Cytoplasmic Domain Of Tissue Factor Research Articles

Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development

AbstractAntiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. Although autoimmune aPL cross react with various coagulation regulatory proteins, lipid-reactive aPL, including those derived from patients with COVID-19, recognize the endolysosomal phospholipid lysobisphosphatidic acid presented by the cell surface–expressed endothelial protein C receptor. This specific recognition leads to complement-mediated activation of tissue factor (TF)–dependent proinflammatory signaling and thrombosis. Here, we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of aPL derived from patients with COVID-19 in mice and the aPL–induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and interferon-α secretion from dendritic cells. Latent infection with murine cytomegalovirus causes TF cytoplasmic domain–dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short-term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic-cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells, and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

Targeting myeloid cell coagulation signaling blocks MAP kinase/TGF-β1-driven fibrotic remodeling in ischemic heart failure.

Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-β1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis

Abstract Introduction The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and implicated in cardiac injuries and dysfunction, the contribution of its cytoplasmic domain in cardiac pathology remains unclear. Purpose We aimed to investigate the contribution of the cytoplasmic domain of TF to post-infarct myocardial injury and adverse left ventricular (LV) remodeling. Methods and results Myocardial infarction was induced by permanent occlusion of the left anterior descending coronary artery. Male mice with C57BL/Jax background were used for the study. Compared with wild-type mice, mice lacking the TF cytoplasmic domain (TFΔCT) had a higher survival rate (90.5% versus 70%, p=0.0298) during a 28-day follow-up after myocardial infarction. Among surviving mice, TFΔCT mice had better cardiac function and less LV remodeling (ESV: 114.5±13.1mL for WT, 67.06±10.8mL for TFΔCT, p<0.001; EDV: 146.6±12.4mL for WT, 99.97±11.71mL for TFΔCT, p<0.001) than wild-type mice. Bone marrow chimerism indicated that deletion of the TF cytoplasmic domain in either bone marrow-derived cells or cardiac resident cells could alleviate post-infarct cardiac dysfunction. Speckle-tracking strain analysis revealed that the overall improvement of post-infarct cardiac performance in TFΔCT mice was attributed to reduced myocardial deformation in the peri-infarct region (strain-%: 11.14±0.97 for WT, 15.34±1.10 for TFΔCT, p=0.007; strain rate-/s: 3.89±0.26 for WT, 5.18±0.21 for TFΔCT, p=0.0005). Histological analysis demonstrated that TFΔCT hearts had in the infarct area greater proliferation of endothelial cells and myofibroblasts accompanied with better scar formation. Compared with wild-type hearts, infarcted TFΔCT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1)-Rac1 axis. Furthermore, infarcted TFΔCT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Conclusions Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): National University Health System of Singapore

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis.

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown.Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF∆CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis.Results: Compared with wild-type mice, TF∆CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF∆CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF∆CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF∆CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF∆CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) - Rac1 axis. In particular, infarcted TF∆CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF∆CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF∆CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps.Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.

Tissue factor cytoplasmic tail regulates myeloid cell derived superoxide formation and TGF-beta 1 driven cardiac remodeling in myocardial infarction

