title: 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 Red-sEPCR indicated that the APC.sEPCR 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.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: P C
authors.initials: PC
authors.lastname: Liaw
authors.forename: P F
authors.initials: PF
authors.lastname: Neuenschwander
authors.forename: M D
authors.initials: MD
authors.lastname: Smirnov
authors.forename: C T
authors.initials: CT
authors.lastname: Esmon
mesh_terms: D001665:Binding Sites; D001779:Blood Coagulation Factors; D002122:Calcium Chloride / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D015943:Factor Va / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D011487:Protein Conformation; D017293:Protein S / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D001779:Blood Coagulation Factors; D008081:Liposomes; D011486:Protein C; D017293:Protein S; D011956:Receptors, Cell Surface; C091963:activated protein C receptor; D015636:Magnesium Chloride; D015943:Factor Va; D002122:Calcium Chloride