Inactivation of Factor VIIa by Antithrombin In Vitro, Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor

SummaryRecent in vitro studies have shown that the zymogen and activated form of factor (F)VII bind to endothelial cell protein C receptor (EPCR). At present, there is no evidence that FVIIa binds to EPCR on vascular endothelium in vivo in the presence of circulating protein C, a primary ligand for EPCR. The present study was carried out to investigate the interaction of murine and human ligands with murine EPCR both in vivo and in vitro. Measurement of endogenous plasma levels of FVII in wild-type, EPCR-deficient and EPCR-over expressing mice showed slightly lower levels of FVII in EPCR-over expressing mice. However, infusion of high concentrations of competing ligands, either human APCi or FVIIai, to EPCR-over expressing mice failed to increase plasma levels of mouse FVII whereas they increased the plasma levels of protein C by two- to three-fold. Examining the association of exogenously administered mouse FVIIa or human FVIIa by immunohistochemistry revealed that human, but not murine FVIIa, binds to the murine endothelium in an EPCR-dependent manner. In vitro binding studies performed using surface plasmon resonance and endothelial cells revealed that murine FVIIa binds murine EPCR negligibly. Human FVIIa binding to EPCR, particularly to mouse EPCR, is markedly enhanced by availability of Mg2+ ions. In summary, our data show that murine FVIIa binds poorly to murine EPCR, whereas human FVIIa binds efficiently to both murine and human EPCR. Our data suggest that one should consider the use of human FVIIa in mouse models to investigate the significance of FVIIa and EPCR interaction.

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Factor VIIa Interaction With Tissue Factor and Endothelial Cell Protein C Receptor on Cell Surfaces

Seminars in Hematology ◽

10.1053/j.seminhematol.2008.03.014 ◽

2008 ◽

Vol 45 ◽

pp. S21-S24 ◽

Cited By ~ 4

Author(s):

Usha R. Pendurthi ◽

L. Vijaya Mohan Rao

Keyword(s):

Endothelial Cell ◽

Tissue Factor ◽

Protein C ◽

Factor Viia ◽

Cell Protein ◽

Cell Surfaces

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O-20 In vitro and in vivo consequences of the sepsis effect on the endothelial cell protein C receptor

Blood Coagulation & Fibrinolysis ◽

10.1097/00001721-200110000-00034 ◽

2001 ◽

Vol 12 (7) ◽

pp. A10-A11

Author(s):

&NA;

Keyword(s):

Endothelial Cell ◽

Protein C ◽

Cell Protein

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Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo

Journal of Thrombosis and Haemostasis ◽

10.1111/jth.12532 ◽

2014 ◽

Vol 12 (5) ◽

pp. 690-700 ◽

Cited By ~ 24

Author(s):

J. Sundaram ◽

S. Keshava ◽

R. Gopalakrishnan ◽

C. T. Esmon ◽

U. R. Pendurthi ◽

...

Keyword(s):

Endothelial Cell ◽

Protein C ◽

Factor Viia ◽

Cell Protein ◽

Barrier Integrity ◽

Vascular Barrier Integrity

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Endothelial cell protein C receptor plays an important role in protein C activation in vivo

Blood ◽

10.1182/blood.v97.6.1685 ◽

2001 ◽

Vol 97 (6) ◽

pp. 1685-1688 ◽

Cited By ~ 188

Author(s):

Fletcher B. Taylor ◽

Glenn T. Peer ◽

Marion S. Lockhart ◽

Gary Ferrell ◽

Charles T. Esmon

Keyword(s):

Monoclonal Antibody ◽

Endothelial Cell ◽

Protein C ◽

Degradation Products ◽

Activated Protein C ◽

Vital Signs ◽

Cell Protein ◽

Increased Risk

Endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex about 5-fold in vitro. Augmentation is EPCR concentration dependent even when the EPCR concentration is in excess of the thrombomodulin. EPCR is expressed preferentially on large blood vessel endothelium, raising questions about the importance of protein C-EPCR interaction for augmenting systemic protein C activation. In these studies, this question was addressed directly by infusing thrombin into baboons in the presence or absence of a monoclonal antibody to EPCR that blocks protein C binding. Activated protein C levels were then measured directly by capturing the enzyme on a monoclonal antibody and assaying with chromogenic substrate. Blocking protein C-EPCR interaction resulted in about an 88% decrease in circulating activated protein C levels generated in response to thrombin infusion. Leukocyte changes, fibrinogen consumption, fibrin degradation products, and vital signs were similar between the animals infused with thrombin alone and those infused with thrombin and the anti-EPCR antibody. The results indicate that EPCR plays a major role in protein C activation and suggest that defects in the EPCR gene might contribute to increased risk of thrombosis.