Abstract Background In the setting of myocardial infarction (MI), patients with coronary no-reflow and/or delayed presentation after onset of symptoms (sub-acute MI) are inflicted by severe thrombo-inflammation and are marked by worse clinical outcome. However, it is unclear whether tissue factor (TF) contributes to outcome post MI solely by the regulatory functions of its cytoplasmic tail independently of its coagulation activity. Purpose We analyzed the role of the TF cytoplasmic domain in the recruitment of myeloid cells into the infarcted myocardium and the consequences on cardiac remodeling, scar formation, development of heart failure and survival post MI. Methods Twelve Patients enrolled in the MICAT (Mainzer Intracoronary Database, ClinicalTrials.gov Identifier: NCT02180178) study were examined. Patients with sub-acute MI and stable coronary artery disease were defined and monocytes were isolated from peripheral blood mononuclear cells (PBMCs). Human heart samples acquired from the left ventricular wall of explanted hearts following cardiac transplantation or obtained during implantation of left ventricular assist device. Samples were investigated for downstream analysis of protein by western blots, RNA quantification and cryo-sectioning. MI was induced in 9 to 12 weeks old male C57BL/6J mice, mice specifically lacking the cytoplasmic tail (CT) of TF (TFΔCT mice) and TFfl/flLysMCre+/− mice by permanent ligation of the left anterior descending artery. Left ventricular function was assessed by High-Frequency Ultrasound System. Infiltration of immune cells into the infarcted myocardium was analyzed by performing flow cytometric analysis after enzymatic digestion of the myocardium. Superoxide levels were quantified by HPLC-based measurement of dihydroethidium derived oxidation product 2-hydroxy ethidium. Results Circulating monocytes in patients with sub-acute MI showed increased nitrosative stress as well as increased phosphorylation of TF CT along with TGF-β1 and NF-kB inflammatory activation, which was recapitulated in cardiac tissue of end-stage heart failure patients with chronic MI. MI results in phosphorylation of the CT of TF within myeloid cells. Using mice with conditional knockout of TF on myeloid cells or TFΔCT mice, we found that this regulatory intracellular domain of TF within myeloid cells is required for cardiac infiltration of inflammatory Ly6Chigh TF+ monocytes, Rac-1 GTPase and superoxide formation of gp91phox + myeloid cells in MI. TGF-β1 dependent SMAD2 activation and cardiac collagen deposition as late sequel of MI was reduced in TFΔCT mice, resulting in attenuated cardiac dysfunction and reduced mortality. Conclusion We conclude that, TF CT drives NADPH-oxidase derived superoxide formation, thrombo-inflammation and cardiac fibrosis. Therefore, it might serve as putative biomarker and risk predictor in MI Funding Acknowledgement Type of funding source: None

Structural and cellular mechanisms of peptidyl-prolyl isomerase Pin1-mediated enhancement of Tissue Factor gene expression, protein half-life, and pro-coagulant activity.

Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regulate Tissue Factor gene expression and activity. Tissue Factor gene expression is regulated by various transcription factors, including activating protein-1 and nuclear factor-κ B. The peptidyl-prolyl isomerase Pin1 is known to modulate the activity of these two transcription factors, and we now show that Pin1 augments Tissue Factor gene expression in both vascular smooth muscle cells and activated endothelial cells via activating protein-1 and nuclear factor-κ B signaling. Furthermore, the cytoplasmic domain of Tissue Factor contains a well-conserved phospho-Ser258-Pro259 amino-acid motif recognized by Pin1. Using co-immunoprecipitation and solution nuclear magnetic resonance spectroscopy, we show that the WW-domain of Pin1 directly binds the cytoplasmic domain of Tissue Factor. This interaction occurs via the phospho-Ser258-Pro259 sequence in the Tissue Factor cytoplasmic domain and results in increased protein half-life and pro-coagulant activity. Taken together, our results establish Pin1 as an upstream regulator of Tissue Factor-mediated coagulation, thereby opening up new avenues for research into the use of specific Pin1 inhibitors for the treatment of diseases characterized by pathological coagulation, such as thrombosis and atherosclerosis.

Cytoplasmic domain of tissue factor promotes liver fibrosis in mice.

