Protease Activated Receptor and Thrombomodulin Signaling by coagulation proteases

Project Summary Thrombomodulin (TM) is an integral membrane receptor on endothelial cells which binds to exosite- 1 of thrombin to switch the specificity of thrombin from a procoagulant to an anticoagulant protease. The interaction with TM enables thrombin to activate protein C to activated protein C (APC). In addition to its anticoagulant function, APC binds to endothelial protein C receptor (EPCR) to cleave protease-activated receptor 1 (PAR1) at Arg46 site to elicit cytoprotective responses in endothelial cells. The exosite-1-dependent interaction of thrombin with TM on endothelial cells inhibits the thrombin recognition of PAR1 and procoagulant substrates. TM exerts direct anti-inflammatory 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 the cytoplasmic domain of TM is linked to the actin cytoskeleton and involved in the regulation of PTEN/AKT/mTOR signaling axis. TM endows a quiescence phenotype to endothelial cells through modulation of this signaling axis under basal and stimulated conditions. We demonstrate that interaction of EGF-like domains of TM with exosite-1 of thrombin and proexosite-1 of prothrombin leads to activation (phosphorylation at Ser172) of AMPK, thereby TM differentially regulating mTORC1 (inhibition) and mTORC2 (activation) signaling. We hypothesize by this signaling mechanism, TM contributes to regulation of the vascular tone, maintenance, and stabilization of the barrier permeability function of the vasculature under steady-state and stimulated conditions. We further demonstrate TM changes the PAR1 cleavage specificity of thrombin from Arg41 site to Arg46 site, thereby switching the PAR1- dependent signaling specificity of thrombin from a proinflammatory response to a cytoprotective one. We have prepared a series of TM receptor constructs, recombinant APC and thrombin derivatives and TM-null and PAR1-null endothelial cells to investigate the following three Specific Aims: Aim 1 will investigate the mechanism by which TM maintains a quiescence phenotype in endothelial cells. Aim 2 will investigate the hypothesis that the cytoplasmic domain of TM is linked to the actin cytoskeleton and involved in regulation of mTOR signaling upon interaction with ligands thrombin and prothrombin. Aim 3 will investigate mechanisms through which EPCR and TM modulate the signaling specificity of PAR1 cleavage by coagulation proteases in endothelial cells.