Metalloproteolytic release of endothelial cell protein C receptor

Previous studies observed that there is about 100 ng/ml soluble endothelial cell protein C receptor (EPCR) in human plasma and that the levels increas e in inflammatory diseases. In this study we examine the potential mechanis ms involved in release of EPCR from cells. We find that EPCR is released fr om the surface of endothelium and transfected 293 cells by a metalloproteas e in a constitutive fashion. The mass of soluble EPCR is 4 kDa less than in tact EPCR. Release is blocked by either the hydroxamic acid based inhibitor , KD-IX-73-4 or by 1,10-phenanthroline, but not by matrix metalloprotease i nhibitors. Release is stimulated by phorbol 12-myristate 13-acetate, thromb in, interleukin-1 beta, and hydrogen peroxide. Stimulation with these agent s reduces EPCR expression levels sufficiently to decrease the rate of prote in C activation to a limited extent. The influence of phorbol 12-myristate 13-acetate on both EPCR release and inhibition of protein C activation are enhanced by microtubule disruption with nocodazole, EPCR release is augment ed by transfection of EPCR expressing 293 cells with caveolin, suggesting t hat release is caveolae dependent. These studies indicate that metalloprote olytic release of EPCR is a highly regulated process that is sensitive to b oth coagulation factors and inflammatory mediators.