AIMTo evaluate the role of tissue factor (TF) and protease activated receptor (PAR)-2 in liver fibrosis.METHODSUsing CCl4 administration for eight weeks, we induced hepatic fibrosis in wild-type C57BL/6 mice and in mice with deletion of the cytoplasmic signalling domain of TF (TF§CT/§CT), deletion of PAR-2 (PAR-2-/-) and combined deletion of TF signalling domain and PAR-2 (TF§CT/§CT/PAR-2-/-). Hepatic fibrosis area was assessed by quantitative imaging of picrosirius red staining. Hepatic collagen content was assessed by hydroxyproline levels. Hepatic stellate cells (αSMA positive) and hepatic macrophages (CD68 positive) were identified by immunohistochemistry. Hepatic gene expression was determined by PCR and liver TGFβ1 content by ELISA.RESULTSCCl4 treated mice with deletion of the PAR-2 gene (PAR-2-/-) and the cytoplasmic domain of TF (TF§CT/§CT) developed significantly less hepatic fibrosis, characterised by reduced liver fibrosis area and hydroxyproline content, compared to control wildtype mice treated with CCl4. The observed reduction in histological fibrosis was accompanied by a significant decrease in the hepatic content of TGFβ, the prototypic fibrogenic cytokine, as well as fewer activated hepatic stellate cells and hepatic macrophages. Deletion of the TF cytoplasmic signalling domain reduced hepatic fibrosis to levels similar to those observed in mice lacking PAR-2 signalling but combined deletion provided no added protection against fibrosis indicating a lack of mutual modulating effects that have been observed in other contexts such as angiogenic responses.CONCLUSIONTissue factor cytoplasmic domain is involved in TF-PAR-2 signalling initiating hepatic fibrosis and is a potential therapeutic target, as its deletion would not impact coagulation.

Identification Of The Coagulation Factor VIIa Integrin Binding Site Essential For Pro-Angiogenic Tissue Factor Signaling

The cellular initiator of the coagulation cascade, tissue factor (TF), plays pivotal roles in primary hemostasis and wound repair. In vitro evidence point to specific functions of the TF-FVIIa complex in inducing a pro-angiogenic and migratory program in keratinocytes dependent on activation of G protein-coupled signaling of protease activated receptor 2 (PAR2). Accordingly, mice with low levels of FVIIa have delayed cutaneous wound healing and pathological angiogenesis in genetic mouse models of cancer progression is impaired in mice that either lack PAR2 or the TF cytoplasmic domain.Our previous data demonstrated that TF cytoplasmic domain signaling regulates α3β1-dependent migration and that FVIIa promotes the association of TF with β1 integrin heterodimers, but the functional contributions of this interaction to TF-dependent signaling remained unclear. Here, we identified a KGE motif in the FVIIa protease domain that is crucial for FVIIa-dependent complex formation of TF with integrins. Mutation of the negatively charged E26 to A affected neither the catalytic activity of FVIIa, nor significantly FXa generation in a purified system and on cell surfaces. Wild-type FVIIa induced co-immunoprecipitation of TF with keratinocyte-expressed α3β1 integrin. In contrast, FVIIa E26A was severely impaired in promoting association of TF with integrins in a pull-down assay with an antibody that recognized the active conformational state of the integrin β1. Previous transcriptome analysis has indicated that TF-FVIIa produces signaling distinct from activation of PAR2 with agonist peptide. In keratinocytes, wild-type FVIIa induced PI-3 kinase-dependent AKT and ERK phosphorylation, whereas ERK was phosphorylated independent of PI-3 kinase in cells stimulated with the PAR2 agonist SLGIRL. Mutation of FVIIa E26 required for integrin ligation abolished AKT phosphorylation and attenuated ERK phosphorylation, indicating that activation of the PI-3 kinase pathway specifically depends on complex formation of TF with integrins in the context of PAR2 cleavage. Consistently, FVIIa E26A inefficiently supported the PI-3 kinase-, ERK- and PAR2-dependent induction of the pro-angiogenic cytokine IL-8.Mutation of FVIIa E26 similarly reduced complex formation with integrin β1 and attenuated PI-3 kinase- and ERK-dependent IL-8 induction in melanoma cells. Furthermore, the mutation impaired stimulation of migration in melanoma and breast cancer cells. In addition, deletion of the cytoplasmic domain-deleted TF transfected into TF-negative melanoma cells resulted in significantly reduced IL-8 induction by FVIIa in comparison to controls. However, the TF cytoplasmic domain was not required for FVIIa-induced complex formation with integrins, demonstrating direct contributions of the TF cytoplasmic domain to pro-angiogenic signaling. Taken together, these data elucidate the molecular details by which ligand binding promotes complex formation between TF and integrins and provide a novel mechanism of protease selective signaling of a promiscuous PAR in angiogenesis and wound repair. Disclosures:Ostergaard:Novo Nordisk A/S: Employment.

