Effect Of Trauma/endotoxin-Induced Inflammatory Reaction On The Initiation Of Coagulation And Regulation Of Anticoagulation Activity

Objective: It is inevitable that patients with systemic inflammatory response syndrome or sepsis induced by severe trauma or infection often have abnormal coagulation, anticoagulation and fibrinolytic function, while disseminated intravascular coagulation (DIC) is regarded as the most severe clinical manifestation. DIC is one of the important causes of death of patients with severe trauma. Due to the complexity of the illnesses in patients with trauma and infection, clinical symptoms of DIC are often neglected at the early stage of DIC (non-overt DIC), however, corresponding treatments are extremely important to improve the survival rate of patients. At present, there is still no sufficient realization of non-overt DIC clinically, as well as poor understanding of the complicated interrelation between various inflammatory products and coagulation during trauma-infection; and there is still unrealistically in application of drug for treatment, especially the extensive application of hemostatic. Therefore, this study adopts the way of combination of impact injury and injection of endotoxin via vein, which comes into being a common factor inducing injury in clinical medicine and construct an experimental non-overt DIC animal model to probe into questions stated above.Methods and results as follows:First part: Construction of rabbit non-overt DIC model induced by combination of trauma and endotoxin.Different injury factors including impact injury, endotoxin and combination of impact injury and endotoxin were created by strike on rabbit chest with Type BIM-II biological impact machine and injection of endotoxin via vein. The following treatment impact parameters were selected in model preparing: 800kpa driving pressure, 30% compression and 1.77cm2 impact area. LPS was injected intravenously as infectious factor. 30 Male New Zealand rabbits were divided into four groups in a completely random design as follows: trauma group(T group), LPS group(L group), T+L group with both trauma and endotoxin treatment (T+L group), and control group.We observed biochemical indexes including coagulation index (prothrombin time-PT, activated partial thromboplastin time-aPTT, thrombin time-TT, fibrinogen-Fib), thrombelastogram (reaction time-R, clotting time-K, maximum amplitude-MA, formation speed of clotαangle), D-dimer, antithrombin-Ⅲ(AT-Ⅲ)as well as histopathologic changes in the important organs. Scoring of coagulation function in the rabbits with trauma and infection was performed according to the changes of the above indexes.1. When the two injury factors were used alone to cause injury, at the early stage(1h)of impact, PT and aPTT were shortened; fibrinogen was increased; and AT-Ⅲactivity decreased obviously. TEG displays that both the R value and K value were shortened, while MA value was increased.2. In animal models co-impacted by the two impact factors, AT-Ⅲactivity was decreased obviously at the early stage, while cogulation indexes indicated that these models were at hypercoagulabale state with more severe injury in lung, kidney and liver than the one impacted by only trauma or endotoxin, quantities of microthrombus presented in important organs, however, their DIC score was less than 5.Second part: The relationship of inflammatory reaction with abnormal coagulation in rabbits with trauma and concurrent infectionThe injure method and groups are the same as the first part.The changes of interleukin-1β(IL-1β), interleukin-6(IL-6), and interleukin-10(IL-10) were observed by enzyme-linked immunosorbent assay. The changes of tissue factor (TF), tissue factor pathway inhibitors(TFPI)were observed by chromogenic substrate. TF and TFPI expression in tissue were detected by in situ hybridization.1. Both IL-1βand IL-6 were elevated significantly in non-overt DIC animal models induced by trauma and infection, while the activity of serum TF was increased. There were positive correlation between IL-1β, IL-6 level and the change of TF activity.2. The amount of IL-10 was obviously increased in serum of injured animals and would be at a high level for 24 hour. 3. TF/TFPI ratio was increased obviously in groups with severe trauma and infection, while it was decreased at the early stage of impact in groups with only trauma, slight injury and high survival rate.4. The expression of TF mRNA and TFPI mRNA was elevated in pulmonary tissue by in situ hybridization; while only the expression of TF mRNA was elevated in kidney, and TFPI mRNA expression was decreased.Third part: Protecting effects of low molecular weight heparin on rabbits with non-overt DIC after trauma and infectionThe injure method is the same as T+L group in first part. 28 Male New Zealand rabbits were divided into four groups in a completely random design as follows: impact injury combined with endotoxin without treatment group, treatment with low molecular weight heparin(LMWH group), treatment with tranexamic acid(TA group), and treatment with low molecular weight heparin and tranexamic acid(L+TA group).The changes of interleukin-1β(IL-1β)and interleukin-6(IL-6)were observed by enzyme-linked immunosorbent assay. The changes of tissue factor (TF), tissue factor pathway inhibitors(TFPI)were observed by chromogenic substrate. TF and TFPI mRNA expression in tissue was detected by in situ hybridization. TFPI mRNA expression of monocyte in blood was detected by real time fluorescent quantitative PCR.1. LMWH reduced the expression of IL-1βand IL-6.2. LMWH could reduce the activity of TF in serum and its expression in the lung and kidney. We found out that LMWH could obviously up-regulated the mRNA expression of TFPI in monocytes at the early stage of impact (1h) by real time fluorescent quantitative PCR. TFPI activity in serum was increased as well, which resulted in a decrease of TF/TFPI ratio and only a few microthrombosis in the organs of LMWH group.3. The results showed that only use of plasminogen activator inhibitor- tranexamic acid increased the frequency of thrombus formation, however, it didn't result in an increased incidences of pulmonary arterial thrombus and more thrombus in the important organs after using tranexamic acid on the basis of treatment with anti-coagulant.Conclusions:This study successfully mimicks non-overt DIC animal model by impact and injection of endotoxin. Pro-inflammatory mediators participate in the development of abnormal coagulation after trauma and infection and promote the release of TF which activated the extrinsic coagulation pathway. There were positive correlation between IL-1β, IL-6 level and the change of TF activity. TF/TFPI ratio was increased in group with severe trauma and endotoxin, suggesting that is related to the severity of injury, while the survival rate of DIC was correlated with the decrease of TF/TFPI ratio due to the increase of TFPI. The inconsistency of TFPI mRNA expression in different organs was related to the severity of injury in the organs. Beside its anticoagulation function, low molecular weight heparin could inhibit the inflammation reaction, reduce the release of TF, and attenuate the coagulation reaction as well. It won't lead to the formation of thrombus by application of tranexamic acid on the basis of treatment with LMWH. The result is opposite to that only use of tranexamic acid. This investigation will provide a theoretical and experimental basis for clinical diagnosis and treatment of non-overt DIC induced by trauma and infection.