The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells

AbstractTissue factor (TF)-positive microvesicles from various sources can promote cellular proliferation or alternatively induce apoptosis, but the determining factors are unknown. In this study the hypothesis that the ratio of fVIIa:TF within microvesicles determines this outcome was examined. Microvesicles were isolated from HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7 cell lines and microvesicle-associated fVIIa and TF antigen and activity levels were measured. Human coronary artery endothelial cells (HCAECs) were incubated with these purified microvesicles, or with combinations of fVIIa-recombinant TF, and cell proliferation/apoptosis was measured. Additionally, by expressing mCherry-PAR2 on HCAEC surface, PAR2 activation was quantified. Finally, the activation of PAR2 on HCAEC or the activities of TF and fVIIa in microvesicles were blocked prior to addition of microvesicles to cells. The purified microvesicles exhibited a range of fVIIa:TF ratios with HepG2 and 786-O cells having the highest (54:1) and lowest (10:1) ratios, respectively. The reversal from proapoptotic to proliferative was estimated to occur at a fVIIa:TF molar ratio of 15:1, but HCAEC could not be rescued at higher TF concentrations. The purified microvesicles induced HCAEC proliferation or apoptosis according to this ruling. Blocking PAR2 activation on HCAEC, or inhibiting fVIIa or TF-procoagulant function on microvesicles prevented the influence on HCAEC. Finally, incubation of HCAEC with recombinant TF resulted in increased surface exposure of fVII. The induction of cell proliferation or apoptosis by TF-positive microvesicles is dependent on the ratio of fVIIa:TF and involves the activation of PAR2. At lower TF concentrations, fVIIa can counteract the proapoptotic stimulus and induce proliferation.

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Abstract 190: Purinergic Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP-1 Site and Negative Regulator

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.190 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Yiwei Liu ◽

Lingxin Zhang ◽

Chuan Wang ◽

Shama Roy ◽

Jianzhong Shen

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Tissue Factor ◽

Promoter Activity ◽

Purinergic Receptor ◽

Consensus Sequence ◽

Negative Regulator ◽

Coagulation Cascade ◽

Human Coronary Artery

Previously we reported that the P2Y2 receptor (P2Y2R) is one of the predominant purinergic receptors expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here we report a role of a newly identified AP-1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP-1 site at -1363 bp of human TF promoter region is highly conserved across multiple species. P2Y2R activation increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this new distal AP-1 site all significantly supressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2 and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2, but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation, leading to Fra-1 activation while JNK activated c-Jun and ATF-2. These findings reveal the basis for P2Y purinergic receptor regulation of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.

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IL-33 / ST2 Signaling Promotes TF Expression by Regulating NF-κB Activation in Coronary Artery Endothelial Microparticles

Archives of Medical Science ◽

10.5114/aoms/137377 ◽

2021 ◽

Author(s):

Yujuan Yuan ◽

Hui Cheng ◽

Jing Tao ◽

Muyesai Nijiati

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Tissue Factor ◽

Stable Coronary Artery Disease ◽

Dimethyl Fumarate ◽

Human Coronary Artery ◽

Roc Curve Analysis ◽

Endothelial Microparticles ◽

Artery Disease ◽

St2 Receptor

IntroductionInterleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial Microparticles(EMPs). Tissue factor (TF) plays a central role in hemostasis and thrombosis.Material and methodsThe study analyzed the coronary blood of level of CD31+EMPs, TF protein and IL-33 protein in Acute Myocardial Infarction (AMI) and stable coronary artery disease (SCAD) patients. Human coronary artery endothelial cells (HCAECs) were treated with IL-33 to obtain EMPs. The TF activity of EMPs was tested by Thermo Fisher by adding the TF antibody. Furthermore, TF and Tissue Factor Pathway Inhibitor (TFPI) protein were tested by ELISA. Finally, NF-κB inhibitor dimethyl fumarate (DMF) and soluble extracellular domain of ST2 coupled to the Fc fragment of human IgG1 (sST2) were added to HCAECs which were treated with IL-33, and the TF protein level was also tested by ELISA.ResultsThe AMI patients had higher level of CD31+EMPs, TF protein and IL-33 protein than the SCAD patients in coronary artery. In AMI patients (N=27), the IL-33 protein positively correlated with CD31+EMPs (r=0.794, p<0.01). According to the ROC curve analysis, the AUC of CD31+EMPs, TF protein and IL-33 protein were 0.888, 0.962 and 0.778 respectively. In the cell culture, the TF activity and TF protein in EMPs increased gradually with time of intervention by the treatment of IL-33. IL-33 binding to the ST2 receptor promoted TF expression by regulating NF-κB activation in EMPs of HCAECs.ConclusionsActivated endothelial cells and EMPs they released simultaneously express TF, which is a risk factor for cardiovascular disease.

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TLR9 signaling regulates tissue factor and tissue factor pathway inhibitor expression and activity in human coronary artery endothelial cells

The FASEB Journal ◽

10.1096/fasebj.26.1_supplement.835.4 ◽

2012 ◽

Vol 26 (S1) ◽

Author(s):

Driss El Kebir ◽

Janos G Filep

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Human Coronary Artery ◽

Tissue Factor Pathway ◽

Tlr9 Signaling ◽

Expression And Activity

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The adipokine visfatin induces tissue factor expression in human coronary artery endothelial cells

Thrombosis Research ◽

10.1016/j.thromres.2012.06.007 ◽

2012 ◽

Vol 130 (3) ◽

pp. 403-408 ◽

Cited By ~ 11

Author(s):

Plinio Cirillo ◽

Vito Di Palma ◽

Fabio Maresca ◽

Francesco Pacifico ◽

Francesca Ziviello ◽

...

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Tissue Factor ◽

Tissue Factor Expression ◽

Human Coronary Artery ◽

Factor Expression

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Adipocytokine resistin induces tissue factor expression in human coronary artery endothelial cells via NF kB-dependent pathway

European Heart Journal ◽

10.1093/eurheartj/eht310.4370 ◽

2013 ◽

Vol 34 (suppl 1) ◽

pp. 4370-4370

Author(s):

P. Calabro ◽

L. Riegler ◽

P. Cirillo ◽

F. Fimiani ◽

G. Limongelli ◽

...

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Tissue Factor ◽

Tissue Factor Expression ◽

Human Coronary Artery ◽

Factor Expression ◽

Dependent Pathway

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Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m115.681163 ◽

2015 ◽

Vol 291 (4) ◽

pp. 1553-1563 ◽

Cited By ~ 6

Author(s):

Yiwei Liu ◽

Lingxin Zhang ◽

Chuan Wang ◽

Shama Roy ◽

Jianzhong Shen

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Tissue Factor ◽

Promoter Activity ◽

Consensus Sequence ◽

Coagulation Cascade ◽

Loss Of Function ◽

P2y2 Receptor ◽

Human Coronary Artery

We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at −1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.

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