Selective but not pan-CDK inhibition abrogates 5-FU-driven tissue factor upregulation in colon cancer.

Thromboembolic events are complications in cancer patients and hypercoagulability has been linked to the tissue factor (TF) pathway, making this an attractive target. Here, we investigated the effects of chemotherapeutics and CDK inhibitors (CDKI) abemaciclib/palbociclib (CDK4/6), THZ-1 (CDK7/12/13), and dinaciclib (CDK1/2/5/9) alone and in combination regimens on TF abundance and coagulation. The human colorectal cancer (CRC) cell line HROC173 was treated with 5-FU or gemcitabine to stimulate TF expression. TF+ cells were sorted, recultured, and re-analyzed. The effect of treatment alone or in combination was assessed by functional assays. Low-dose chemotherapy induced a hypercoagulable state and significantly upregulated TF, even after reculture without treatment. Cells exhibited characteristics of epithelial-mesenchymal transition, including high expression of vimentin and mucin. Dinaciclib and THZ-1 also upregulated TF, while abemaciclib and palbociclib downregulated it. Similar results were observed in coagulation assays. The same anticoagulant activity of abemaciclib was seen after incubation with peripheral immune cells from healthy donors and CRC patients. Abemaciclib reversed 5-FU-induced TF upregulation and prolonged clotting times in second-line treatment. Effects were independent of cytotoxicity, senescence, and p27kip1 induction. TF-antibody blocking experiments confirmed the importance of TF in plasma coagulation, with Factor XII playing a minor role. Short-term abemaciclib counteracts 5-FU-induced hypercoagulation and eventually even prevents thromboembolic events.

PDIA2 has a dual function in promoting androgen deprivation therapy induced venous thrombosis events and castrate resistant prostate cancer progression.

Androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer (PCa) that effectively delays the tumor progression. However, it also increases the risk of venous thrombosis event (VTE) in patients, a leading cause of mortality. How a pro-thrombotic cascade is induced by ADT remains poorly understood. Here, we report that protein disulfide isomerase A2 (PDIA2) is upregulated in PCa cells to promote VTE formation and enhance PCa cells resistant to ADT. Using various in vitro and in vivo models, we demonstrated a dual function of PDIA2 that enhances tumor-mediated pro-coagulation activity via tumor-derived extracellular vehicles (EVs). It also stimulates PCa cell proliferation, colony formation, and xenograft growth androgen-independently. Mechanistically, PDIA2 activates the tissue factor (TF) on EVs through its isomerase activity, which subsequently triggers a pro-thrombotic cascade in the blood. Additionally, TF-containing EVs can activate the Src kinase inside PCa cells to enhance the AR signaling ligand independently. Androgen deprivation does not alter PDIA2 expression in PCa cells but enhances PDIA2 translocation to the cell membrane and EVs via suppressing the clathrin-dependent endocytic process. Co-recruitment of AR and FOXA1 to the PDIA2 promoter is required for PDIA2 transcription under androgen-deprived conditions. Importantly, blocking PDIA2 isomerase activity suppresses the pro-coagulation activity of patient plasma, PCa cell, and xenograft samples as well as castrate-resistant PCa xenograft growth. These results demonstrate that PDIA2 promotes VTE and tumor progression via activating TF from tumor-derived EVs. They rationalize pharmacological inhibition of PDIA2 to suppress ADT-induced VTE and castrate-resistant tumor progression.

Impact of tissue factor expression and administration routes on thrombosis development induced by mesenchymal stem/stromal cell infusions: re-evaluating the dogma.

