scholarly journals Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor

2003 ◽  
Vol 373 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Laurent O. MOSNIER ◽  
John H. GRIFFIN
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protease Activated Receptor 1 ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Dose Dependent

In a model of staurosporine-induced apoptosis using EAhy926 endothelial cells, inhibition of apoptosis by activated protein C was dose-dependent and required the enzyme's active site, implicating activated protein C-mediated proteolysis. Consistent with this implication, both protease-activated receptor-1 (PAR-1) and endothelial cell protein C receptor (EPCR) were required for the anti-apoptotic effects of activated protein C.

Signaling by Activated Protein C in Endothelial Cells Is PAR1 Dependent but Distinct from Thrombin Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 221-221 ◽  
Author(s):  
Matthias Riewald ◽  
Clemens Feistritzer ◽  
Wolfram Ruf
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Biological Effects ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protein Levels ◽  
Thrombospondin 1 ◽  
Anti Inflammatory

Abstract Thrombin initiates fibrin formation and platelet activation, and activates protein C, generating activated protein C (APC) that inhibits blood coagulation by a negative feedback loop. Thrombin has also proinflammatory effects through activation of cellular protease activated receptor-1 (PAR1). Endothelial cell protein C receptor (EPCR) can bind both protein C and APC and activation of EPCR-bound protein C is enhanced. Results from animal models and clinical trials indicate that APC has potent protective effects in systemic inflammation that are independent from its well established anticoagulant function and recombinant APC was recently approved to treat patients with severe sepsis. The molecular basis for APC’s anti-inflammatory effects is incompletely understood. We have identified PAR1 and EPCR as part of a novel APC signaling pathway in quiescent endothelial cells, raising the question how the same receptor PAR1 can mediate both pro- and anti-inflammatory effects. In an overexpression system in PAR-deficient fibroblasts, wildtype PAR2 but not a PAR2 variant with an Arg36 to Ala substitution at the P1 position was activated by APC, indicating that APC can activate PAR2 in addition to PAR1 through a canonical cleavage mechanism. Therefore, we tested whether endothelial cell PAR2 can be activated by APC under conditions where endogenous PAR2 expression is upregulated. Even when PAR2 expression was highly upregulated in inflammatory cytokine-stimulated human umbilical endothelial cells (HUVECs), signaling by APC was strictly dependent on PAR1 cleavage and signaling. Consistent with these results in HUVECs, intravenous injection of APC in wildtype, PAR1−/−, and PAR2−/− mice demonstrated that PAR1 is the major murine receptor that mediates induction of the transcript for monocyte chemoattractant protein-1 in the lung in response to APC. This indicates that indeed the same receptor PAR1 mediates signaling by APC and thrombin both in vitro and in vivo. To test the possibility that APC diminishes proinflammatory thrombin-PAR1 signaling by downregulating cellular levels of functional PAR1, we tested whether preincubation with APC can desensitize Erk1/2 phosphorylation by thrombin. Phospho-Erk1/2 was induced by APC dependent upon PAR1 cleavage, but APC-pretreated cells still responded to PAR1-dependent thrombin signaling, suggesting that only a fraction of the cellular PAR1 is subject to cleavage by APC. These results indicate that APC does not block thrombin signaling by desensitation at the receptor level. Large-scale gene expression profiling demonstrated that APC and thrombin had specific effects on gene expression in tumor necrosis factor α (TNFα )-perturbed endothelial cells that were not detected in quiescent cells. Transcripts for several proapoptotic genes including p53 and thrombospondin-1 were downregulated by APC but not by thrombin or PAR1 agonist peptides in TNFα-stimulated HUVECs. Western blotting confirmed that in TNFα-perturbed HUVECs pretreatment with APC significantly reduced the increase in cellular p53 protein levels in response to the cytotoxic doxorubicin. This APC effect was dependent on EPCR binding and PAR1 cleavage. Thrombospondin-1 protein levels were similarly downregulated by APC but upregulated by thrombin. Both down- and upregulation by APC and thrombin, respectively, were PAR1 dependent. These findings demonstrate that the same receptor on the same cell type can mediate opposite biological effects and they suggest that EPCR cosignaling may modify PAR1-dependent APC signaling in endothelial cells.

scholarly journals Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3199-3208 ◽  
Author(s):  
Prosenjit Sen ◽  
Ramakrishnan Gopalakrishnan ◽  
Hema Kothari ◽  
Shiva Keshava ◽  
Curtis A. Clark ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Signaling ◽  
Protective Effect ◽  
Protein C ◽  
Factor Viia ◽  
Mitogen Activated Protein Kinase ◽  
Cell Protein ◽  
Mapk Activation ◽  
Protease Activated Receptor 1

Abstract Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.

