein. Our study indicates involvement of platelet TLR-4 in enhancing the secretion of antimicrobial peptides from neutrophils. While platelet aggregation can be due to a variety of mechanisms in inflammatory disease, the mutual activation of platelets and neutrophils may augment host defense.

Hemopexin Gene Therapy Inhibits Inflammation and Vaso-Occlusion in Transgenic Sickle Cell Mice

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). Hemolysis releases free hemoglobin (Hb) and Hb-containing microparticles into the vasculature that upon oxidation to methemoglobin frees heme from the globin, which in turn can promote oxidative stress and activate toll-like receptor 4 (TLR4) signaling. Hemopexin (HPX), a plasma β1-glycoprotein, binds heme with a very high affinity (Kd < 10-12 M), and transports it to the liver for catabolism via CD91 receptor-mediated uptake. SCD patients have low serum HPX levels likely due to chronic hemolysis leading to increased HPX catabolism with insufficient compensatory increase in synthesis. We and others have shown that HPX transports heme to the liver, and inhibits heme toxicity and the activation of endothelial, leukocyte and platelet TLR4 signaling. Acute studies have shown HPX infusion prior to a heme challenge protects sickle mice from vaso-occlusion and developing acute pulmonary injury while chronic HPX infusion therapy modified heme toxicity to endothelium. We hypothesize that in SCD mice, hepatic overexpression of HPX will bind the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis (vaso-occlusion). To examine the protective role of HPX in SCD, we transplanted bone marrow from NY1DD SCD mice into HPX-/- or normal C57BL/6 mice. After 12 weeks, conversion to the HbS phenotype was confirmed by isoelectric focusing. Dorsal skin fold chambers (DSFC) were implanted in week 13 and microvascular stasis (% non-flowing venules) assessed in response to heme (3.2 µmol/kg) infusion. HPX-/- sickle mice had 34% ± 3% and 24% ± 2% at 1h and 4h post heme, significantly greater than HPX+/+ C57BL/6 sickle mice which had 21% ± 5% and 13% ± 8% at 1 and 4 h, (mean ± SD, p<.05), demonstrating the protective role of HPX in SCD. To further test our hypothesis, we utilized Sleeping Beauty (SB) transposon-mediated gene therapy to overexpress rat HPX in NY1DD and Townes-SS SCD mice. Rat HPX plasmid (pT2/Caggs-HPX) was delivered with an SB transposase plasmid (pK/CMV-SB 100X) and luciferase (LUC) plasmid (pT2/Caggs-LUC, as tracer) in trans into NY1DD or Townes-SS SCD mice by hydrodynamic tail vein injections. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or LUC plasmid with SB transposase plasmid in trans. One week later, the mice LUC bioluminescence imaging showed the liver was the primary location of expression. Four weeks later, the HPX SCD mice had marked increases in hepatic rat HPX mRNA (300-2000 copies/5ng total RNA) comparing to LRS and SB-LUC controls (0-44 copies/5ng total RNA). Plasma and hepatic HPX were significantly increased compared to LRS and SB-LUC controls. In vitro expression of the rat HPX plasmid in Chinese Hamster Ovary cells, and protein purification confirmed heme binding activity by spectroscopic scan absorbance shifts of rat HPX-heme complexes at 414nm.DSFCs were implanted 4 weeks after plasmid infusion and microvascular stasis was assessed in response to heme (3.2 µmol/kg) infusion. NY1DD and Townes-SS mice overexpressing rat HPX (SB-HPX) had significantly less stasis than LRS or SB-LUC treated SCD mice (Figure 1A and B). HPX overexpression markedly increased nuclear Nrf2 expression in the livers of sickle mice, presumably by promoting delivery of heme to the liver and activating the Keap1-Nrf2 axis. In addition, hepatic HO-1 activity and protein and CD91 protein were increased in sickle mice overexpressing HPX and NF-ĸB activation was markedly decreased as assessed by nuclear phospho-p65-NF-ĸB expression on western blots demonstrating the anti-inflammatory properties of HPX in sickle mice. In conclusion, supplementing HPX levels in transgenic sickle mice via gene therapy activates the Nrf2 anti-oxidant axis and ameliorates inflammation and vaso-occlusion. We speculate that plasma HPX supplementation may be beneficial in SCD especially during hemolytic crises or acute chest syndrome. [Display omitted] DisclosuresVercellotti:Cydan: Research Funding; CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding. Belcher:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding.

