Protective Signaling by Protein C in Endothelial Cells

The anticoagulant activated protein C (APC) has major anti-inflammatory effects and its therapeutic efficiency to reduce mortality in patients with severe sepsis led to its recent approval for the treatment of this condition in adults. While anticoagulatory functions are well investigated, the molecular basis for APC`s anti-inflammatory effects is incompletely understood. Aim of the study: Endothelial cell PC receptor (EPCR) can bind both PC and APC and activation of EPCR-bound PC is enhanced. Moreover, the protease activated receptor-1 (PAR-1) and EPCR have been identified as part of a novel pathway for APC signaling in endothelial cells. The overall hypothesis is that PAR-dependent signaling of the PC system in endothelial cells limits inflammatory responses and reduces lethality in sepsis. Methods: The role of PAR1 and EPCR in suppressive effects of APC on the gene expression profiles in human umbilical vein endothelial cells will be defined using real time PCR. Data will be confirmed on protein levels using highly sensitive antibodies in westen blot assays and expression of the EPCR on the cell surface will be detected using flow cytometry. Adenovirus-mediated gene transfer will be used to introduce mutant EPCR molucules into endothelial cells and responses to variants of the EPCR intracellular domain will be tested. In transendothelial migration assays using Transwell-Filters we will establish whether APC signaling in endothelial cells counteracts chemotactic transmigration of monocytic cells. Summary: These experiments will elucidate anti- inflammatory effects and signaling mechanisms of APC in consideration of the influence of EPCR and PAR-1.