The Effects of Heparin Therapy in a Mouse Model of E. coli pneumonia

尽管使用有效的抗生素与心肺支持相结合，但在过去的三十年中，脓毒症和脓毒性休克的死亡率仍然很高（分别为25 - 30%和40 - 70%）。此外，脓毒症和脓毒性休克的发生率似乎正在增加。新出现的数据表明，激活凝血和抑制纤维蛋白溶解有助于脓毒症期间的炎症和血管损伤。基于此，抗凝剂已被提议用于治疗致命综合征。尽管几种促进纤维蛋白溶解或抑制凝血酶的药物在临床上没有显示出令人信服的益处，但人们对使用肝素治疗脓毒症越来越感兴趣。然而，在我们在小鼠模型肺炎脓毒症模型中进行的早期研究中，肝素在广泛的剂量范围内没有益处，并且在最高剂量下是有害的。在机制研究中，当我们比较未感染和感染动物时，肝素对循环白色血细胞、血小板和一组13种细胞因子的影响明显不同。然而，在这些原始研究时，我们完成凝血试验的尝试并不成功。然而，在肝素缺乏生存效应的情况下，这些试验对于确认肝素具有其主要预期药理学作用非常重要。目前，我们的实验室已经获得了在啮齿动物中进行凝血试验（PT、aPTT和纤维蛋白原）的必要设备。PT和aPTT试验基于双相透射率。然后将用3.2%柠檬酸钠（1：9）抗凝的血浆与包括促凝血酶原激酶和氯化钙或鞣花酸和氯化钙的试剂混合。然后分析达到透射率降低50%的时间。在我们实验室中，使用该系统对小鼠、大鼠和犬进行的PT和aPTT测量结果在每次测量中保持一致，与文献中报告的水平接近但不相同。见附录1。然而，本研究的主要目的是使用该设备比较肝素与安慰剂在患有和不患有肺炎的小鼠中的作用。TATc的测量将使用市售小鼠ELISA试剂盒进行。 这项研究将分两部分进行。研究的第一部分将研究3种肝素剂量（100、500和2500 u/kg，基于我们的既往研究和人体给药）或安慰剂对正常动物12小时内凝血指标（PT、aPTT和TATc）的影响。 剂量100和500 u/kg接近患者在12 h治疗性肝素期间接受的剂量。将研究2500 u/kg剂量，以确定早期研究中观察到的有害趋势是否与过度抗凝相关。在本研究的这一部分，动物将仅接受单次肝素给药，目的是显示使用肝素时发生的可测量的凝血变化及其时程。如果在研究的第一部分中观察到可测量的变化，则在第二部分中，将使用早期研究的给药方案（q12 h）对100和500 u/kg剂量进行检测，并在肠内出血后24和48 h进行凝血试验。大肠杆菌或生理盐水挑战。 在这部分研究中，我们还将评估E. coli对凝血参数的影响。

Despite the use of effective antibiotics in combination with cardiopulmonary support, the mortality rate from sepsis and septic shock for the last three decades has remained high (2530% and 4070% respectively. Furthermore, the incidence of sepsis and septic shock appear to be increasing. Emerging data has suggested that activation of coagulation and suppression of fibrinolysis contributes to inflammation and vascular injury during sepsis. Based on this anticoagulants have been proposed for the treatment of deadly syndrome. Although several agents that promote fibrinolysis or inhibit thrombin have not shown convincing benefit clinically, there is increasing interest in the use of therapeutic heparin treatment for sepsis. However in early studies we performed in a mouse model pneumonia sepsis model, heparin, was not beneficial over a wide range of doses and at the highest dose was harmful. In mechanism studies it was evident that heparin was having significantly different effects on circulating white blood cells, platelets and a panel of 13 cytokines, when we compared non-infected and infected animals. However, at the time of these original studies, our attempts at completing coagulation assays were unsuccessful. In the face of absent survival effects with heparin, these tests are important however to confirm that heparin was having its primary intended pharmacologic effects. At the present time, our laboratory has obtained the necessary equipment to perform coagulation assays (PT, aPTT and fibrinogen) in rodent. The PT and aPTT assays are based on biphasic transmittance. Plasma anticoagulated with 3.2% sodium citrate (1:9) is then mixed with reagents including thromboplastin and calcium chloride or ellagic acid and calcium chloride. Then the time to reach a 50% reduction in transmittance is analyzed. Measurements of PT and aPTT in mice, rats and dogs in our laboratory with this system have been consistent from measurement to measurement and are close but not the same as reported levels in the literature. See appendix 1. However the primary goal of the present study is to use this equipment to compare the effects of heparin to a placebo in mice with and without pneumonia. Measures of TATc will be performed with commercially available mouse ELISA kits. This study will be done in two parts. The first part of the study will investigate the effects of 3 heparin doses (100, 500, and 2500 u/kg, based on our prior studies and human dosing) or placebo on coagulation measures (PT, aPTT and TATc) over 12 h in normal animals. Doses 100 and 500 u/kg are close to the doses patients receive during 12 h of therapeutic heparin. The dose 2500 u/kg will be studied to determine whether the harmful trend noted with it in earlier studies was related to excessive anticoagulation. In this part of the study animals will only receive a single heparin dose and the goal will be to show that measurable changes in coagulation occur with heparin and what their time course is. If measurable changes are observed in the first part of the study, then in the second part doses 100 and 500 u/kg will be tested using the treatment regimen from earlier studies (q12 h) and coagulation tests will be performed 24 and 48 h after intratracheal E. coli or normal saline challenges. During this part of the study we will also assess the effects of E. coli on coagulation parameters.