Cellular fibronectin containing extra domain A promotes arterial thrombosis in mice through platelet Toll-like receptor 4

AbstractCellular fibronectin containing extra domain A (Fn-EDA+), which is produced in response to tissue injury in several disease states, has prothrombotic activity and is known to interact with Toll-like-receptor 4 (TLR4). The underlying mechanism and cell types involved in mediating the prothrombotic effect of Fn-EDA+ still remain unknown. Using intravital microscopy, we evaluated susceptibility to carotid artery thrombosis after FeCl3-induced injury in mice expressing Fn lacking EDA (Fn-EDA−/− mice) or Fn containing EDA (Fn-EDA+/+ mice). Fn-EDA−/− mice exhibited prolonged times to first thrombus formation and complete occlusion and a significant decrease in the rate of thrombus growth (P < .05 vs Fn-EDA+/+ mice). Genetic deletion of TLR4 reversed the accelerated thrombosis in Fn-EDA+/+ mice (P < .05) but had no effect in Fn-EDA−/− mice. Bone marrow transplantation experiments revealed that TLR4 expressed on hematopoietic cells contributes to accelerated thrombosis in Fn-EDA+/+ mice. In vitro studies showed that cellular Fn-EDA+ interacts with platelet TLR4 and promotes agonist-induced platelet aggregation. Finally, Fn-EDA+/+ mice specifically lacking platelet TLR4 exhibited prolonged times to first thrombus formation and complete occlusion (P < .05 vs Fn-EDA+/+ mice containing platelet TLR4). We conclude that platelet TLR4 contributes to the prothrombotic effect of cellular Fn-EDA+, suggesting another link between thrombosis and innate immunity.

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

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. 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. 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.

Genetic Deletion of Toll-Like Receptor 4 on Platelets Attenuates Experimental Pulmonary Hypertension

Recent studies demonstrate a role for toll-like receptor 4 (TLR4) in the pathogenesis of pulmonary hypertension (PH); however, the cell types involved in mediating the effects of TLR4 remain unknown. The objective of this study was to determine the contribution of TLR4 expressed on nonparenchymal cells to the pathogenesis of PH. TLR4 bone marrow chimeric mice revealed an equal contribution of TLR4 on nonparenchymal and parenchymal cells in the pathogenesis of PH as determined by measuring right ventricular (RV) systolic pressure and RV hypertrophy. However, the deletion of TLR4 from myeloid lineage cells had no effect on the development of PH because we found no difference in RV systolic pressure or RV hypertrophy in wild-type versus LysM-TLR4(-/-) mice. To explore the potential role of platelet TLR4 in the pathogenesis of PH, platelet-specific TLR4(-/-) mice were generated (PF4-TLR4(-/-) mice). TLR4(-/-) platelets from either global TLR4(-/-) or PF4-TLR4(-/-) mice were functional but failed to respond to lipopolysaccharide, demonstrating a lack of TLR4. PF4-TLR4(-/-) mice demonstrated significant protection from hypoxia-induced PH, including attenuated increases in RV systolic pressure and RV hypertrophy, decreased platelet activation, and less pulmonary vascular remodeling. The deletion of TLR4 from platelets attenuated serotonin release after chronic hypoxia, and lipopolysaccharide-stimulated platelets released serotonin and promoted pulmonary artery smooth muscle cell proliferation in a serotonin-dependent manner. Our data demonstrate that TLR4 on platelets contributes to the pathogenesis of PH and further highlights the role of platelets in PH.

