Summary

Prostacyclin (PGI² ) has beneficial cytoprotective properties, is apotent inhibitor of platelet aggregation and has been reportedto improve microcirculatory blood flow during sepsis. Theformation of PGI² in response to proinflammatory cytokines iscatalysed by the inducible cyclooxygenase (COX) isoformCOX-2. Recombinant human activated protein C (rhAPC, drotrecoginalfa (activated)) was shown to have multiple biologicalactivities in vitro and to promote resolution of organ dysfunctionin septic patients.Whether rhAPC exerts its beneficial effects bymodulating prostanoid generation is unknown up to now. It wastherefore the aim of the study to examine the in vitro effect ofrhAPC on COX-2-mRNA-expression and PGI2 release fromhuman umbilical vein endothelial cells (HUVEC).We found thatrhAPC, at supra-therapeutical concentrations (500ng/ml-20µ g/ml), upregulated the amount of COX-2-mRNA in HUVEC att=3-9h and caused a time- and dose-dependent release of6-keto PGF1 a , the stable hydrolysis product of prostacyclin.RhAPC further increased the stimulating effect of tumor necrosis factor- α (TNF- α ) and thrombin on COX-2-mRNA-levels.Transcript levels of cyclooxygenase-1 (COX-1) and prostaglandinI2 synthase, however, were unaffected by the stimulation withrhAPC or thrombin.The upregulatory effect on COX2-mRNAlevels was specific for rhAPC since the zymogen protein C inequimolar concentrations had no effect on COX-2-mRNA-levelsor 6keto PGF_1α -release.Western Blot analysis revealed anincrease of COX-2-protein content in HUVEC after treatmentwith rhAPC. As shown by experiments using monoclonal antibodiesagainst the thrombin receptor PAR-1 (mAb=ATAP2) andagainst the endothelial protein C receptor (EPCR;mAb=RCR-252), the effect of rhAPC on COX-2-mRNA upregulationwas mediated by binding to the EPCR-receptor andsignaling via PAR-1.These results demonstrate that induction ofCOX-2-expression is an important response of HUVEC tostimulation with rhAPC and may represent a new molecularmechanism, by which rhAPC promotes upregulation of prostanoidproduction in human endothelium.