A novel protein C–factor VII chimera provides new insights into the structural requirements for cytoprotective protease‐activated receptor 1 signaling

Gleeson, Eimear M.; McDonnell, Cormac J.; Soule, Erin E.; Fox, Orla Willis; Rushe, H.; Rehill, Aisling M.; Smith, Owen; O’Donnell, James S.; Preston, Roger J. S.

doi:10.1111/jth.13807

Cited by 8 publications

(17 citation statements)

References 35 publications

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“…Although aPC bound to EPCR is the prototypical PAR1 activator and the FVIIa light chain cannot substitute for the same region in aPC in signaling , FVIIa in complex with EPCR also elicits TF‐independent PAR1 signaling in endothelial cells . In contrast, the binary TF‐FVIIa complex activates PAR2 and has been linked to a variety of pathological conditions.…”

Section: Tf‐fviia and Intracellular Signalingmentioning

confidence: 99%

Tissue factor at the crossroad of coagulation and cell signaling

Zelaya

Rothmeier

Ruf

2018

Journal of Thrombosis and Haemostasis

117

115

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The tissue factor (TF) pathway plays a central role in hemostasis and thrombo-inflammatory diseases. Although structure-function relationships of the TF initiation complex are elucidated, new facets of the dynamic regulation of TF's activities in cells continue to emerge. Cellular pathways that render TF non-coagulant participate in signaling of distinct TF complexes with associated proteases through the protease-activated receptor (PAR) family of G protein-coupled receptors. Additional co-receptors, including the endothelial protein C receptor (EPCR) and integrins, confer signaling specificity by directing subcellular localization and trafficking. We here review how TF is switched between its role in coagulation and cell signaling through thiol-disulfide exchange reactions in the context of physiologically relevant lipid microdomains. Inflammatory mediators, including reactive oxygen species, activators of the inflammasome, and the complement cascade play pivotal roles in TF procoagulant activation on monocytes, macrophages and endothelial cells. We furthermore discuss how TF, intracellular ligands, co-receptors and associated proteases are integrated in PAR-dependent cell signaling pathways controlling innate immunity, cancer and metabolic inflammation. Knowledge of the precise interactions of TF in coagulation and cell signaling is important for understanding effects of new anticoagulants beyond thrombosis and identification of new applications of these drugs for potential additional therapeutic benefits.

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Section: Tf‐fviia and Intracellular Signalingmentioning

confidence: 99%

Tissue factor at the crossroad of coagulation and cell signaling

Zelaya

Rothmeier

Ruf

2018

Journal of Thrombosis and Haemostasis

117

115

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“…24 Furthermore, residues in the GLA and EGF1 domain of APC were implicated in the interaction of APC with PAR1. 31 Thus, extending the scope of APC's interactive surfaces beyond its protease domain seems well warranted and one region of APC that has been relatively overlooked for structure-function analysis is the C-terminal region of the light chain.…”

Section: Introductionmentioning

confidence: 99%

C‐terminal residues of activated protein C light chain contribute to its anticoagulant and cytoprotective activities

Yamashita

Zhang

Sanner

et al. 2020

Journal of Thrombosis and Haemostasis

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Background: Activated protein C (APC) is an important homeostatic blood coagulation protease that conveys anticoagulant and cytoprotective activities. Proteolytic inactivation of factors Va and VIIIa facilitated by cofactor protein S is responsible for APC's anticoagulant effects, whereas cytoprotective effects of APC involve primarily the endothelial protein C receptor (EPCR), protease activated receptor (PAR)1 and PAR3.Objective: To date, several binding exosites in the protease domain of APC have been identified that contribute to APC's interaction with its substrates but potential contributions of the C-terminus of the light chain have not been studied in detail. Methods:Site-directed Ala-scanning mutagenesis of six positively charged residues within G142-L155 was used to characterize their contributions to APC's anticoagulant and cytoprotective activities.Results and Conclusions: K151 was involved in protein S dependent-anticoagulant activity of APC with some contribution of K150. 3D structural analysis supported that these two residues were exposed in an extended protein S binding site on one face of APC. Both K150 and K151 were important for PAR1 and PAR3 cleavage by APC, suggesting that this region may also mediate interactions with PARs. Accordingly, APC's cytoprotective activity as determined by endothelial barrier protection was impaired by Ala substitutions of these residues. Thus, both K150 and K151 are involved in APC's anticoagulant and cytoprotective activities. The differential contribution of K150 relative to K151 for protein S-dependent anticoagulant activity and PAR cleavage highlights that binding exosites for protein S binding and for PAR cleavage in the C-terminal region of APC's light chain overlap. K E Y W O R D S activated protein C, anticoagulant activity, cytoprotective activity, protease activated receptor, protein S S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section.