P38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles.

Inflammation, Obesity, and Thrombosis

Abstract SCI-45Clinical and epidemiological studies have long established a connection between obesity and thrombosis leading to increased cardiovascular complications, in part attributed to elevated expression of the prothrombotic molecules plasminogen activator inhibitor 1 and circulating tissue factor (TF). Obesity also increases the risk for metabolic dysfunction, and emerging evidence indicates that coagulation proteases offer a range of signaling pathways that control multiple aspects of the metabolic syndrome, including weight gain, insulin resistance, type 2 diabetes, and hepatic steatosis. While adipose inflammation due to recruitment and activation of macrophages and secretion of inflammatory cytokines promotes systemic hypercoagulability and insulin resistance, pathways that sustain adipose macrophage inflammation in obesity are unclear. Our studies suggest that TF signaling via the G-protein-coupled receptor, protease activated receptor 2 (PAR2), represents a novel link between obesity, coagulation, and associated adipose inflammation and insulin resistance. Mice lacking the cytoplasmic domain of TF or PAR2 were protected from high fat diet-induced weight gain, adipose inflammation, and insulin resistance. Genetic loss of TF cytoplasmic domain and PAR2 in myeloid cells attenuated adipose macrophage inflammation and increased insulin sensitivity, an effect that was also achieved by acute pharmacologic inhibition of TF-PAR2 signaling in macrophages. In contrast, TF signaling in nonhematopoietic cells specifically promoted obesity via its effects on energy metabolism. In adipocytes, TF-VIIa signaling suppressed basal and insulin-mediated AKT phosphorylation, with concordant transcriptional changes in genes regulating lipid and glucose metabolism. Thus, the procoagulant state induced by obesity can in fact contribute to the metabolic syndrome, and adipose TF signaling may be at the intersection between obesity, inflammation, and thrombosis. Disclosures:No relevant conflicts of interest to declare.

Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.

Tissue factor–protease-activated receptor 2 signaling promotes diet-induced obesity and adipose inflammation

Tissue factor (TF), the initiator of the coagulation cascade, mediates coagulation factor VIIa-dependent activation of protease activated receptor-2 (PAR2). Here we delineate an unexpected role for coagulation signaling in obesity and its complications. Mice lacking PAR2 (F2rl1) or the cytoplasmic domain of TF (F3) are protected from high fat diet (HFD) induced weight gain and insulin resistance. In hematopoietic cells, genetic deletion of TF-PAR2 signaling reduces adipose tissue macrophage inflammation and specific pharmacological inhibition of macrophage TF signaling rapidly ameliorates insulin resistance. In contrast, non-hematopoietic cell TF-VIIa-PAR2 signaling specifically promotes obesity. Mechanistically, adipocyte TF cytoplasmic domain dependent VIIa signaling suppresses Akt phosphorylation with concordant adverse transcriptional changes of key regulators of obesity and metabolism. Pharmacological blockade of adipocyte TF in vivo reverses these effects of TF-VIIa signaling and rapidly improves energy expenditure. Thus, TF signaling is a potential therapeutic target to improve impaired metabolism and insulin resistance in obesity.

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF.

