Abstract
Previous studies observed that there is about 100 ng/ml soluble endothelial cell protein C receptor (EPCR) in human plasma and that the levels increase in inflammatory diseases. In this study we examine the potential mechanisms involved in release of EPCR from cells. We find that EPCR is released from the surface of endothelium and transfected 293 cells by a metalloprotease in a constitutive fashion. The mass of soluble EPCR is 4 kDa less than intact 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 inhibitors. Release is stimulated by phorbol 12-myristate 13-acetate, thrombin, interleukin-1beta, and hydrogen peroxide. Stimulation with these agents reduces EPCR expression levels sufficiently to decrease the rate of protein 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 augmented by transfection of EPCR expressing 293 cells with caveolin, suggesting that release is caveolae dependent. These studies indicate that metalloproteolytic release of EPCR is a highly regulated process that is sensitive to both coagulation factors and inflammatory mediators.
Highlights
Protein C, a critical zymogen of the natural anticoagulantactivated protein C, is activated on the surface of the endothelium by a complex between thrombin and the cell surface receptor thrombomodulin
Previous studies demonstrated that human plasma contains about 100 ng/ml soluble endothelial cell protein C receptor (EPCR) that is selectively processed to yield a form fully capable of binding protein C/activated protein C [8]
We examined the conditioned medium from HUVEC, the EA.hy924 endothelial cell line and 293 cells stably transfected with wild type EPCR for the presence of soluble EPCR
Summary
(Received for publication, December 1, 1999, and in revised form, December 10, 1999). Release is stimulated by phorbol 12-myristate 13-acetate, thrombin, interleukin-1, and hydrogen peroxide Stimulation with these agents reduces EPCR expression levels sufficiently to decrease the rate of protein C activation to a limited extent. An endothelial cell protein C receptor (EPCR) was identified [2, 3], cloned [2], and shown to facilitate protein C activation by the thrombin thrombomodulin complex [4]. Isolation of the plasma EPCR revealed that it has a mass similar to intact EPCR as assessed by SDS-gel electrophoresis and that it retains its affinity for protein C and activated protein C [8] Many cellular receptors, such as L-selectin [6], and biological response modifiers, such as tumor necrosis factor ␣ [11, 12], are released from cells by metalloproteases. We find that release of soluble EPCR is mediated by a metalloprotease and its activity is inducible by inflammatory mediators and thrombin
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