Recombinant human activated protein C upregulates the release of soluble fractalkine from human endothelial cells

Fractalkine is a unique endothelial cell-derived chemokine that functions both as a chemoattractant and as an adhesion molecule. Recent findings suggest that fractalkine plays an important role in inflammatory diseases by modulating leucocyte endothelial cell interactions. A modulating effect on the immune system in severe sepsis has been suggested for recombinant human activated protein C (rhAPC). However, a little is known about the effect of rhAPC on the endothelial release of soluble fractalkine. The effect of rhAPC (50 ng/ml to 10 mu g/ml) and protein C (in equimolar concentrations) on the synthesis of fraktalkine-mRNA and release of soluble protein in human umbilical vein endothelial cells (HUVEC) was determined by reverse transcription-polymerase chain reaction and by an enzyme-linked immunosorbent assay. rhAPC at supra-pharmacological concentrations (1-10 mu g/ml) stimulated fractalkine-messenger RNA-gene transcription and release of soluble fractalkine in a time- and dose-dependent manner, whereas the zymogen protein C was ineffective. As shown by experiments using monoclonal antibodies against the thrombin receptor, protease-activated receptor-1 (PAR-1), PAR-2 and against the endothelial protein C receptor (EPCR), the effect of rhAPC on fractalkine upregulation was mediated by binding to the EPCR-receptor and signalling via PAR-1. These in vitro data demonstrate that induction of fractalkine release is an important response of HUVEC to stimulation with rhAPC and may lead to a better understanding of the molecular pathways involved in the mode of action of rhAPC. Further clinical trials are needed to confirm the in vivo relevance of these data.