The effect of TLR4 expression in platelets and activation of platelets on the pathogenesis of thrombocytopenia induced by lipopolysaccharide in mice

Objective To determine the role of the platelet activation and the expression of platelet Toll-like receptor 4 (TLR4) in thrombocytopenia induced by lipopolysaccharide (LPS) in mice.Methods ICR mice were randomly (random number) divided into control group,model 3,6,12,24,48,72h groups and neutrocytopenia syndrome (NEP) group.The blood samples of mice in model groups were detected 3,6,12,24,48,and 72 hours after intravenous injection of LPS respectively.Anti- neutrophil monoclonal antibody was administered in the mice of NEP group 24 h before injection of LPS,and blood samples were collected 24 h after injection of LPS.The determination of platelet count (PC),mean platelet volume (MPV) and platelet distribution width (PDW) was carried out with full automatic hemocyte analyzer.The plasma tumor necrosis factor- α (TNF- α),macrophage colony stimulating factor (M-CSF) and soluble CD40 ligand (sCD40L) levels were measured by using ELISA.The rate of platelet TLR4 expression was detected by flow cytometry.Comparison between groups was carried out by using ANOVA statistical methods.Results PC was reduced by 30％ 3 h after intravenous injection of LPS,and reached the lowest level within 24 h (P ＜0.01 ).MPV and PDW were increased compared with healthy controls (P ＜0.01 ).Plasma TNF - α,M - CSF and sCD40L were significantly increased after LPS stimulation (P ＜0.01 ),and reached the peak during 6 ～ 24 h after LPS administration.PC had correlation with plasma levels of M - CSF ( r =- 0.746) and sCD40L ( r =- 0.573 ).The platelet TLR4 expression was significantly increased 6 h after LPS stimulation.The platelet TLR4 expression in NEP group was significantly lower than that in 24 h model group ( P ＜ 0.01 ).Conclusions The excessive activation platelet represented by increase in sCD40L,MPV,PDW and high levels of M-CSF in macrophages along with platelet TLR4 expression participate in the pathogenesis of thrombocytopenia.The up - regulation of the platelet TLR4 expression is dependent on neutrophils in case of thrombocytopenia induced by LPS. Key words: Lipopolysaccharides; Endotoxemia; Thrombocytopenia; Platelet activation; Toll like receptor 4; Macrophage colony- stimulating factor; Soluble CD40 ligand; Neutrophils; Mean platelet volume; Platelet distribution width

The role of platelet TLR4 expression in the pathogensis of thrombocytopenia in sepsis

Objective To unravel the relationship between Toll-like receptor 4 (TLR4) expression in platelets and the activation of platelet in thrombocytopenia (TCP) in case of sepsis. Method A total of 64 patients with sepsis were prospectively studied with control. Platelet count (PC), mean platelet volume (MPV),platelet distribution width (PDW),platelet TLR4 expression, platelet PAC-1 expression, sCD40L and TNF-α concentrations were compared between healthy control group (15 cases) and sepsis group on admission and 3 d,Sd,and 9 days after admission. The changes of MPV and PDW in TCP and non-TCP subgroups of sepsis before and after treatment were recorded. Prognostic index was analyzed. Results The PC was lower in sepsis group (P=0.006), and MPV and PDW were higher in sepsis group than those in healthy control group (P = 0.046, P =0.001). Platelet TLR4 and PAC-1 expressions, and sCD40L and TNF-α levels also increased more significantly in sepsis group (P ＜ 0.001). PAC-1 expression and TNF- level were higher in TCP group than those in non-TCP group before and after treatment (P = 0.023, P = 0.011). The sCD40L concentration and platelet TLR4 expression were significantly higher in treated TCP groups than in non-TCP group (P = 0.047, P = 0.001). Compared with non-TCP,the rate of bleeding was higher (P = 0.024) and the length of ICU stay was longer (P = 0.013).The APACHE Ⅱ score and the 28-day mortality were higher in TCP group (P ＜ 0.01, P = 0.048). Conclusions The elevation of platelet TLR4 expression in sepsis along with platelet activation is closely related to the incidence of thrombocytopenia. The occurrence of TCP is a poor prognosis sign of sepsis. Key words: Sepsis; Thrombocytopenia; Toll-like receptor; Platelet activation; Glycoprotein Ⅱb/Ⅲa; Soluble CD40 ligand ; β-Thromboglobulin; Tumor necrosis factor-α; Interleukin-8

LPS Mediates the Activation of Platelet by Toll-Like Receptor 4.