Cooperation of tissue factor cytoplasmic domain and PAR2 signaling in breast cancer development

AbstractConstitutive expression of tissue factor (TF) by cancer cells triggers local activation of the coagulation cascade and promotes breast cancer progression through cell signaling involving protease activated receptor (PAR)2. In human breast cancer, TF and PAR2 are up-regulated and TF cytoplasmic domain phosphorylation is correlated with relapse. Here we show that cancer cell PAR2 signaling promotes angiogenesis independent of PAR2 phosphorylation at the recognized β-arrestin recruitment site. Similar to PAR2−/− mice, TF cytoplasmic domain–deleted (TFΔCT) mice have delayed spontaneous breast cancer development in the polyoma middle T model. Simultaneous deletion of PAR2 in TFΔCT mice did not further delay tumor appearance, consistent with overlapping roles of TF and PAR2 in promoting the angiogenic switch in early stages of breast cancer. In advanced carcinomas, tumor-associated macrophages were reduced in TFΔCT and TFΔCT/PAR2−/− mice, and increased tumor vessel diameters of TFΔCT mice were partially reversed by PAR2-deficiency, indicating that the TF cytoplasmic domain has additional roles that are interdependent with PAR2 signaling in regulating host angiogenic responses. These experiments demonstrate a crosstalk of tumor cell TF cytoplasmic domain and PAR2 signaling and provide a possible mechanism for the close correlation between TF phosphorylation and cancer recurrence of TF and PAR2-positive clinical breast cancer.

Calcium Ionophore-Induced Tissue Factor (TF) Decryption Induces TF Immobilization Into Lipid Rafts and Negative Regulation of TF Procoagulant Activity.

AbstractAbstract 1131Cell exposed tissue factor (TF), the physiologic initiator of blood coagulation, is normally expressed in a low procoagulant, or cryptic conformation, and requires activation, or decryption, to fully exhibit its procoagulant potential. TF decryption is not fully understood and multiple decrypting mechanisms have been proposed including phosphatidylserine (PS) exposure, TF monomerization, association with lipid rafts and redox modulation of TF. Calcium ionophores have been extensively used as TF decrypting agents, and both PS-dependent and independent mechanisms have been associated with ionophore-induced TF decryption. In the present study we analyzed the changes that occur in the lateral mobility of cell exposed TF during calcium ionophore-induced decryption, using a TF chimera with monomeric yellow fluorescent protein (YFP-TF). The YFP-TF expressed by endothelial cells (EC) retains TF procoagulant activity, is mainly exposed on the cell surface and can be decrypted similarly with endogenous TF by the calcium ionophore ionomycin. We analyzed the changes in TF membrane mobility during decryption using live cell imaging of YFP-TF expressed in EC. Fluorescence recovery after photobleaching (FRAP) analysis revealed a decreased mobility of TF in EC treated with the decrypting agent ionomycin. The YFP-TF fluorescence in the region of interest was more easily bleached in ionomycin–treated cells as compared with controls. The observed maximum recovery (Rmax) of YFP-TF fluorescence in the bleached region of interest was significantly higher in control cells (80.84% recovery) as compared with ionomycin treated EC (39.29% recovery). These correlated with a decrease in YFP-TF mobile fraction from 50% for the control cells to 18% for the ionomycin treated EC. The lateral diffusion of the YFP-TF mobile fraction was similar between the two conditions, with halftime of fluorescence recovery of 7.69 sec in ionophore-treated cells and 10.69 sec in controls. These results suggest an immobilization of YFP-TF during decryption, which can be achieved by either lipid raft translocation or cytoskeleton floating. Similar to previous observations where TF cytoplasmic domain did not influence TF decryption, deletion of the TF cytoplasmic domain did not affect the lateral mobility of YFP-TF in FRAP analysis. To analyze decryption-induced changes in TF association with lipid domains, membrane fractions were isolated on a discontinuous Opti-Prep density gradient. Ionomycin treatment induced YFP-TF translocation from higher density, non-raft membrane fractions toward higher-buoyancy, raft fractions. Furthermore, the observed TF translocation into lipid rafts occurs without the formation of the quaternary complex with coagulation factors FVIIa, FXa and tissue factor pathway inhibitor (TFPI), as previously described. To address the functional modulation of TF procoagulant potential in response to lipid raft translocation, cell membrane cholesterol was either depleted with methyl-β-cyclodextrine (MβCD) or supplemented from an aqueous mixture of cholesterol-MβCD. Membrane cholesterol depletion decrypted TF in EC, likely through PS exposure, while also enhancing the procoagulant potential of ionomycin-decrypted TF. In contrast, cholesterol supplementation decreases the procoagulant potential of ionomycin-decrypted TF. Taken together, these observations support the model of tonic inhibition of TF procoagulant activity by the lipid raft environment. In conclusion, by live cell imaging we show that TF membrane mobility changes during calcium-ionophore induced decryption resulting in an immobilization of TF in lipid rafts. The immobilization is not influenced by the cytoplasmic domain of TF and does not require the formation of the TF-FVIIa-FXa-TFPI quaternary complex. Translocation into lipid rafts provides tonic inhibition of TF procoagulant potential and, as a consequence, we show for the first time that decrypting agents can also initiate negative regulation of TF procoagulant function. This negative feedback loop may help convert the decrypted TF back to its cryptic, low coagulant form. Disclosures:No relevant conflicts of interest to declare.