BACKGROUND: Hyperactive coagulation might cause dangerous complications such as portal vein thrombosis and pulmonary embolism after mesenchymal stem/stromal cell (MSC) therapy. Tissue factor (TF), an initiator of the extrinsic coagulation pathway, has been suggested as a predictor of this process. METHODS: The expression of TF and other pro- and anticoagulant genes was analyzed in xeno- and serum-free manufactured MSCs. Furthermore, culture factors affecting its expression in MSCs were investigated. Finally, coagulation tests of fibrinogen, D-dimer, aPPTs, PTs, and TTs were measured in patient serum after umbilical cord (UC)-MSC infusions to challenge a potential connection between TF expression and MSC-induced coagulant activity.  RESULTS: Xeno- and serum-free cultured adipose tissue and UC-derived MSCs expressed the highest level of TF, followed by those from dental pulp, and the lowest expression was observed in MSCs of bone marrow origin. Environmental factors such as cell density, hypoxia, and inflammation impact TF expression, so in vitro analysis might fail to reflect their in vivo behaviors. MSCs also expressed heterogeneous levels of the coagulant factor COL1A1 and surface phosphatidylserine and anticoagulant factors TFPI and PTGIR. MSCs of diverse origins induced fibrin clots in healthy plasma that were partially suppressed by an anti-TF inhibitory monoclonal antibody. Furthermore, human umbilical vein endothelial cells exhibited coagulant activity in vitro despite their negative expression of TF and COL1A1. Patients receiving intravenous UC-MSC infusion exhibited a transient increase in D-dimer serum concentration, while this remained stable in the group with intrathecal infusion. There was no correlation between TF expression and D-dimer or other coagulation indicators. CONCLUSIONS: The study suggests that TF cannot be used as a solid biomarker to predict MSC-induced hypercoagulation. Local administration, prophylactic intervention with anticoagulation drugs, and monitoring of coagulation indicators are useful to prevent thrombogenic events in patients receiving MSCs. Trial registration NCT05292625. Registered March 23, 2022, retrospectively registered, https://www. CLINICALTRIALS: gov/ct2/show/NCT05292625?term=NCT05292625&draw=2&rank=1 . NCT04919135. Registered June 9, 2021, https://www. CLINICALTRIALS: gov/ct2/show/NCT04919135?term=NCT04919135&draw=2&rank=1 .

Cancer-associated thrombosis: What about microRNAs targeting the tissue factor coagulation pathway?

Cancer patients are often diagnosed with venous thromboembolism (VTE), a cardiovascular disease that substantially decreases their quality of life and survival rate. Haemostasis in these patients is deregulated, which is reflected in the common presentation of a blood hypercoagulation state. Despite the inconsistent results, existing evidence suggests that the expression of microRNAs (miRNAs) is deregulated in the context of venous thrombogenesis in the general population. However, few miRNAs are known to be linked to cancer-associated VTE due to the lack of studies with oncological patients. Parallelly, coagulation factor III, also known as tissue factor (TF), tissue factor pathway inhibitor 1 (TFPI1) and tissue factor pathway inhibitor 2 (TFPI2) have been proposed to have a central role in cancer-associated VTE and tumour progression. Yet, contrary to what was expected, the role of miRNAs targeting the TF coagulation pathway (or extrinsic coagulation pathway) is poorly explored in cancer-induced thrombogenesis. In this review, in addition to miRNAs implicated in VTE, TF and TFPI1/2-targeting miRNAs were revised. Future studies should clarify the implications of these non-coding RNAs in tumour coagulome.

EML4-ALK fusion protein in Lung cancer cells enhances venous thrombogenicity through the pERK1/2-AP-1-tissue factor axis.

BACKGROUND: Accumulating evidence links the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangement to venous thromboembolism (VTE) in non-small cell lung cancer (NSCLC) patients. However, the corresponding mechanisms remain unclear. METHOD: High-throughput sequencing analysis of H3122 human ALK-positive NSCLC cells treated with ALK inhibitor/ dimethyl sulfoxide (DMSO) was performed to identify coagulation-associated differential genes between EML4-ALK fusion protein inhibited cells and control cells. Sequentially, we confirmed its expression in NSCLC patients' tissues and in the plasma of a subcutaneous xenograft mouse model. An inferior vena cava (IVC) ligation model was used to assess clot formation potential. Additionally, pathways involved in tissue factor (TF) regulation were explored in ALK-positive cell lines H3122 and H2228. Statistical significance was determined by Student t-test and one-way ANOVA using SPSS. RESULTS: Sequencing analysis identified a significant downregulation of TF after inhibiting EML4-ALK fusion protein activity in H3122 cells. In clinical NSCLC cases, TF expression was increased especially in ALK-positive NSCLC tissues. Meanwhile, H3122 and H2228 with high TF expression exhibited shorter plasma clotting time and higher TF activity versus ALK-negative H1299 and A549 in cell culture supernatant. Mice bearing H2228 tumor showed a higher concentration of tumor-derived TF and TF activity in plasma and the highest adjusted IVC clot weights. Limiting EML4-ALK protein phosphorylation downregulated extracellular regulated protein kinases 1/2 (ERK1/2)-activating the protein-1(AP-1) signaling pathway and thus attenuated TF expression. CONCLUSION: EML4-ALK fusion protein may enhance venous thrombogenicity by regulating coagulation factor TF expression. There was potential involvement of the pERK1/2-AP-1 pathway in this process.

