A Novel Mechanism of Endothelial Protein C Receptor Release, in Microparticulate Form, by Activated Protein C.

Activated protein C (APC) administration is now used for treating patients with severe sepsis. We investigated its effect on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release from the cell surface. Exposure of human umbilical vein endothelial cells or monocytes to APC (from physiological levels of 0.5 up to 100nM) resulted in the increasing release of EPCR-containing microparticles (EPCR-MP), as demonstrated by confocal microscopy. Further characterisation through flow cytometry showed a concomitant fall in EPCR levels from the cell surface. This release of EPCR could not be inhibited by the metalloproteinase inhibitors 1, 10-phenanthroline or Ro31-9790, unlike soluble EPCR (sEPCR) that is metalloproteinase cleaved at the cell surface following thrombin or pro- inflammatory cytokine stimulation. Western blotting confirmed the molecular weight of EPCR-MP to be identical to the full-length membrane form (49 kD) and different from sEPCR (45 kDa). APC was also bound to EPCR-MP and could be quantified by ELISA using EPCR capture and APC detection by chromogenic substrate, S2366. Using an initial factor Va incubation step followed by a prothrombinase assay, the APC bound to EPCR-MP could significantly reduce thrombin generation. This was abrogated in the presence of excess α₁-antitrypsin, an APC inhibitor. By contrast, APC bound to sEPCR could no longer inactivate factor Va. Further characterisation showed the APC induction of EPCR-MP to be time dependent with increasing release over 24 hours, as quantified by ELISA. The phenomenon also required the active site of APC. Neither protein C, heat-inactivated or D-Phe-Pro-Arg-chloromethylketone-blocked APC could induce EPCR-MP formation. Co-incubation with hirudin (6mM) did not alter the APC effect and excluded any role of contaminating thrombin. This novel observation provides new insights into the consequences of APC therapy in the septic patient as well as demonstrating for the first time that there can be 2 circulating forms of EPCR. Unlike sEPCR however, EPCR-MP can facilitate and potentially disseminate the anticoagulant activity of bound APC.