The Interaction between the Endothelial Cell Protein C Receptor and Protein C Is Dictated by the γ-Carboxyglutamic Acid Domain of Protein C

Regan, Lisa M.; Mollica, Jeffery S.; Rezaie, Alireza R.; Esmon, Charles T.

doi:10.1074/jbc.272.42.26279

Cited by 77 publications

(72 citation statements)

References 54 publications

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“…It is also possible that there may be an alternative activated protein C-binding receptor on the surface of monocytes (51). Using Ca 2ϩ -free conditions (which prevents activated protein C-EPCR interactions) (52) or mAbs that block activated protein C-EPCR interactions (53), we observed that recombinant activated protein C retained the ability to bind to the monocyte cell surface (unpublished observations). In contrast, the binding of recombinant activated protein C to endothelial cell surfaces is blocked by anti-EPCR Abs as well as by the removal of calcium in the binding buffer (unpublished observations).…”

Section: Figurementioning

confidence: 92%

Activated Protein C Up-Regulates IL-10 and Inhibits Tissue Factor in Blood Monocytes

Toltl¹,

Beaudin

Liaw

2008

The Journal of Immunology

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The protective effect of recombinant activated protein C therapy in patients with severe sepsis likely reflects the ability of recombinant activated protein C to modulate multiple pathways implicated in sepsis pathophysiology. In this study, we examined the effects of recombinant activated protein C on the anti-inflammatory cytokine IL-10 and on the procoagulant molecule tissue factor (TF) in LPS-challenged blood monocytes. Treatment of LPS-stimulated monocytes with recombinant activated protein C resulted in an up-regulation of IL-10 protein production and mRNA synthesis. The up-regulation of IL-10 required the serine protease activity of recombinant activated protein C and was dependent on protease-activated receptor-1, but was independent of the endothelial protein C receptor. At the intracellular level, p38 MAPK activation was required for recombinant activated protein C-mediated up-regulation of IL-10. We further observed that incubation of LPS-stimulated monocytes with recombinant activated protein C down-regulated TF Ag and activity levels. This anticoagulant effect of recombinant activated protein C was dependent on IL-10 since neutralization of endogenously produced IL-10 abrogated the effect. In patients with severe sepsis, plasma IL-10 levels were markedly higher in those treated with recombinant activated protein C than in those who did not receive recombinant activated protein C. This study reveals novel regulatory functions of recombinant activated protein C, specifically the up-regulation of IL-10 and the inhibition of TF activity in monocytes. Our data further suggest that these activities of recombinant activated protein C are directly linked: the recombinant activated protein C-mediated up-regulation of IL-10 reduces TF in circulating monocytes.

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Section: Figurementioning

confidence: 92%

Activated Protein C Up-Regulates IL-10 and Inhibits Tissue Factor in Blood Monocytes

Toltl¹,

Beaudin

Liaw

2008

The Journal of Immunology

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“…The ability to inhibit protein C activation over quiescent HUVEC with the anti-EPCR mAb, but not with annexin V, argues strongly that the protein C interaction with the cell surface that augments activation is mediated primarily if not exclusively by EPCR rather than by negatively charged phospholipids. This provides selectivity for protein C activation because the other vitamin K-dependent proteins would compete effectively for the phospholipid surface and inhibit protein C activation, but these factors do not compete effectively for binding to EPCR (34).…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation