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“…However, studies conducted in EA.hy26 cells failed to show that FVIIa could prevent thrombin-induced enhanced permeability. 15 Recent studies by Gleeson et al 16 showed that an APC chimeric with an FVIIa-gla domain failed to mediate the EPCR-and PAR1dependent barrier protective effect, indicating that amino acid residues other than the EPCR binding site in APC were necessary for cytoprotective PAR1 signaling. It is unclear at present the reason for these differences in FVIIa's ability to provide a barrierprotective effect in the above studies, but it may reflect differences in endothelial cell types or reagents used in these studies.…”

Section: Introductionmentioning

confidence: 99%

Factor VIIa induces anti-inflammatory signaling via EPCR and PAR1

Kondreddy

Wang

Keshava

et al. 2018

Blood

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Recent studies show that endothelial cell protein C receptor (EPCR) interacts with diverse ligands, in addition to its known ligands protein C and activated protein C (APC). We showed in earlier studies that procoagulant clotting factor VIIa (FVIIa) binds EPCR and downregulates EPCR-mediated anticoagulation and induces an endothelial barrier protective effect. Here, we investigated the effect of FVIIa's interaction with EPCR on endothelial cell inflammation and lipopolysaccharide (LPS)-induced inflammatory responses in vivo. Treatment of endothelial cells with FVIIa suppressed tumor necrosis factor α (TNF-α)- and LPS-induced expression of cellular adhesion molecules and adherence of monocytes to endothelial cells. Inhibition of EPCR or protease-activated receptor 1 (PAR1) by either specific antibodies or small interfering RNA abolished the FVIIa-induced suppression of TNF-α- and LPS-induced expression of cellular adhesion molecules and interleukin-6. β-Arrestin-1 silencing blocked the FVIIa-induced anti-inflammatory effect in endothelial cells. In vivo studies showed that FVIIa treatment markedly suppressed LPS-induced inflammatory cytokines and infiltration of innate immune cells into the lung in wild-type and EPCR-overexpressing mice, but not in EPCR-deficient mice. Mechanistic studies revealed that FVIIa treatment inhibited TNF-α-induced ERK1/2, p38 MAPK, JNK, NF-κB, and C-Jun activation indicating that FVIIa-mediated signaling blocks an upstream signaling event in TNFα-induced signaling cascade. FVIIa treatment impaired the recruitment of TNF-receptor-associated factor 2 into the TNF receptor 1 signaling complex. Overall, our present data provide convincing evidence that FVIIa binding to EPCR elicits anti-inflammatory signaling via a PAR1- and β-arrestin-1 dependent pathway. The present study suggests new therapeutic potentials for FVIIa, which is currently in clinical use for treating bleeding disorders.

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A novel protein C–factor VII chimera provides new insights into the structural requirements for cytoprotective protease‐activated receptor 1 signaling

Cited by 8 publications

References 35 publications

Tissue factor at the crossroad of coagulation and cell signaling

Tissue factor at the crossroad of coagulation and cell signaling

C‐terminal residues of activated protein C light chain contribute to its anticoagulant and cytoprotective activities

Factor VIIa induces anti-inflammatory signaling via EPCR and PAR1

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