Thrombin and activated protein C inhibit the expression of secretory group IIA phospholipase A₂ in the TNF-α-activated endothelial cells by EPCR and PAR-1 dependent mechanisms

Introduction: Thrombin and tumor necrosis factor (TNF)-α up-regulate the expression of proinflammatory molecules in human umbilical vein endothelial cells (HUVECs). However, activated protein C (APC) down-regulates the expression of the same molecules. The expression level of secretory group IIA phospholipase A₂ (sPLA₂-IIA) is known to be elevated in inflammatory disorders including in sepsis. Here, we investigated the effects of APC and thrombin on the expression of sPLA₂-IIA and extracellular signal-regulated kinase (ERK) in HUVECs. Materials and methods: The expression level of sPLA₂-IIA was quantitatively measured by an enzyme-linked-immunosorbent-assay following stimulation of HUVECs with either thrombin or TNF-α in the absence and presence of the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 and the cholesterol-depleting drug methyl-β-cyclodextrin (MβCD). Results and conclusions: Thrombin had no effect on the expression of sPLA₂-IIA in HUVECs, however, TNF-α potently induced its expression. The prior treatment of cells with APC inhibited expression of sPLA₂-IIA through the EPCR-dependent cleavage of PAR-1. Further studies revealed that if HUVECs were pretreated with the zymogen protein C to occupy EPCR, thrombin also inhibited the TNF-α-mediated expression of sPLA₂-IIA through the cleavage of PAR-1. The EPCR-dependent cleavage of PAR-1 by both APC and thrombin increased the phosphorylation of ERK 1/2. Pretreatment of cells with either LY294002 or MβCD abolished the inhibitory activity of both APC and thrombin against sPLA₂-IIA expression, suggesting that the protein C occupancy of EPCR confers a PI3-kinase dependent protective activity for thrombin such that its cleavage of the lipid-raft localized PAR-1 inhibits the TNF-α-mediated expression of sPLA₂-IIA in HUVECs.