Tissue factor in cancer progression and angiogenesis

Constitutive expression of tissue factor (TF) by cancer cells triggers local and systemic activation of the coagulation cascade and is a major cause of cancer-associated thrombosis. Primary breast cancer biopsies show a marked upregulation of TF and protease activated receptor (PAR) 2, as well as increased TF cytoplasmic domain phosphorylation that is correlated with cancer relapse. TF signaling involving PAR2 and integrins has multiple effects on angiogenesis and tumor progression. The non-coagulant, alternatively spliced form of TF retains an integrin-binding site and, upon deposition into the tumor stroma, stimulates angiogenesis by ligating endothelial integrins α vβ 3 and α 6β 1. On tumor cells, full-length TF is constitutively associated with laminin-binding β 1 integrins that support TF-VIIa-PAR2 signaling leading to upregulation of pro-angiogenic and immune modulatory cytokines and growth factors. Deficiency of PAR2, but not of the thrombin receptor PAR1, delays spontaneous breast cancer development and the angiogenic switch in mice. In addition, human xenograft breast cancer growth and angiogenesis is suppressed by selective antibody inhibition of TF-VIIa-PAR2 signaling, but not by blocking TF initiated coagulation. Thus, interruption of TF signaling represents a potential anti-angiogenic strategy that does not carry an increased risk of bleeding associated with prolonged inhibition of the TF coagulation pathway.

Evidence for tissue factor phosphorylation and its correlation with protease‐activated receptor expression and the prognosis of primary breast cancer

Tissue factor (TF)-mediated protease-activated receptor (PAR)-2 signaling is associated with a promigratory, invasive and proangiogenic phenotype in experimental models of breast cancer and has been mechanistically coupled to phosphorylation of the TF cytoplasmic domain (pTF). However, the clinical relevance of these findings is unknown. Here, we provide the first in vivo evidence of TF phosphorylation in experimental as well as clinical breast cancer tumors. pTF was demonstrated in MDA-MB-231 xenografts and in tumors from the MMTV-PyMT transgene model of spontaneous murine breast adenocarcinoma. Tumors from PAR-2-deficient transgenic mice were negative for pTF, thus linking pTF to PAR-2 signaling. The clinical correlation between TF, pTF, PAR-1, PAR-2 and vascular endothelial growth factor (VEGF)-A was determined by immunohistochemistry on tumors from a cohort of 172 consecutive primary breast cancer patients, with a median follow-up time of 50 months. In 160 evaluable patient tumors, pTF was associated with TF (p = 0.01) and cancer cell expression of PAR-1 (p = 0.001), PAR-2 (p = 0.014) and VEGF-A (p = 0.003) using chi(2) test. PAR-2 and VEGF-A were coexpressed (p = 0.013) and associated with a more aggressive phenotype. Interestingly, all patients experiencing recurrences had tumors expressing pTF and PAR-2, and pTF alone as well as coexpression of pTF and PAR-2 were significantly correlated with shorter recurrence-free survival (log rank test, p = 0.04 and p = 0.02, respectively). This study provides the first evidence to link PAR-2 expression and TF phosphorylation to clinical data in human breast cancer. In conjunction with experimental tumor models, these data support an important role of TF-PAR-2 signaling in breast cancer recurrence.

