A role for proteinase-activated receptor 2 and PKC-ε in thrombin-mediated induction of decay-accelerating factor on human endothelial cells

Thrombin, an important mediator of thrombosis and inflammation, may also enhance vascular cytoprotection. Thus thrombin induces expression of the complement-inhibitory protein decay-accelerating factor (DAF) in human umbilical vein endothelial cells (HUVECs), thus increasing protection against complement-mediated injury. Using PKC isozyme-specific peptide antagonists and adenoviral constructs, we have shown in the present study that PKC-epsilon is the primary isozyme involved in DAF induction by thrombin. Experiments with proteinase-activated receptor-1 (PAR(1)) and PAR(2) activating peptides (APs) showed that DAF expression induced by PAR(1)-AP was PKC-alpha-dependent; in contrast, PAR(2)-AP induction of DAF required activation of PKC-epsilon. PAR(1)-AP and PAR(2)-AP in combination exerted an additive effect on DAF protein expression, which was equivalent to that observed with thrombin alone. These data implied a specific role for PAR2 in DAF induction, which was supported by the observation that upregulation of endothelial cell (EC) PAR(2)-enhanced DAF induction by thrombin. ERK1/2, p38, and JNK MAPK were also involved in thrombin-induced DAF upregulation, with evidence of interdependence between ERK1/2 and JNK. A role for transactivation of PAR(2) by PAR(1) was suggested by partial inhibition of thrombin-induced DAF expression by the PAR(1) signaling antagonists BMS200261 and SCH79797, whereas inhibition of thrombin-induced cleavage of PAR(1) by specific MAbs or hirudin completely abrogated the response. Together, these data imply that the predominant pathway for thrombin-induced DAF expression involves transactivation of PAR(2) by PAR(1) and signaling via PKC-epsilon/MAPK. This may represent an important, novel pathway for endothelial cytoprotection during inflammation and angiogenesis and suggests that PAR(2) may play a central role in some thrombin-induced responses.