LPS-induced Disseminated Intravascular Coagulation Model Research Articles

Recombinant human erythropoietin attenuates hepatic dysfunction by suppressing hepatocellular apoptosis in lipopolysaccharide-induced disseminated intravascular coagulation in rats.

The aim of the present study was to clarify the effect of recombinant human erythropoietin (EPO) and low molecular weight heparin (LMWH) on a rat model of lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC). Experimental DIC was induced by sustained infusion of 5 mg/kg LPS for 4 h. EPO or LMWH was then administered to the LPS-induced DIC model. LPS-induced consumption coagulopathy, hemostatic activation and plasma TNF elevation remained unaltered in the LPS+EPO group, except for the D-dimer levels, and these abnormalities were significantly improved in the LPS+LMWH group. Plasma alanine aminotransferase (ALT) levels were markedly reduced in the LPS+EPO group, accompanied by a significant suppression of hepatocellular apoptosis. In the LPS+LMWH group, plasma creatinine levels and glomerular fibrin deposition were significantly attenuated, along with plasma ALT levels and hepatocellular apoptosis. Thus, a single administration of EPO may improve hepatic dysfunction by primarily exerting an anti-apoptotic, not anticoagulant, effect in the LPS-induced DIC model.

Tetrahydropalmatine has a therapeutic effect in a lipopolysaccharide-induced disseminated intravascular coagulation model.

ObjectivesThe aim of this study was to determine the therapeutic effects of tetrahydropalmatine (Tet) on disseminated intravascular coagulation (DIC) by exploring the role of Tet using a lipopolysaccharide (LPS)-induced DIC model.Methods/Materials: We established a mouse DIC model by injecting LPS. Hematoxylin-eosin (HE) staining was performed to detect liver and kidney damage. Blood samples were obtained to determine liver and kidney injury indexes, coagulation indexes, and inflammatory cytokines. An in vitro cell inflammation model was also established. Tumor necrosis factor-α (TNF-α) levels and nuclear factor kappa B (NF-κB) signaling pathway activation were determined by western blot.ResultTet ameliorated the damage to organ tissues, improved coagulation indexes, and reduced the inflammatory cytokine production in LPS-induced mouse DIC. Tet also inhibited TNF-α expression by suppressing NF-κB signaling pathway activation in an in vitro LPS model using RAW 264.7 macrophages.ConclusionsTet has a mitigating and therapeutic effect on the LPS-induced DIC model via anticoagulant and anti-inflammatory effects, showing its potential as an adjunct to DIC treatment.

BMSCs pre-treatment ameliorates inflammation-related tissue destruction in LPS-induced rat DIC model

This study aimed to investigate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on disseminated intravascular coagulation (DIC) model rats and to further explore the underlying mechanism. A rat model of lipopolysaccharide (LPS)-induced DIC was successfully established, as indicated by impaired plasma hemostatic parameters and damaged organ functions in rats. Importantly, pre-treatment with rat allogeneic BMSCs before LPS injection significantly alleviated systemic intravascular coagulation, reduced plasma levels of organ dysfunction indicators and pro-inflammatory cytokines, suppressed fibrin microthrombi formation, ameliorated liver, heart, and renal injuries, and increased 24-hour survival rates in LPS-induced DIC rats. The protection of BMSCs against DIC was in a moderately dose-dependent manner. Further investigation revealed that BMSCs co-cultured with peripheral blood mononuclear cells (PBMCs) significantly inhibited the LPS-stimulated PBMCs proliferation and the release of pro-inflammatory cytokines from PBMCs. Of note, upregulation of immunosuppressive factors including indoleamine 2,3-dioxygenase and interleukin-10, which was induced by interferon-γ, contributed to BMSCs-mediated inhibition of LPS-stimulated PBMCs proliferation. These effects do not depend on the direct cell–cell contact. In conclusion, BMSCs pre-treatment ameliorates inflammation-related tissue destruction in LPS-induced DIC model rats. The protection of BMSCs may be attributed to their anti-inflammatory and immunomodulatory properties, which render BMSCs a promising source for stem cell-based therapeutic approaches in inflammation-related DIC.

Atractylenolide Ⅰ protects against lipopolysaccharide-induced disseminated intravascular coagulation by anti-inflammatory and anticoagulation effect.

