Regulation of tissue factor-mediated initiation of the coagulation cascade by cell surface Grp78

Objective - To test the hypothesis that Grp78 negatively regulates cell surface tissue factor ( TF) procoagulant activity and whether this is mediated by physical interaction. Methods and Results - Biopanning with phage- displayed peptidyl libraries has identified peptide probes that bind selectively in vivo to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds 78- kDa glucose regulated protein ( Grp78). Grp78 participates in numerous pathological processes, including the regulation of the coagulation cascade, but the mechanism of Grp78 regulation of coagulation is unknown. To characterize this function, we analyzed the effect of Grp78 on TF- mediated procoagulant activity on murine brain endothelial cells ( bEND. 3) and macrophage- like ( RAW) cells, which are relevant in mediation of atherothrombosis. We show that Grp78 is present on the surface of endothelium and monocyte/ macrophage- like cells in atherosclerotic lesions. Inhibition of Grp78 resulted in increased procoagulant activity. We demonstrate that Grp78 negatively regulates procoagulant activity by interacting physically with the TF extracellular domain on the cell surface. Conclusions - The evidence indicates that Grp78 negatively regulates TF functional activity via direct binding to and functional inhibition of TF. Identification of the mechanism by which Grp78 regulates TF function may advance insight into the pathobiology of atherosclerosis and associated arterial thrombosis.