Bacterial endotoxin activates several mammalian biological cascades, including the coagulation, complementation and kallikrein systems. However, the mechanism(s) by which endotoxin generates enzymatically active components of these host cascades is unknown. Limulus polyphemus, the horseshoe crab, will be used to study the interactions between endotoxin and a host cascade system. Endotoxin activates a proteolytic blood coagulation that frequently accompanies sepsis (or endotoxemia) in man. The hypothesis to be tested is that a single, specific endotoxin- sensitive factor in Limulus is activated after interaction with bacterial endotoxin, with formation of an endotoxin-coagulation factor complex. This complex subsequently produces activation of the remaining factors of the blood coagulation cascade. To test the hypothesis, we will: 1. Identify which of the Limulus coagulation factors interacts directly with endotoxin and determine whether a stable complex is formed between endotoxin and the factor which interacts with endotoxin. If stable, the structure and stoichiometry of this complex will be determined. 2. Determine the structural changes (conformational, proteolytic of subunit changes) that are associated with activation of the endotoxin- sensitive coagulation factor as a result of its interactions with endotoxin. 3. Identify the chemical components of endotoxin that are required for interaction with the endotoxin-sensitive protein and those that are required for activation of this protein.
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