scholarly journals Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 895-901 ◽  
Author(s):  
GM Rodgers ◽  
MT Conn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Vascular Endothelial Cell ◽  
Activated Protein C ◽  
Cell Protein ◽  
Blood Levels ◽  
Factor V ◽  
Elevated Blood ◽  
Cultured Endothelial Cells

Abstract Elevated blood levels of homocysteine are associated with atherosclerosis and thrombotic disease. We previously reported that treatment of cultured endothelial cells with homocysteine increased endogenous factor V activity by activation of the cofactor. Because endothelial cell-associated factor Va would be regulated by the protein C mechanism, the ability of homocysteine-treated arterial and venous endothelial cells to activate protein C was investigated. Both arterial and venous endothelial cells activated protein C; 0.6 mmol/L homocysteine reduced endothelial cell protein C activation by 12%. Maximal inhibition (90%) of protein C activation occurred with 7.5 to 10 mmol/L homocysteine after 6 to 9 hours of incubation. Metabolism of homocysteine was not accelerated by cultured endothelial cells. Investigation of the mechanism(s) by which homocysteine reduced protein C activation indicated that the metabolite did not induce an inhibitor to activated protein C, but in low concentrations acted as a competitive inhibitor to thrombin. These data suggest that perturbation of the vascular endothelial cell protein C mechanism by homocysteine may contribute to the thrombotic tendency seen in patients with elevated blood levels of this metabolite.

scholarly journals Endothelial cell protein C receptor: a multiliganded and multifunctional receptor

Blood ◽  
2014 ◽  
Vol 124 (10) ◽  
pp. 1553-1562 ◽  
Author(s):  
L. Vijaya Mohan Rao ◽  
Charles T. Esmon ◽  
Usha R. Pendurthi
Keyword(s):  
Endothelial Cell ◽  
Protein C ◽  
Factor Viia ◽  
Activated Protein C ◽  
Cell Receptor ◽  
Cell Protein ◽  
Future Research ◽  
Proinflammatory Response ◽  
Therapeutic Modalities ◽  
Protease Activated Receptor 1

Abstract Endothelial cell protein C receptor (EPCR) was first identified and isolated as a cellular receptor for protein C on endothelial cells. EPCR plays a crucial role in the protein C anticoagulant pathway by promoting protein C activation. In the last decade, EPCR has received wide attention after it was discovered to play a key role in mediating activated protein C (APC)-induced cytoprotective effects, including antiapoptotic, anti-inflammatory, and barrier stabilization. APC elicits cytoprotective signaling through activation of protease activated receptor-1 (PAR1). Understanding how EPCR-APC induces cytoprotective effects through activation of PAR1, whose activation by thrombin is known to induce a proinflammatory response, has become a major research focus in the field. Recent studies also discovered additional ligands for EPCR, which include factor VIIa, Plasmodium falciparum erythrocyte membrane protein, and a specific variant of the T-cell receptor. These observations open unsuspected new roles for EPCR in hemostasis, malaria pathogenesis, innate immunity, and cancer. Future research on these new discoveries will undoubtedly expand our understanding of the role of EPCR in normal physiology and disease, as well as provide novel insights into mechanisms for EPCR multifunctionality. Comprehensive understanding of EPCR may lead to development of novel therapeutic modalities in treating hemophilia, inflammation, cerebral malaria, and cancer.

Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 895-901 ◽  
Author(s):  
GM Rodgers ◽  
MT Conn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Vascular Endothelial Cell ◽  
Activated Protein C ◽  
Cell Protein ◽  
Blood Levels ◽  
Factor V ◽  
Elevated Blood ◽  
Cultured Endothelial Cells

Elevated blood levels of homocysteine are associated with atherosclerosis and thrombotic disease. We previously reported that treatment of cultured endothelial cells with homocysteine increased endogenous factor V activity by activation of the cofactor. Because endothelial cell-associated factor Va would be regulated by the protein C mechanism, the ability of homocysteine-treated arterial and venous endothelial cells to activate protein C was investigated. Both arterial and venous endothelial cells activated protein C; 0.6 mmol/L homocysteine reduced endothelial cell protein C activation by 12%. Maximal inhibition (90%) of protein C activation occurred with 7.5 to 10 mmol/L homocysteine after 6 to 9 hours of incubation. Metabolism of homocysteine was not accelerated by cultured endothelial cells. Investigation of the mechanism(s) by which homocysteine reduced protein C activation indicated that the metabolite did not induce an inhibitor to activated protein C, but in low concentrations acted as a competitive inhibitor to thrombin. These data suggest that perturbation of the vascular endothelial cell protein C mechanism by homocysteine may contribute to the thrombotic tendency seen in patients with elevated blood levels of this metabolite.

scholarly journals Activated protein C up-regulates procoagulant tissue factor activity on endothelial cells by shedding the TFPI Kunitz 1 domain