Esmon

1999

Journal of Biological Chemistry

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Blocking protein C binding to the endothelial cell protein C receptor (EPCR) on the endothelium is known to reduce protein C activation rates. Now we isolate human EPCR and thrombomodulin (TM) and reconstitute them into phosphatidylcholine vesicles. The EPCR increases protein C activation rates in a concentration-dependent fashion that does not saturate at 14 EPCR molecules/TM. Without EPCR, the protein C concentration dependence fits a single class of sites (K m ‫؍‬ 2.17 ؎ 0.13 M). With EPCR, two classes of sites are apparent (K m ‫؍‬ 20 ؎ 15 nM and K m ‫؍‬ 3.2 ؎ 1.7 M). Increasing the EPCR concentration at a constant TM concentration increases the percentage of high affinity sites. Holding the TM: EPCR ratio constant while decreasing the density of these proteins results in a decrease in the EPCR enhancement of protein C activation, suggesting that there is little affinity of the EPCR for TM. Negatively charged phospholipids also enhance protein C activation. EPCR acceleration of protein C activation is blocked by anti-EPCR antibodies, but not by annexin V, whereas the reverse is true with negatively charged phospholipids. Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates 7-fold over these cells, whereas annexin V is ineffective, indicating that EPCR rather than negatively charged phospholipid provide the surface for protein C activation. EPCR expression varies dramatically among vascular beds. The present results indicate that the EPCR concentration will determine the effectiveness of the protein C activation complex.

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“…16 As demonstrated here for PS, the Gla domains of other vitamin K-dependent proteins have been implicated in proteinprotein and protein-membrane interactions. 30,[47][48][49][50][51] Interestingly, a chimeric protein C species in which the Gla domain was replaced by the prothrombin Gla domain has been found to be insensitive to PS in a plasma clotting assay 45 ; in addition, PS dependence is associated with the C-terminal part of the protein C Gla domain. 45 Thus, it was suggested that PS functions in plasma are mainly dependent on interaction with APC and that this interaction is disrupted by replacing the Gla domain of protein C with that of prothrombin.…”

Section: Discussionmentioning

confidence: 99%

The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding

Saller

Villoutreix

Amelot

et al. 2005

Blood

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We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin ␥-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (Gla FII -PS) or in PS deleted of the thrombinsensitive region (TSR) (Gla FII -⌬TSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solventexposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into Gla FII -PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC. IntroductionProtein S (PS) is a vitamin K-dependent protein involved in regulating the anticoagulant activity of activated protein C (APC). In purified systems, PS functions as a nonenzymatic cofactor for APC in proteolytic inactivation of activated factor V (FVa) and activated factor VIII (FVIIIa), 1 reflecting the ability of PS to interact with APC. However, these effects are relatively weak and are inconsistent with the major physiologic role of PS. Indeed, the importance of PS as a natural anticoagulant is clearly demonstrated by the occurrence of severe thrombotic complications in infants with homozygous hereditary PS deficiency and by a mild thrombotic tendency in heterozygous subjects. 2 This suggests that additional components may determine the expression of the APC cofactor activity of PS in plasma. Thus, activated factor X (FXa) and activated factor IX (FIXa) have been shown to protect FVa and FVIIIa from inactivation by APC, and, in this system, PS is able to abolish these protective effects. [3][4][5] Another determinant is FV, which acts in synergy with PS in FVIIIa inactivation by APC in purified systems. 6 PS also demonstrates APC-independent anticoagulant activity by inhibiting prothrombinase and tenase activities, 7,8 and this may contribute to the overall anticoagulant activity of PS in plasma.PS is a single-chain, 635-amino acid glycoprotein composed of an N-terminal domain rich in ␥-carboxyglutamic acid (Gla domain; amino acids 1-45), a thrombin-sensitive region (TSR; amino acids 46-74), 4 epidermal growth factor (EGF)-like domains (amino acids 75-242), 9 and a large C-terminal sex hormone-binding globulin (SHBG)-like region (amino acids 243-635). 10 The SHBGlike domain of PS binds tightly to C4b...

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The Interaction between the Endothelial Cell Protein C Receptor and Protein C Is Dictated by the γ-Carboxyglutamic Acid Domain of Protein C

Cited by 77 publications

References 54 publications

Activated Protein C Up-Regulates IL-10 and Inhibits Tissue Factor in Blood Monocytes

Activated Protein C Up-Regulates IL-10 and Inhibits Tissue Factor in Blood Monocytes

Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation

The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding

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