To investigate whether atractylenolide Ⅰ (ATL-Ⅰ) has protective effect on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) invivo and invitro, and explore whether NF-κB signaling pathway is involved in ATL-Ⅰ treatment. New Zealand white rabbits were injected with LPS through marginal ear vein over a period of 6hat a rate of 600μg/kg (10mL/h). Similarly, in the treatment groups, 1.0, 2.0, or 5.0mg/kg ATL-Ⅰ were given. Both survival rate and organ function were tested, including the level of alanine aminotransferase (ALT), blood urine nitrogen (BUN), and TNF-α were examined by ELISA. Also hemostatic and fibrinolytic parameters in serum were measured. RAW 264.7 macrophage cells were administered with control, LPS, LPS+ATL-Ⅰ and ATL-Ⅰ alone, and TNF-α, phosphorylation (P)-IκBα, phosphorylation (P)-NF-κB (P65) and NF-κB (P65) were determined by Western blot. The administration of LPS resulted in 73.3% mortality rate, and the increase of serum TNF-α, BUN and ALT levels. When ATL-Ⅰ treatment significantly increased the survival rate of LPS-induced DIC model, also improved the function of blood coagulation. And protein analysis indicated that ATL-I remarkably protected liver and renal as decreasing TNF-α expression. Invitro, ATL-I obviously decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65), with the decrease of P-IκBα. ATL-Ⅰ has protective effect on LPS-induced DIC, which can elevate the survival rate, reduce organ damage, improve the function of blood coagulation and suppress TNF-α expression by inhibiting the activation of NF-κB signaling pathway.

Effect of activated human protein C on disseminated intravascular coagulation induced by lipopolysaccharide in rats.

Protein C is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating coagulation and fibrinolysis by inactivating not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). The aim of the present study was to examine the effect of a human APC product (designated as CTC-111), compared with that of heparin, on the disseminated intravascular coagulation (DIC) induced by lipopolysaccharide (LPS) in rats. LPS (1 mg/kg/h) infusion was performed through a femoral vein for 4 h. One-fifth amount of the total dosage of CTC-111 or heparin was injected into the other femoral vein, followed by a 4-h infusion of the remainder. Both CTC-111 (10,000-100,000 U/kg) and heparin (400-800 IU/kg) inhibited the decrease in platelet count and fibrinogen level equally. The prolonged activated partial thromboplastin time and prothrombin time observed in DIC rats were further elongated in both CTC-111- and heparin-treated rats. But, this prolongation was less in CTC-111-treated rats than in the heparin-treated ones. Heparin inhibited the increase in fibrin and fibrinogen degradation products more prominently than CTC-111. On the other hand, CTC-111 strongly inhibited the increase in PAI-1 activity but heparin did not. These results suggest that CTC-111 may enhance fibrinolysis through its direct inhibitory effect on PAI-1. The parameters for liver or renal damage, i.e., plasma glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine (Cre) and blood urea nitrogen (BUN), were significantly increased by LPS infusion. Both CTC-111 (100,000 U/kg) and heparin (800 IU/kg) decreased the increase in GOT and GPT levels significantly, whereas neither affected the increase in Cre or BUN. From these results, the activation of the blood coagulation system might partially contribute to the progression of liver damage caused by LPS, and might be less involved in the progression of renal damage in this model. In conclusion, CTC-111 showed both anticoagulant and profibrinolytic activity in the LPS-induced DIC model without excessive prolongation of coagulation time. From these results, CTC-111 is expected to be a useful remedy for DIC without the risk of bleeding.

Immunoglobulin preparations attenuate organ dysfunction and hemostatic abnormality by suppressing the production of cytokines in lipopolysaccharide-induced disseminated intravascular coagulation in rats*