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6338-6346 ◽  
Author(s):  
Reto A. Schuepbach ◽  
Kara Velez ◽  
Matthias Riewald
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tissue Factor ◽  
Protein C ◽  
Vascular System ◽  
Apical Cell ◽  
Activated Protein C ◽  
Fine Tuning ◽  
Cell Protein ◽  
Apical Cell Membrane

AbstractThrombin and activated protein C (APC) signaling can mediate opposite biologic responses in endothelial cells. Given that thrombin induces procoagulant tissue factor (TF), we examined how TF activity is affected by APC. Exogenous or endogenously generated APC led to increased TF-dependent factor Xa activity. Induction required APC's proteolytic activity and binding to endothelial cell protein C receptor but not protease activated receptors. APC did not affect total TF antigen expression or the availability of anionic phospholipids on the apical cell membrane. Western blotting and cell surface immunoassays demonstrated that APC sheds the Kunitz 1 domain from tissue factor pathway inhibitor (TFPI). A TFPI Lys86Ala mutation between the Kunitz 1 and 2 domains eliminated both cleavage and the enhanced TF activity in response to APC in overexpression studies, indicating that APC up-regulates TF activity by endothelial cell protein C receptor-dependent shedding of the Kunitz 1 domain from membrane-associated TFPI. Our results demonstrate an unexpected procoagulant role of the protein C pathway that may have important implications for the regulation of TF- and TFPI-dependent biologic responses and for fine tuning of the hemostatic balance in the vascular system.

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

2020 ◽  
Vol 40 (5) ◽  
pp. 1275-1288 ◽  
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.

In Vitro Protection of Human Endothelial Cells from Adenovirus Vector Toxicity by Activated Protein C.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3694-3694
Author(s):  
Jay Nelson Lozier ◽  
Felice D’Agnillo
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Endothelial Cell ◽  
Protein C ◽  
First Generation ◽  
Activated Protein C ◽  
Adenovirus Vector ◽  
Dose Dependent

Abstract High dose adenovirus vector administration in vivo has been associated with toxicity toward many cell types, including endothelial cells. Some of the prominent pathological features of an adenovirus vector death in a gene therapy trial included capillary leak syndrome and disseminated intravascular coagulation (DIC). We investigated the hypothesis that activated protein C (APC) might have a protective effect on primary human microvascular endothelial cells exposed to a first-generation adenovirus vector. We exposed primary human endothelial cells to a first-generation (E1, E3 deleted) adenovirus vector, AVC3FIX5 at concentrations ranging from 103 to 105 vector particles per cell and showed dose-dependent cell death as early as 6 hours (40% cell death at the highest dose). Phase contrast and immunofluorescence microscopy revealed that some cells died rapidly by primary necrosis while others died by apoptosis over a longer time course. By 40 hours, only 40% of the cells were viable. We then tested the effect of pretreatment of endothelial cells with APC concentrations ranging from 1 nM to 100 nM. Dose-dependent protection was seen in which cell death was reduced to 9 and 2 % at APC concentrations of 50 and 100 nM, respectively. We also tested the effect of timing of the APC treatment and showed that 1 hour pre-treatment or concurrent APC treatment were protective, but APC administered one hour after the adenovirus exposure was substantially less protective. This suggested that APC exerts its protective actions on endothelial cells either by interfering with early steps in the interaction of the vector with the cells, (e.g., vector entry) or by modulating death signaling pathways. It has been proposed that APC protects against cell damage in sepsis by interaction with the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1) on the endothelial cell surface to induce MCP-1 and other immunomodulatory genes by proteolytic signaling (Riewald et al., Science296:1880–1882, 2002). Other investigators have shown protective effects of APC for endothelial cells subjected to hypoxia through normalization of levels of p53, Bax, and Bcl-2 gene expression (Cheng et al., Nat Med9:338–342, 2003). The APC concentrations in our experiments that were maximally protective (50–100 nM) were of the same order of magnitude as was shown to be protective in vitro by these investigators. If APC can be shown to have a protective effect against adenovirus-induced endothelial cell toxicity and DIC in vivo, this may be a useful therapeutic strategy to explore as treatement of gene therapy vector toxicity. Cell Viability vs. APC Concentration Cell Viability vs. APC Concentration

Activated Protein C Cytoprotective Signaling in Endothelial Cells Involves apoER2 and Disabled-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1102-1102
Author(s):  
Ranjeet Kumar Sinha ◽  
Xia Yang ◽  
Laurent O. Mosnier ◽  
John H. Griffin
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Signaling Pathway ◽  
Protein C ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Activated Protein C ◽  
Adaptor Protein ◽  
Src Family Kinases ◽  
Cell Protein

