The responses associated with E. coli sepsis and shock are numerous making it difficult to single out the important mediators and target tissues from epiphenomena. Three recent observations, however, provide an opportunity to study further, mechanisms and regulation of septic shock. First, Beutler et. al showed that antibody against tumor necrosis factor (TNF) protect a significant number of mice from endotoxin shock. Second, we showed that activated protein C also completely protects baboons from endotoxin/E. coli shock. Third, we showed that cultured endothelial cells have the protein S cofactor necessary for expression of the anticoagulant activity of activated protein C, and that exposure of these cells to TNF completely suppresses this protein S cofactor (anticoagulant) activity while expressing tissue factor (coagulation) activity. Since protein S on the endothelial cell is influenced favoring coagulation by TNF and favoring anticoagulation by activated protein C, we hypothesize that the coagulopathic and lethal effects of endotoxin are mediated through TNF and that they might be blocked by activated protein C acting through the endothelial cell-protein S assembly.
Our first aim, therefore, is to determine if the protective effects, like the anticoagulant effects, are receptor mediated and require protein S. We also will determine if aortic endothelium in response to E. coli loses protein S while gaining tissue factor activity at the time systemic coagulopathy is most severe. We will then determine if activated protein C prevents both the systemic coagulopathy and the change in the activities of aortic endothelium. Finally, we will determine how late following E. coli infusion, activated protein C can be started and still protect the animals from shock. We want to know if failure to protect coincides with loss of S cofactor activity of aortic endothelium.
Our second aim i s to study the role of TNF in E. coli shock and determine if activated protein C inhibits any of its effects as it does those of E. coli. We will determine if TNF levels rise following E. coli infusion and if activated protein C blocks this rise. We will than infuse TNF directly, compare its effects with those of E. coli and find which of these effects are inhibited by activated protein C. Finally, assuming that activated protein C not only acts to prevent perturbation of endothelium by TNF, but also inhibits its synthesis, we will study the in vitro effect of activated protein C on endotoxin induced elaboration of TNF by cultures of monocyte/macrophages.
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