We attempted to clarify the effect of immunoglobulin concentrates on the rat lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) model. Prospective, comparative, experimental study. Laboratory at a university hospital. Male Wistar rats, aged 6 to 7 wks and weighing 160 to 170 g. Two kinds of experiments were performed. In the first, experimental DIC was induced by sustained infusion of 30 mg/kg LPS for 4 hrs via the tail vein, and two doses of immunoglobulin (25 or 100 mg/kg/4.5 hrs) were administered to rats 30 mins before infusion of LPS, after which immunoglobulin infusion was continued for a further 4 hrs. In the second, experimental DIC was induced by sustained infusion (5 mg/kg/1 hr) of LPS for 1 hr, and one dose of immunoglobulin (100 mg/kg/4 hrs) was administered to rats after LPS induction. The parameters were estimated at 4 hrs and 8 hrs in the first experiment and at 1, 5, and 10 hrs in the second one. Similar results were observed in the two experiments. Consumption coagulopathy and hemostatic activation were attenuated, especially when immunoglobulin was administered before LPS infusion. Plasma levels of creatinine and alanine aminotransferase were significantly depressed by coadministration of immunoglobulin. Marked glomerular fibrin deposition was observed in the LPS-induced DIC model, but this deposition was reduced by immunoglobulin. In the first stage of the experiment, plasma levels of tumor necrosis factor (TNF) and interleukin (IL)-6 were suppressed by coadministration of immunoglobulin. In the second, plasma levels of IL-6 were significantly suppressed by immunoglobulin. It was concluded that plasma levels of TNF and IL-6 could be significantly suppressed by immunoglobulin in the LPS-induced DIC model. Moreover, hemostatic abnormality, organ dysfunction, and glomerular fibrin deposition in this model were all ameliorated by immunoglobulin.

Recombinant human antithrombin expressed in Chinese hamster ovary cells shows in vivo efficacy on rat DIC model similarly to plasma-derived antithrombin regardless of different N-glycosylation

Plasma-derived human antithrombin (pAT) is used for the treatments of disseminated intravascular coagulation (DIC) and hereditary antithrombin deficiencies. We expressed recombinant human antithrombin (rAT) in Chinese hamster ovary (CHO) cells. The purified rAT is composed of 55% α-isoform and 45% β-isoform. The structure of the N-linked oligosaccharides of rAT is the same biantennary complex type as previously found in pAT with less sialylated on the non-reducing ends. Most of the oligosaccharides of rAT are fucosylated at the reducing ends of N-acetylglucosamine, while those of pAT are not fucosylated. Despite of the difference in sialylation and fucosylation of the oligosaccharide units, rAT and pAT showed indistinguishable heparin cofactor and progressive activities, and they bound to thrombin in a one-to-one stoichiometric manner. In lipopolysaccharide (LPS)-induced and thromboplastin-induced DIC rat models, rAT reduced fibrinogen and platelet consumption to a similar extent with pAT. In LPS-induced DIC model, both ATs similarly restrained the increase of alanine aminotransferase and aspartate aminotransferase activities. Finally, pharmacokinetic analysis showed that both ATs had similar half-lives in the circulation of normal rats. Together, the present study demonstrated that rAT prepared in CHO cells has potential for a substitute of pAT in therapeutic use.

Selective inducible nitric oxide synthase inhibition attenuates organ dysfunction and elevated endothelin levels in LPS‐induced DIC model rats

We examined the role of nitric oxide (NO) produced by an inducible isoform of NO synthase (iNOS) using N[6]-(iminoethyl)-lysine (L-NIL), a selective iNOS inhibitor, in the rat model of lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) and investigated changes in organ function, plasma levels of NOX (metabolites of NO) and endothelin. We induced experimental DIC by the sustained infusion of 30 mg kg(-1) LPS for 4 h via the tail vein. We then investigated the effect of L-NIL (6 mg kg(-1), from - 0.5 to 4 h) on LPS-induced DIC. Blood was withdrawn at 4 and 8 h, and all four groups (LPS with or without L-NIL at 4 and 8 h) consisted of eight rats. Three of the animals in the 8-h LPS group died, and we examined blood samples from five rats in this group. None of the other rats died. The LPS-induced elevation of creatinine, alanine aminotransferase, glomerular fibrin deposition and plasminogen activator inhibitor was significantly suppressed by L-NIL coadministration, although L-NIL did not affect the platelet count, fibrinogen concentration or the level of thrombin-antithrombin complex. Moreover, plasma levels of the D-dimer that reflect the lysis of cross-linked fibrin were significantly increased by L-NIL coadministration in the LPS-induced DIC model. Plasma levels of NOX and endothelin were obviously increased by LPS infusion. However, both levels were significantly suppressed in the LPS + L-NIL group, when compared with the LPS group. Although mean arterial pressure (MAP) was significantly decreased between 2 and 8 h compared with the control in the LPS group, this depression was significantly attenuated in the LPS + L-NIL group. Our results suggest that NO induced by iNOS contributes to hypotension (depressed MAP), the progression of hepatic and renal dysfunction, microthrombus deposition and elevated endothelin levels in the rat model of LPS-induced DIC.