Abstract 3001Poster Board II-978Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) on various tissues or cells in host. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (CD62P, P-selectin) and CD40L on platelet are the indexes to determine platelet activation. The presentexperiment was designed to investigate the expression of Toll-like receptor 4 (TLR4) on platelet and to determine whether platelet TLR4 involves in platelet activation induced by Lipopolysaccsharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy people were pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4,CD62P and CD40L on platelets were detected through flow cytometry, and platelet TLR4 was further determined by performing western blot analysis. The results show that the percentage of TLR4-positive platelet induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, P<0.01). TLR4 expression on platelets treated with LPS was remarkedly elevated in the presence or absent of thrombin. However, the expression level was much higher in presence of both than thrombin alone( 39.16%,P<0.01). Moreover, the similar results were found in Western blot analysis. Synchronously, the expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin(42.68% and 14.80%) and LPS respectively, and the increase of CD62P was more significant when stimulated with both of LPS and thrombin(63.03%). Although anti-TLR4 antibody inhibited significantly the increases of TLR4, CD62P and CD40L on platelets induced by LPS, it didn't effect their increases induced by thrombin. The experiment results support the evidence that functional TLR4 can be expressed on human platelet. It may involve in platelet activation as an important mediator of LPS- induced CD62P and CD40L expressions on platelets. Disclosures:No relevant conflicts of interest to declare.

Toll‐like receptor 4 ligand can differentially modulate the release of cytokines by human platelets

Blood platelets link the processes of haemostasis and inflammation. This study examined the immunomodulatory factors released by platelets after Toll-Like Receptor 4 (TLR4) engagement on their surfaces. Monoclonal anti-human FcgammaRII Ab (IV.3)-treated human platelets were cultured with TLR4 ligands in the presence or absence of blocking monoclonal antibody to human TLR4. The release of sCD62p, epidermal growth factor (EGF), transforming growth factor beta (TGFbeta), interleukin (IL)-8, platelet activating factor 4 (PAF4), platelet-derived growth factor, alpha, beta polypeptide (PDGF-AB), Angiogenin, RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted) and sCD40L were measured by specific enzyme-linked immunosorbent assay. TLR4 ligand [Escherichia coli lipopolysaccharide (LPS)] bound platelet TLR4, which differentially modulates the release of cytokines by platelets. It was noted that (i) sCD62p, IL-8, EGF and TGFbeta release were each independent of platelet activation after TLR4 engagement; (ii) RANTES, Angiogenin and PDGF-AB concentration were weaker in platelet supernatant after TLR4 engagement; (iii) sCD40L and PAF4 are present in large concentration in the releaseate of platelets stimulated by TLR4 ligand. The effects of LPS from E. coli on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody, consistent with the immunomodulation being specifically mediated by the TLR4 receptor. We propose that platelets adapt the subsequent responses, with polarized cytokine secretion, after TLR4 involvement.

Role for platelet toll-like receptor 4 (TLR4) in the formation of Neutrophil Extracellular Traps (NETs) in sepsis (44.22)

Abstract It has been known for many years that LPS, neutrophils and platelets all participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. Using flow chambers in vitro we suggest a novel innate immune response leading to enhanced trapping of bacteria in blood vessels. The mechanism involved platelet TLR4 detecting LPS in blood and inducing a unique response, specifically platelet binding to adherent neutrophils, but not platelet aggregation or P-selectin expression. Subsequently, the platelets stimulated very robust neutrophil activation leading to formation of NETs. These NETs retained their integrity under flow conditions and functioned to ensnare bacteria within the vasculature. Plasma from severely septic patients also induced TLR4-dependent platelet-neutrophil interactions leading to the production of NETs. We propose that this novel bacterial trapping mechanism would only occur under extreme conditions such as severe sepsis and platelet TLR4 (not leukocyte TLR4) functioned as the threshold switch for this innate immune response to occur. With the advent of antibiotics perhaps reducing the need for NET formation, we would propose that inhibiting platelet activation with TLR4 inhibitors may inhibit NET formation and reduce inadvertent tissue injury. This work was funded by: CIHR and AHFMR