Thrombomodulin (TM) is a type-1 integral membrane receptor which was identified and cloned as a receptor for thrombin on endothelial cells. It is a multi-domain glycoprotein comprised of five distinct domains that include an N-terminal lectin-like domain, followed by six epidermal growth factor (EGF)-like domains, a membrane proximal Ser/Thr rich domain harboring chondroitin sulfate glycosaminoglycans, a single spanning transmembrane domain and a cytoplasmic tail. TM switches the specificity of thrombin from a procoagulant to an anticoagulant protease by binding to exosite-1 of thrombin via its EGF-like domains 4, 5, and 6 (TM456), thereby enabling thrombin to activate protein C to activated protein C (APC). The exosite-1-dependent interaction of thrombin with TM on endothelial cells can inhibit the thrombin recognition of protease-activated receptor 1 (PAR1) and procoagulant substrates. TM also exerts antiinflammatory functions through its lectin-like domain by unknown mechanisms. Recent results have indicated an association between loss of TM expression and uncontrolled cell proliferation and metastasis. Based on our preliminary data in this application, we hypothesize that thrombin can function as a true ligand for TM to elicit direct exosite-1-dependent antiinflammatory signaling responses in vascular endothelial cells. We hypothesize that this function of TM not only plays a cytoprotective role in response to inflammatory stimuli, but it may also contribute to regulation of the vascular tone, maintenance and stabilization of the barrier permeability function of the vasculature under steady-state conditions. We further hypothesize that TM prevents thrombin from interacting with PAR1 on microvascular endothelial cells, thereby TM directly transmitting the protective signaling function of thrombin via its cytoplasmic domain. We have prepared a series of TM receptor constructs, recombinant APC and thrombin derivatives and TM-null endothelial cells to investigate the following three Specific Aims:
Aim 1 will investigate the hypothesis that thrombin can function as a true ligand for TM to elicit direct cytoprotective signaling responses in endothelial cells.
Aim 2 will investigate the mechanism by which components of the thrombin-TM-APC signaling axis inhibit the proinflammatory signaling function of high mobility group box 1 (HMGB1) in endothelial cells.
Aim 3 will develop a chlorhexidine gluconate (CG)-induced peritoneal injury model and evaluate the mechanism of the cytoprotective function of the thrombin- TM-protein C signaling axis in this model.
(relevance of this research to public health): The studies of this application will investigate the mechanism by which components of the natural anticoagulant pathways, thrombin-thrombomodulin-protein C-EPCR initiate cytoprotective signaling responses in order to maintain the integrity of blood vessels. The mis-regulation of these signaling systems can impair vascular function and lead to debilitating thrombotic and inflammatory disorders including heart attack, stroke and severe sepsis. A better understanding of signaling mechanisms that modulate vascular functions as outlined in this application is essential for discovering new drugs potentially useful for treating ever-increasing inflammatory and thrombotic disorders.
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