Abstract
We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.
Highlights
Grants HL 101917 and HL 68571 from the NHLBI
We recently demonstrated that when endothelial protein C receptor (EPCR) on endothelial cells is occupied by either protein C or activated protein C (APC), the activation of protease-activated receptor 1 (PAR-1) by thrombin leads to initiation of protective cellular responses and down-regulation of the same proinflammatory molecules that are thought to be targets for the protective effects of APC [15, 16]
In a series of studies, we showed that both EPCR and PAR-1 are colocalized within lipid rafts and that the unbound EPCR exists in association with caveolin-1 in endothelial cells [15,16,17]
Summary
Construction, Expression, and Purification of Recombinant Proteins—Construction and expression of the Ser-195 3 Ala (FX-S195A) (chymotrypsinogen numbering [24]) and the Gla domainless mutant of FX (GDFX) in human embryonic kidney (HEK293) cells has been described [25, 26]. Following twice washing of neutrophils (1.5 ϫ 106/ml, 200 l/well), they were resuspended in the adhesion medium (RPMI containing 2% fetal bovine serum and 20 mM HEPES) and added to confluent monolayers of EA.hy926 cells in 96-well plates, which were treated for 4 h with 20 nM of either APC or thrombin Ϯ FX-S195A zymogen (175 nM) followed by TNF-␣ (10 ng/ml for 4 h). Beads were washed, resuspended in loading buffer, and proteins were separated on 12% SDS-PAGE followed by transferring to a PVDF membrane and Western blotting using an anti-RhoA monoclonal antibody as described by the manufacturer. The NF-B pathway activation in the conditioned cell lysates (treated with different PAR-1 agonists and activated by TNF-␣ as described under figure legends) was monitored by Western blotting employing two antibodies that are specific for either NF-B p65 (as an index of total cellular NF-B p65) or its phosphorylated form (as an index of the NF-B pathway activation) as described [16]. The immunoreactive protein bands were visualized by SuperSignal West Pico (Pierce, Rockford, IL)
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