Platelet Tissue Factor: Expression of Pro-Coagulant Activity depends on Gpibα Activation and Signaling through Lyn-Mediated Phosphorylation. A Platelet-Based Model of Hemostasis

Human platelets synthesize and store functionally silent tissue factor (TF) which has procoagulant activity (PCA) after platelet activation. We now explored the location of inactive and active forms of TF and mechanisms of activation. We reported that resting, non-permeabilized human platelets express scant surface TF, which was strikingly enhanced and co-localized with GPIbα after stimulation (Blood 2007; 109:5242). The externalization of TF was confirmed by immunoprecipitation (Ip) from biotinylated membranes before and after platelet activation. Moreover, TF and GPIbα co-precipitated in Ip assays and both glycoproteins were prominently found in lipid rafts (Lr) domains. TF-dependent PCA, assessed by FXa generation, was negligible or absent in resting, leukocyte-free platelet preparations. Interestingly, FVII was found in platelet membranes (western blot) and no exogenous FVIIa was needed to trigger TF-dependent FXa generation from washed platelets. Platelet responses to activation (TF-dependent PCA, platelet aggregation and secretion) depended on the agonist used. With 1IU/mL VWF + 1.2 mg/mL Ristocetin (Ris) PCA increased 5 to 10-fold 2 min after stimulation and platelets formed large aggregates with null 14C-serotonin secretion. In contrast, both platelet aggregation and secretion were normal 2 min after activation with 1IU thrombin, 10μM TRAP or 2μg/mL collagen; and PCA induced by thrombin was only ≈1/2 of that elicited by VWF+Ris, whereas 2 min after TRAP or collagen stimulation no PCA induction was detected. Removal of membrane cholesterol (by methyl-β-cyclo-dextrin) or disruption of Lr (with filipin III) abolishes the VWF-Ris-induced PCA. Lr's from resting platelets contain TF of Mr ≈60kDa. Five min after activation an increase in Lr's TF was observed, but its Mr depended on the agonist: species of ≈47kDa and ≈60kDa were found after VWF-Ris and TRAP activation, respectively. Given that GPIbα signals through Lyn, member of the Src family, inhibition of signaling with PP2 resulted in 80% fall of VWF-Ris-induced PCA. Ip assays revealed that Lyn co-precipitated with both GPIbα and TF in VWF-Ris activated, but not resting platelets. The phosphokinase activity of Lyn on TF was tested. A polyclonal antibody raised against the phosphorylated (Ser253/Ser258) cytoplasmic domain of TF (Dr. W. Ruf, La Jolla) recognized membrane TF only in activated, not in resting platelets. These findings indicate that VWF-induced activation of GPIbα, subsequent signaling with Lyn-induced serine phosphorylation, along with a change of TF to a ≈47kDa species, triggers human platelets PCA. The previously described role of phospho-disulphide isomerases (PDI) in TF activation through SH groups oxidation was also explored. TF and PDI co-precipitated in resting and activated platelet membranes and antagonizing PDI with bacitracin inhibited TF-dependent PCA. After platelet activation and labeling with MPB, a sulfhydryl-specific probe, TF protein (with PCA) was detected on the plasma membrane, denoting presence of reduced thiols. Furthermore, platelet incubation with phenylarsine oxide, a blocking reagent of vicinal SH groups, or HgCl2, a potent oxidant of thiol groups, had no effect on platelet PCA. Thus, it seems unlikely that TF activation depends on SH oxidation. Finally, we found that platelet TF was sufficient to speed up the clotting of plasma. In fact, clotting time of PRP (2 × 108 platelets mL−1) incubated with ionophore A23187 (2 min, 37°C) and then re-calcified, was 59 ± 6 sec, whereas clotting time in re-calcified PRP without previous activation was 137 ± 19 sec (n=19, p<0.0001). Taken together, our results highlight the crucial role of platelets, not only in assembling clotting complexes and reactions on their surface, but also providing enough TF to trigger the whole process. This novel, comprehensive understanding of hemostasis, (“platelet-based hemostasis model”), unifies primary and secondary hemostasis around the platelets, which would be able to synchronize and modulate the times of both processes ensuring a confined thrombin generation and adequate deposit of fibrin when and where it is needed. It also emphasizes the self-sufficiency of intravascular components to carry out both normal hemostasis and thrombus formation. In this context, platelet PCA may become a central pharmacological target for preventing or managing bleeding and thrombotic disorders.

