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

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Abstract
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-ε is the primary isozyme involved in DAF induction by thrombin. Experiments with proteinase-activated receptor-1 (PAR1) and PAR2activating peptides (APs) showed that DAF expression induced by PAR1-AP was PKC-α-dependent; in contrast, PAR2-AP induction of DAF required activation of PKC-ε. PAR1-AP and PAR2-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 PAR2in DAF induction, which was supported by the observation that upregulation of endothelial cell (EC) PAR2-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 PAR2by PAR1was suggested by partial inhibition of thrombin-induced DAF expression by the PAR1signaling antagonists BMS-200261 and SCH79797 , whereas inhibition of thrombin-induced cleavage of PAR1by specific MAbs or hirudin completely abrogated the response. Together, these data imply that the predominant pathway for thrombin-induced DAF expression involves transactivation of PAR2by PAR1and signaling via PKC-ε/MAPK. This may represent an important, novel pathway for endothelial cytoprotection during inflammation and angiogenesis and suggests that PAR2may play a central role in some thrombin-induced responses.

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