Mechanisms by Which Soluble Endothelial Cell Protein C Receptor Modulates Protein C and Activated Protein C Function

Abstract

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl 2 and 0.6 mM MgCl 2 , sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K i ‫؍‬ 40 ؎ 7 and 33 ؎ 4 nM, respectively). Without MgCl 2 , the K i values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas RedsEPCR indicated that the APC⅐sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K i ‫؍‬ 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K d ‫؍‬ 27 ؎ 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 ؎ 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

Thyroid hormonal axis regulates protein C anticoagulation pathway in rats

“…EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of this receptor (sEPCR) circulates in plasma and has been shown to bind protein C and aPC with an affinity A B C similar to that of intact membrane-bound EPCR [21]. In contrast to membrane-bound EPCR, sEPCR inhibits protein C activation over large vessel endothelium.…”

Cudratricusxanthone A inhibits endothelial protein C receptor shedding in vitro and in vivo

“…sEPCR circulates in plasma, retaining its ability to bind both PC and APC, but does not enhance protein C activation (Kurosawa et al 1997). It inhibits anticoagulant activity of APC by formation of a complex, which does not bind to phospholipid membranes (Liaw et al 2000). sEPCR can be detected in plasma, resulting from shedding of membrane EPCR, with a plasma concentration of approximately 100 ng/mL; high levels of sEPCR in systemic inflammatory diseases have been reported (Kurosawa et al 1998).…”

Endothelial cell protein C receptor and the risk of venous thrombosis

“…Like the membrane-associated form, sEPCR binds PC and APC with similar affinity. However, its binding to APC inhibits the anticoagulant activity of APC by abrogating its ability to inactivate factor Va, and its binding to PC precludes PC activation by thrombin-TM complexes 16 ( Figure 2). Soluble EPCR has mainly procoagulant properties.…”