Beneficial effects of urokinase on lipopolysaccharide-induced disseminated intravascular coagulation in rats

In a rat model of lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC), we used urokinase (UK) in an attempt to clarify the role of fibrinolysis and to investigate changes in plasma endothelin levels. Two kinds of experiment were performed. The first one: experimental DIC was induced by sustained infusion of 30 mg/kg LPS for 4 h via the tail vein, and two doses of UK (2.0 or 10.0 IU/g/4.5 h) were administered to rats 30 min before infusion of LPS, after which UK infusion was continued for a further 4 h. The second one: experimental DIC was induced by sustained infusion of 1 mg/kg/10 min LPS for 10 min, and two doses of UK (2.0 or 10.0 IU/g/4 h) were administered to rats at 30 min after LPS infusion. The parameters described below were determined at 4 h in the first experiment, at 4 h and 8 h in the second one. The similar results were observed in both kinds of experiment. There were no significant differences in plasma thrombin-antithrombin complex, fibrinogen or platelet number among the three DIC groups, in both kinds of experiment. Plasma levels of D-dimer were significantly increased in the LPS + higher dose of UK group when compared with the LPS group. The increased plasma plasminogen activator inhibitor (PAI) activity seen in the LPS group was significantly suppressed in the groups receiving UK (especially higher dose of UK). In addition, the increased plasma levels of creatinine and alanine aminotransferase seen in the LPS group were significantly suppressed in the groups receiving UK (especially higher dose of UK). Plasma levels of endothelin, known to be a potent vasoconstrictive agent, were markedly elevated by LPS infusion, and were significantly suppressed in the groups receiving UK of both kinds of experiment, in a dose-dependent fashion compared with LPS group. Glomerular fibrin deposition was significantly suppressed in the groups receiving UK when compared with the LPS group. No manifestations of bleeding were observed in any of the groups. Enhanced fibrinolysis and depressed endothelin induced by UK thus appear to play an important role in preventing the development of organ failure in the LPS-induced DIC model.

Relationship between endothelin and the pathophysiology of tissue factor-induced and lipopolysaccharide-induced disseminated intravascular coagulation in rats: a study examining the effect of an endothelin receptor antagonist.

We investigated the relationship between endothelin, a potent vasoconstrictor peptide, and the pathophysiology of disseminated intravascular coagulation (DIC), using two models of DIC. Experimental DIC was induced by sustained infusion of 50 mg/kg lipopolysaccharide (LPS), or 3.75 U/kg thromboplastin, for 4 h via the rat tail vein. The effect of administration of a non-selective endothelin receptor antagonist (TAK-044) (2, 10, or 50 mg/kg, from -0.5 to 4 h) on thromboplastin-induced DIC was not significant. However, LPS-induced elevation of alanine aminotransferase, creatinine and glomerular fibrin deposition was significantly suppressed by co-administration of TAK-044 in a dose-dependent manner, although no effect of TAK-044 was observed on the platelet count, fibrinogen concentration or the level of thrombin-antithrombin complex. Moreover, plasma levels of D-dimer, which reflect the grade of fibrinolysis of cross-linked fibrin, were significantly increased by co-administration of each dose of TAK-044 in the LPS-induced DIC model in rats. Our results suggest that vasoconstriction, as well as depressed fibrinolysis, contribute to severe organ dysfunction in LPS-induced, but not thromboplastin-induced, DIC, and that endothelin plays a role in the development of organ injury in LPS-induced DIC in rats.

Significance of decreased plasma D-dimer levels following lipopolysaccharide-induced disseminated intravascular coagulation in rats.

