Factor Xa activates endothelial cells by a receptor cascade between EPR-1 and PAR-2

In addition to its pivotal role in hemostasis, factor Xa binds to human umb ilical vein endothelial cells through the recognition of a protein called e ffector cell protease receptor (EPR-1). This interaction is associated with signal transduction, generation of intracellular second messengers, and mo dulation of cytokine gene expression. Inhibitors of factor Xa catalytic act ivity block these responses, thus indicating that the factor Xa-dependent e vent of local proteolysis is absolutely required for cell activation. Becau se EPR-1 does not contain proteolysis-sensitive sites, we investigated the possibility that signal transduction by factor Xa requires proteolytic acti vation of a member of the protease-activated receptor (PAR) gene family. Ca talytic inactivation of factor Xa by DX9065 suppressed factor Xa-induced in crease in cytosolic free Ca2+ in endothelial cells (IC50=0.23 mu mol/L) but failed to reduce ligand binding to EPR-1. In desensitization experiments, trypsin or the PAR-2-specific activator peptide, SLIGKV, ablated the Ca2+ s ignaling response induced by factor Xa. Conversely, pretreatment of endothe lial cells with factor Xa blocked the PAR-2-dependent increase in cytosolic Ca2+ signaling, whereas PAR-1-dependent responses were unaffected. Direct cleavage of PAR-2 by factor Xa on endothelial cells was demonstrated by cle avage of a synthetic peptide duplicating the PAR-2 cleavage site and by imm unofluorescence with an antibody to a peptide containing the 40-amino acid PAR-2 extracellular extension. These data suggest that factor Xa induces en dothelial cell activation via a novel cascade of receptor activation involv ing docking to EPR-1 and local proteolytic cleavage of PAR-2.