Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor that is activated by extracellular proteolytic cleavage at the N terminus. PAR1 is activated by a diverse array of serine proteases, including thrombin and activated protein C (APC), and by the zinc-dependent collagenase, matrix metalloprotease 1 (MMP1). PAR1 is expressed highly on endothelial cells and acts as a major regulator of vascular integrity and coagulation. Sepsis is a deadly complication of infection and is characterized by an excessive inflammatory response and aberrant activation of the coagulation cascade. A key early event in sepsis is loss of vascular integrity, causing leakage of vascular fluid and increased tissue factor generation. PAR1 plays a key role in this endothelial retraction and pharmacologic blockade of PAR1 early in sepsis reduces vascular leakage and improves survival in mouse models. However, the upstream PAR1 activation cascade in early sepsis is poorly understood. Lipopolysaccharide (LPS), a component of the Gram negative outer cell membrane, is an important initiator of the inflammatory response in Gram negative sepsis. Macrophages are known to secrete MMP1, a PAR1 agonist, in response to LPS. This led to the hypothesis that MMP1 signaling through PAR1 in early sepsis leads to loss of vascular integrity and activation of the coagulation cascade. Preliminary results indicate that endothelial cells secrete MMP1 in response to LPS stimulation and that pharmacologic blockade of MMP1 in vivo leads to improved survival in mouse models of sepsis. The purpose of this work is to determine the effect of MMP1-PAR1 signaling on vascular integrity and coagulation during sepsis. MMP1-PAR1 signaling in response to LPS will be studied on endothelial cells using in vitro assays of vascular leakage, actin retraction, and Rho-GTP generation. The role of MMP1-PAR1 signaling in vivo will also be studied by examining the effects of pharmacologic MMP1 blockade on clinical markers of vascular function and coagulation in a mouse cecal ligation and puncture (CLP) model of sepsis. Additionally, the enzymatic activity and expression of the putative mouse homologue of MMP1 and the hypothesized PAR1 agonist in vivo, mColA, has not been fully characterized. The collagenolytic and PAR1 agonist activity profile of mColA and other secreted mouse collagenases will be determined in vitro and plasma expression levels and activity of these collagenases during sepsis progression will be quantified in vivo. Ideally, this work will delineate a novel mechanism endothelial function regulation and will identify novel approaches to sepsis treatment.
Sepsis is a deadly disease that occurs when an infection triggers an excessively severe inflammatory response. Current treatments for sepsis are inadequate and over 120,000 people died from sepsis in the US in 2000. This proposal seeks to better understand the early stages of sepsis with the hope that this knowledge will help identify novel sepsis therapeutics.
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