Sharing Tissue Factor

Existence of a close relationship between increased clotting and malignancy has been clearly recognized for over a century. Many tumor cell types express tissue factor (TF), a procoagulant protein, on their cell surfaces. Tissue factor is a transmembrane cellular receptor for coagulation factor VII/VIIa. Binding of factor VIIa to TF triggers the activation of coagulation cascade leading ultimately to the generation of thrombin which in turn stimulates platelet activation and cleaves fibrinogen. Thus, there is no surprise that the activation of coagulation by tumor cell TF or by membrane fragments shed from tumor cells carrying TF contributes to cancer-associated thrombotic disorders.1,2 The close association between cancer and TF has also raised the possibility that TF may contribute to the pathogenesis of cancer beyond thrombosis. In fact, there are a number of reports in the literature showing that TF expressed by tumor cells plays an important role in both primary tumor growth and metastasis (see reviews3–5). The prometastatic effect of TF involves both activation of the coagulation pathway (eg, mechanisms linked to thrombin generated by TF) and effects mediated through the direct signaling activity of TF, probably via the cytoplasmic domain of TF.6 Recent studies have shown that direct TF-FVIIa protease-induced cell signaling is a major contributor to tumor growth in breast cancer.7 However, there are a couple of convincing reports in the literature that provide evidence that is contrary to the general belief that tumor-derived TF is crucial for primary tumor growth or metastasis. Toomey et al,8 using embryonic stem (ES) cells that do not express TF, showed that tumor-derived TF was not required for either tumor growth or metastasis. In a more recent study, Palumbo et al9 showed that tumor TF was neither required for primary tumor growth nor necessary for the initial …

Neutrophil activation by the tissue factor/Factor VIIa/PAR2 axis mediates fetal death in a mouse model of antiphospholipid syndrome

Women with antiphospholipid syndrome (APS), a condition characterized by the presence of antiphospholipid antibodies (aPL), often suffer pregnancy-related complications, including miscarriage. We have previously shown that C5a induction of tissue factor (TF) expression in neutrophils contributes to respiratory burst, trophoblast injury, and pregnancy loss in mice treated with aPL. Here we analyzed how TF contributes to neutrophil activation and trophoblast injury in this model. Neutrophils from aPL-treated mice expressed protease-activated receptor 2 (PAR2), and stimulation of this receptor led to neutrophil activation, trophoblast injury, and fetal death. An antibody specific for human TF that has little impact on coagulation, but potently inhibits TF/Factor VIIa (FVIIa) signaling through PAR2, inhibited aPL-induced neutrophil activation in mice that expressed human TF. Genetic deletion of the TF cytoplasmic domain, which allows interaction between TF and PAR2, reduced aPL-induced neutrophil activation in aPL-treated mice. Par2-/- mice treated with aPL exhibited reduced neutrophil activation and normal pregnancies, which indicates that PAR2 plays an important role in the pathogenesis of aPL-induced fetal injury. We also demonstrated that simvastatin and pravastatin decreased TF and PAR2 expression on neutrophils and prevented pregnancy loss. Our results suggest that TF/FVIIa/PAR2 signaling mediates neutrophil activation and fetal death in APS and that statins may be a good treatment for women with aPL-induced pregnancy complications.