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) on HUVEC, so increasing protection against complement-mediated injury. Using protein kinase C (PKC) isozyme-specific peptide antagonists and adenoviral constructs, we show that PKC is the primary isozyme involved in DAF induction by thrombin. Experiments with proteinase-activated receptor (PAR)₁ and PAR₂ activating peptides (AP) showed that DAF expression induced by PAR₁-AP was PKC-dependent whilst in contrast, PAR₂-AP induction of DAF required activation of PKC. PAR₁ and PAR₂-AP in combination exerted an additive effect on DAF protein expression, which was equivalent to that seen with thrombin alone. These data implied a specific role for PAR₂ in DAF induction, which was supported by the observation that upregulation of EC PAR2 enhanced DAF induction by thrombin. ERK1/2, p38 and JNK MAPK were also involved in thrombin-induced DAF upregulation, with evidence for interdependence between ERK1/2 and JNK. A role for transactivation of PAR₂ by PAR₁ was suggested by partial inhibition of thrombin-induced DAF expression by the PAR₁ signaling antagonists BMS-200261 and SCH79797, whereas inhibition of thrombin-induced cleavage of PAR₁ 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 PAR₂ by PAR₁ and signaling via PKC/MAPK. This may represent an important novel pathway for endothelial cytoprotection during inflammation and angiogenesis, and suggests that PAR₂ may play a central role in some thrombin-induced responses.