These studies focus on the role of endothelial protein C receptor (EPCR) and thrombin activatable fibrinolytic inhibitor (TAFI) as regulators of the neutrophil/endothelial interaction induced by E. coli. All the inflammatory and hemostatic events studied in the baboon model of E. coil sepsis culminate in an aberrant neutrophil/endothelial interaction leading to increased permeability and coagulation disorders. EPCR and thrombomodulin (TM) are at the point of attack and therefore are both targets and regulators of this interaction through activation of protein C and TAFI by the TM/thrombin complex and through release of soluble EPCR by endothelial-derived metalloproteases. We postulate that TAFla (procarboxypeptidase beta) attenuates neutrophil activation by inactivating C5a, and that soluble EPCR attenuates subsequent tight binding of neutrophils to endothelium. The close association of EPCR and TAFla with the endothelium and neutrophils favor these actions. Two general questions are: What is the response and distribution of EPCR t and TAFI between endothelium and neutrophils? Can the information be used to design and time intervention using soluble EPCR and TAFla that would improve the efficacy of activated protein C? To study such questions we have adopted the sublethal model of E. coil sepsis, because the otherwise lethal events are stretched out over time into an initial host (stage 1) and ischemia reperfusion (stage 2) responses. This model allows one to track and intervene with the responses of these regulatory components at critical points of the response to E. coil. Specific questions include what are the timing and distribution of EPCR and TAFI between endothelium and neutrophils with respect to mediators (e.g., C5a) and adherence of neutrophils to the microvascular endothelium? Are there critical events involving these regulators in stage 1 that determine subsequent stage 2 events and whether the response becomes lethal? Can APC and either sEPCR or TAFI be used together to better regulate the neutrophil/endothelial response to E. coil? How critical is timing of intervention in determining whether it is beneficial or harmful? Changes in the expression and distribution of EPCR, TAFI, protein C, thrombomodulin, tissue factor (etc.) on the endothelium, perivascular tissues and neutrophils will be assessed using immunohistochemical and confocal imaging techniques. This includes FACS analysis, and determination of neutrophil half-life. The responses of plasma factors will be followed with ELISAs. Standard physiological parameters will be followed (e.g., temperature, CBC, blood pressure and global assays of hemostatic function) ? ?
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