Abstract Abstract 1102 Activated protein C (APC), a plasma serine protease, exerts both anticoagulant and cytoprotective signaling in vitro and in vivo. APC protects cells from apoptosis, stress, and inflammation; it also can reduce or prevent endothelial barrier leakage as well as murine death from lethal levels of radiation. APC induces selective alterations of gene expression, provides neuroprotection following ischemic stroke, and minimizes damage in chronic neurodegenerative disease animal models. When given as bolus doses, APC reduces death in animals from severe sepsis. Receptors that mediate APC-induced beneficial signaling include endothelial cell protein C receptor (EPCR), PAR1, PAR3, integrins and Tie2. We discovered that apolipoprotein E receptor 2 (ApoER2) (aka LRP8), a lipoprotein receptor, which is well known for its effects in the Reelin-signaling pathway for neurons, has remarkable APC-dependent cytoprotective actions for the monocytic U937 cell line. In neurons, ligation of ApoER2 triggers phosphorylation of disabled-1 (Dab1) by Src family kinases that further promulgate signaling via PI3K, Akt and GSK3beta. Thus, here we report studies that test and support the hypothesis that ApoER2 mediates APC-induced cytoprotective signaling in EA.hy926 endothelial cells, which involves the adaptor protein, Dab1, as well as Src family members, Akt, and GSK3beta. We used siRNA and inhibitors to probe requirements for specific receptors for phosphorylation of key signaling pathway components. APC treatment of endothelial cells induced a 2 to 3-fold increase in phosphorylation of Akt at Ser473 and GSK3beta at Ser9 over 30 to 180 min after APC treatment, as well as a substantial rapid increase in ERK1/2 phosphorylation within 5 min of APC treatment. In purified systems, APC binds with good affinity to ApoER2. APC treatment of endothelial cells caused phosphorylation of Dab1 at Tyr232, a phosphorylation known to trigger Dab1-mediated signaling in other types of cells. Knocking down of ApoER2 using siRNA reduced GSK3beta and ERK1/2 phosphorylation by circa 30–40%. Combined knockdown of EPCR and ApoER2 reduced GSK3beta and ERK1/2 phosphorylation by 50–75%. Additional studies implicated involvement of PI3K and Src kinases. When cells were pretreated with PP2, a Src kinase inhibitor, Y232-Dab1 phosphorylation was reduced by > 50%, consistent with the primary paradigm for apoER2-mediated signaling involving phosphorylations of Dab1 and Src-family kinases. Moreover, the PAR1 antagonist SCH79797 markedly reduced APC-induced phosphorylation of Ser473-Akt, Ser9-GSK3beta and ERK1/2, showing that PAR1 was also required for the full extent of these effects. Hence, both PAR1 proteolysis by APC and ligation of ApoER2 by APC appear to contribute significantly and simultaneously to signaling in this endothelial model system. In summary, these data indicate that apoER2 is an endothelial cell receptor that contributes, with other better recognized receptors, to the spectrum of APC-induced signaling that is integrated by the endothelial cell system to achieve one or more of the multiple cytoprotective effects of APC. Thus, there is a remarkable ensemble of potential endothelial receptors for APC that could variably mediate a spectrum of APC's cytoprotective effects. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distribution of Endothelial Cell Protein C/Activated Protein C Receptor (EPCR) During Mouse Embryo Development

2002 ◽  
Vol 88 (08) ◽  
pp. 259-266 ◽  
Author(s):  
James Crawley ◽  
Jian-Ming Gu ◽  
Gary Ferrell ◽  
Charles Esmon
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Activated Protein C ◽  
Fluorescein Isothiocyanate ◽  
Giant Cells ◽  
Maternal Blood ◽  
Cell Protein ◽  
Aortic Endothelial Cells ◽  
Adult Mice

SummaryThe endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombomodulin•thrombin complex. Deletion of the EPCR gene in mice has been reported to lead to embryonic lethality before embryonic day 10 (E10.0). To identify potential mechanisms responsible for this lethality, we performed an immunohistological analysis of EPCR distribution during mouse embryogenesis. EPCR was detected in the trophoblast giant cells at the feto-maternal boundary from E7.5 and at later time points in the trophoblasts of the placenta, suggesting a role in the haemostatic regulation of the maternal blood that irrigates these surfaces. In the embryo, EPCR was weakly detected in aortic endothelial cells from E13.5. Thereafter, EPCR levels increased in certain large blood vessel endothelial cells suggesting that the specificity of EPCR to large vessels is conferred in utero. However, not until postnatal day 7 did the intensity and distribution of EPCR staining mimic that observed in adult mice.Abbreviations: APC – activated protein C, AP – alkaline phosphatase, EC – endothelial cell, EPCR – endothelial cell protein C/activated protein C receptor, FITC – fluorescein isothiocyanate, HRP – horseradish peroxidase, PBS – phosphate-buffered saline, TM – thrombomodulin, WT – wild–type

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