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FVIIa (Factor VIIa) Induces Biased Cytoprotective Signaling in Mice Through the Cleavage of PAR (Protease-Activated Receptor)-1 at Canonical Arg41 (Arginine41) Site

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314244 ◽

2020 ◽

Vol 40 (5) ◽

pp. 1275-1288 ◽

Cited By ~ 3

Author(s):

Vijay Kondreddy ◽

Usha R. Pendurthi ◽

Xiao Xu ◽

John H. Griffin ◽

L. Vijaya Mohan Rao

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Protein C ◽

Factor Viia ◽

Vascular Endothelial Cell ◽

Cell Protein ◽

Mouse Strains ◽

Biased Agonism ◽

Protease Activated Receptor 1

Objective: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1–mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1. Conclusions: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.

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The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000116 ◽

2012 ◽

Vol 82 (3) ◽

pp. 228-232 ◽

Cited By ~ 7

Author(s):

Mauro Serafini ◽

Giuseppa Morabito

Keyword(s):

Antioxidant Capacity ◽

Dietary Intervention ◽

Ex Vivo ◽

The Body ◽

Dietary Polyphenols ◽

Enzymatic Antioxidant ◽

Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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Organische Nitrate und Thrombozytenfunktion

Hämostaseologie ◽

10.1055/s-0038-1659867 ◽

1988 ◽

Vol 08 (02) ◽

pp. 90-99 ◽

Cited By ~ 7

Author(s):

H. Schröder ◽

K. Schrör

Keyword(s):

Endothelial Cell ◽

Angina Pectoris ◽

Ex Vivo

ZusammenfassungOrganische Nitrate unterschiedlicher chemischer Struktur sowie Nitroprussidnatrium und Molsidomin (bzw. ihre biologisch aktiven Metaboliten) können die (primäre) Aggregation und Sekretion von Humanthrombozyten in vitro und ex vivo hemmen. Eine solche Wirkung wird für Molsidomin (SIN-1) und Nitroprussidnatrium in vitro in Konzentrationen beobachtet, die in der gleichen Größenordnung liegen wie die vasodilatierenden Effekte der Substanzen. Dagegen sind für eine direkte Antiplättchenwirkung organischer Nitrate (Glyzeryltrinitrat, Isosorbiddinitr at, Isosorbidmononitrate, Teopranitol) in vitro Konzentrationen erforderlich, die ca. 100- bis 1000fach höher sind als die Plasmaspiegel der Substanzen nach therapeutischer Dosierung bzw. die Konzentrationen, die isolierte Gefäßstreifen relaxieren. Als gemeinsamer Wirkungsmechanismus der direkten thrombozy-tenfunktionshemmenden und gefäßerweiternden Wirkung all dieser Substanzen kann heute eine Stickoxid-(NO)-vermittelte Stimulation der cGMP-Bildung angenommen werden, das aus organischen Nitraten als »Pro-drug« entsteht. Die Freisetzung von NO, eines »endothelial cell-derived relaxing factors« (EDRF) aus Nitroprussidnatrium und SIN-1 erfolgt spontan. Dagegen erfordert die Freisetzung von NO aus organischen Nitraten einen enzymatischen Stoffwechselweg, der in isolierten Thrombozyten nicht vorhanden ist. Eine Antiplättchenwirkung organischer Nitrate in vivo bzw. ex vivo wird daher über die Stimulation eines endothelialen, thrombozyteninhibitorischen Faktors erklärt. Hierbei sind Prostazyklin sowie ein bisher unbekannter Endothel-zellfaktor neben einer synergistischen Wirkung organischer Nitrate mit endogenem Prostazyklin in Diskussion. Eine thrombozytenfunktionshemmen-de Wirkung organischer Nitrate könnte in Kombination mit ihren hämody-namischen Effekten auch für die an-tianginöse Wirkung in der Klinik bedeutsam sein, insbesondere zur Verhinderung vasospastischer Zustände bei der instabilen Angina pectoris.

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