Identification of functional endothelial protein C receptor in human plasma.

Kurosawa, Shinichiro; Stearns-Kurosawa, Deborah J.; Hidari, Noriko; Esmon, Charles T.

doi:10.1172/jci119548

Cited by 156 publications

(135 citation statements)

References 48 publications

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“…ELISA for EPCR-The assay was modified from Kurosawa et al (23). Microtiter plates were coated overnight at 4°C with 50 l of 5 g/ml anti-EPCR mAb 1494 in 15 mM Na 2 CO 3 , 35 mM NaHCO 3 , pH 9.6.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…Assays were based on experiments done previously [5,[34][35][36], with modifications. Briefly, either HUVEC or MDA-MB-231 cells were grown to confluency in a 96-well plate.…”

Section: Generation Of Apc On a Cell Monolayermentioning

confidence: 99%

“…3B) of HUVEC, as the positive control, and both breast cancer cell lines. A blocking antibody (JNK 1494) to the PC/APC binding site on EPCR [5,[34][35][36] was used in a 12-h transwell chemotaxis assay with the MDA-MB-231 cells. Initially, it was necessary to determine the optimal concentra tion of EPCR blocking antibody to use with the MDA-MB-231 cells.…”

Section: Apc Binding To Epcr Is Necessary To Increase Chemotaxis Of Tmentioning

confidence: 99%

“…Initially, it was necessary to determine the optimal concentra tion of EPCR blocking antibody to use with the MDA-MB-231 cells. HUVEC, as a control, and MDA-MB-231 cells were treated with increasing concentrations of the EPCR blocking antibody to block the generation of APC on the cell surface [5,[34][35][36]. Figs.…”

Section: Apc Binding To Epcr Is Necessary To Increase Chemotaxis Of Tmentioning

confidence: 99%

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Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

Beaulieu

Church

2007

Experimental Cell Research

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Activated protein C (APC) is a serine protease that regulates thrombin (IIa) production through inactivation of blood coagulation factors Va and VIIIa. APC also has non-hemostatic functions related to inflammation, proliferation, and apoptosis through various mechanisms. Using two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, we investigated the role of APC in cell chemotaxis and invasion. Treatment of cells with increasing APC concentrations (1-50 μg/ml) increased invasion and chemotaxis in a concentration-dependent manner. Only the active form of APC increased invasion and chemotaxis of the MDA-MB-231 cells when compared to 3 inactive APC derivatives. Using a modified "checkerboard" analysis, APC was shown to only affect migration when plated with the cells; therefore, APC is not a chemoattractant. Blocking antibodies to endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) attenuated the effects of APC on chemotaxis in the MDA-MB-231 cells. Finally, treatment of the MDA-MB-231 cells with the proliferation inhibitor, Na butyrate, showed that APC did not increase migration by increasing cell number. Therefore, APC increases invasion and chemotaxis of cells by binding to the cell surface and activating specific signaling pathways through EPCR and PAR-1.

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“…sEPCR is present in normal plasma (ϳ100 ng/ml) (11). It is released from the endothelium by a metalloproteinase (12).…”

mentioning

confidence: 99%

Disruption of the Endothelial Cell Protein C Receptor Gene in Mice Causes Placental Thrombosis and Early Embryonic Lethality

Crawley

Ferrell

et al. 2002

Journal of Biological Chemistry

131

110

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The endothelial cell protein C receptor (EPCR) is a type 1 transmembrane protein found primarily on endothelium that binds both protein C and activated protein C with similar affinity. EPCR augments the activation of protein C by the thrombin-thrombomodulin complex. To determine the physiological importance of EPCR, we generated EPCR-deficient mice by homologous targeting in embryonic stem cells. Genotyping of progeny obtained from EPCR ؉/؊ interbreeding indicated that EPCR ؊/؊ embryos died on or before embryonic day 10.5 (E10.5). Reverse transcriptase-PCR confirmed the absence of EPCR mRNA in EPCR ؊/؊ embryos. EPCRembryos removed from extra-embryonic membranes and tissues at day E7.5 and cultured in vitro developed beyond E10.5, suggesting a role for EPCR in the normal function of the placenta and/or at the materno-embryonic interface. Immunohistochemistry revealed the lack of EPCR in trophoblast giant cells of EPCR ؊/؊ embryos. These cells, which normally express EPCR, are in direct contact with the maternal circulation and its clotting factors. In EPCR ؊/؊ embryos, greatly increased fibrin deposition was detected around these cells. To prevent this fibrin deposition, EPCR ؉/؊ -crossed female mice received a daily subcutaneous injection of enoxaparin through pregnancy. Although some EPCR ؊/؊ embryos were rescued from midgestational lethality, this regimen yielded no EPCR ؊/؊ pups. We conclude that EPCR is essential for normal embryonic development. Moreover, EPCR plays a key role in preventing thrombosis at the maternal-embryonic interface.

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Identification of functional endothelial protein C receptor in human plasma.

Cited by 156 publications

References 48 publications

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

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

Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

Disruption of the Endothelial Cell Protein C Receptor Gene in Mice Causes Placental Thrombosis and Early Embryonic Lethality

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