Plasma D-dimer (DD) is considered to be one of the most useful markers in the diagnosis and assessment of disseminated intravascular coagulation (DIC). The present study was performed to clarify the role of DD in a rat model of lipopolysaccharide (LPS)-induced DIC in which low-molecular-weight heparin (LMWH) and tranexamic acid (TA) were used. We investigated whether a relationship exists between plasma DD levels and severity of DIC. Experimental DIC was induced in rats by a sustained 4-hour infusion of 30 mg/kg LPS administered via the tail vein (LPS group). Rats received either LPS alone (LPS group) or LPS combined with 200 U/kg LMWH (LPS+LMWH group) or 50 mg/kg TA (LPS+TA group) from -30 minutes to 4 hours. Blood was drawn from each rat at 4, 8, and 12 hours. Plasma levels of thrombin-antithrombin complex (TAT) and creatinine were suppressed in the LPS+LMWH group, and less glomerular fibrin deposition was observed compared with the LPS group. On the other hand, an increased level of creatinine and increased glomerular fibrin deposition were observed in the LPS+TA group compared with the LPS group. LMWH demonstrated a protective effect against LPS-induced DIC, resulting in increased survival at 12 hours, whereas TA had the opposite effect. From these results, it appears that LMWH protects against LPS-induced DIC, but TA exacerbates LPS-induced DIC. It was interesting that plasma levels of DD were almost completely suppressed by concurrent administration of either TA or LMWH in this LPS-induced DIC model. This finding suggested that plasma levels of DD were suppressed by inhibition of coagulation (reduced deposition of fibrin) in the LPS+LMWH group and that DD levels were also suppressed by inhibition of fibrinolysis (reduced degradation of fibrin by plasmin) in the LPS+TA group. Thus care should be taken when evaluating the significance of plasma DD levels, because suppressed levels can occur with progressive fibrin deposition and worsening organ dysfunction or improvement in the course of DIC.

Beneficial effect of JTV-803, a new synthetic inhibitor of activated factor X, against both lipopolysaccharide-induced and tissue factor-induced disseminated intravascular coagulation in rat models.

We examined whether JTV-803, a specific activated factor X inhibitor independent of antithrombin III (ATIII), is effective against disseminated intravascular coagulation (DIC) in rat models induced by tissue factor (TF) or lipopolysaccharides (LPS). In male Wistar rats, DIC was induced by a 4 h infusion of thromboplastin (3.75 U/kg) or LPS (50 mg/kg). The rats were given JTV-803 (0.3 or 3 mg/kg, bolus intravenously) (JTV-803 groups) or low molecular weight heparin (LMWH groups) (200 U/kg, bolus intravenously) prior to an injection of TF or LPS. The results showed that JTV-803 was dose-dependently effective against DIC in both TF-induced and LPS-induced rat models. This anti-DIC effect of JTV-803 at higher doses was almost equivalent to that of LMWH in both types of DIC. Plasma ATIII activity was more prominent in the group treated with JTV-803 than in that treated with LMWH. None of rats died in the TF-induced DIC model with or without drug administration. On the contrary, seven of 22 rats died (mortality rate, 31.8%) in the LPS-induced DIC model without drug administration. Although the mortality rate of rats induced with LPS and treated with LMWH was quite high (6/16, 37.5%), none of the LPS-induced rats treated with JTV-803 died. These findings suggested that JTV-803 can treat both TF-induced and LPS-induced DIC models, and that this drug has greater potential in preserving ATIII and in improving the prognosis of DIC.

Expression of tissue factor and tissue factor pathway inhibitor in rat lungs with lipopolysaccharide-induced disseminated intravascular coagulation

Disseminated intravascular coagulation (DIC) is a frequent complication of endotoxin shock, and modulation of endothelial cell hemostatic properties has been proposed to play an important role in its onset. We examined the in vivo expression of tissue factor (TF) and TF pathway inhibitor (TFPI) in rat lungs of a lipopolysaccharide (LPS)-induced DIC model. Light and electron microscopic studies showed that fibrin-rich thrombi were present in the pulmonary microvasculature 3 hours after intraperitoneal injection of LPS (7.5 mg/kg) and increased in number at 6 hours. In an immunohistochemical study, an increase in number of monocytes in the microvasculature was observed after LPS injection, and many of these cells (> 90%) were positive for TF antigen. However, no TF expression in endothelial cells was detected. Pulmonary endothelial cells showed positive reaction for TFPI antigen before LPS injection, but TFPI-positive endothelial cells markedly decreased in number after LPS injection. mRNA expression of TF increased and that of TFPI decreased in the lung tissue 3 and 6 hours after LPS injection. High values of TF activity were detected in the lung tissue and plasma, whereas TFPI activities decreased after LPS injection. These results indicate that imbalance between TF and TFPI, overexpression of TF, and underexpression of TFPI in the lung may contribute to thrombus formation in this LPS-induced DIC model.