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

Clinical Center

Investigators

Abstract

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. [unreadable] [unreadable] 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.