Coagulation factor VIIa enhances programmed death-ligand 1 expression and its stability in breast cancer cells to promote breast cancer immune evasion.

BACKGROUND: Immunotherapy for breast cancer has not gained significant success. Coagulation factor VIIa (FVIIa)-tissue factor (TF) mediated activation of protease-activated receptor 2 (PAR2) is shown to promote metastasis and secretion of the immune-modulatory cytokines but the role of FVIIa in cancer immunology is still not well understood. OBJECTIVES: Here, we aim to investigate whether FVIIa protects breast cancer cells from CD8 T-cell-mediated killing. METHODS: Peripheral blood mononuclear cell-derived CD8 T cells were cocultured with vehicle or FVIIa pretreated MDAMB468 cells. The proliferation and activity of CD8 T cells were measured by flow cytometry and ELISA. An allograft model, using wild-type or TF/PAR2-deleted 4T1 cells, was employed to determine the effect of FVIIa on breast cancer immune evasion in vivo. RESULTS: Here, we demonstrate that TF-FVIIa induces programmed death-ligand 1 (PD-L1) in breast cancer cells by activating PAR2. PAR2 activation triggers large tumor suppressor kinase 1 (LATS1) inactivation leading to loss of yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) phosphorylation and subsequent nuclear localization of YAP/TAZ. YAP/TAZ inhibition reduces PD-L1 expression and increases CD8 T-cell activity. We further demonstrate that, apart from transcriptional induction of PD-L1, PAR2 activation also increases PD-L1 stability by enhancing its glycosylation through N-glycosyltransferases STT3A and STT3B. CONCLUSION: In a mouse model of breast cancer, tumor cell-specific PAR2 depletion leads to PD-L1 downregulation and increases anti-PD-1 immunotherapy efficacy. In conclusion, we showed that FVIIa-mediated signaling cascade in cancer cells serves as a tumor intrinsic mechanism of immunosuppression to promote cancer immune evasion.

Tissue factor overexpression in triple-negative breast cancer promotes immune evasion by impeding T-cell infiltration and effector function.

Triple-negative breast cancer (TNBC) remains a most deadly human malignancy with limited response to chemotherapy, targeted therapy and immunotherapy. Tumor immunoenvironment plays an increasingly important role in therapy outcome. Tissue factor (TF) is the target of the FDA-approved ADC Tivdak. HuSC1-39 is the parent antibody of MRG004A, a clinical stage TF-ADC (NCT04843709). Here, we employed HuSC1-39 (termed "anti-TF") to investigate the role of TF in regulating immune-tolerance in TNBC. We found that patients with aberrant TF expression had a poor prognosis and low immune effector cell infiltration, characterizing as "cold tumor". In the 4T1 TNBC syngeneic mouse model, knockout of tumor cell TF inhibited tumor growth and increased tumor infiltration of effector T cell, which was not dependent on the clotting inhibition. In an immune-reconstituted M-NSG mouse model of TNBC, anti-TF inhibited tumor growth, which was further enhanced by a dual-targeting anti-TF&TGFßR fusion protein. There were diminished P-AKT and P-ERK signaling and profound tumor cell death in treated tumors. Transcriptome analyses and immunohistochemistry revealed a dramatically improved tumor immunoenvironment including the increase of effector T cells, decrease of Treg cells and the transformation of tumor into "hot tumor". Moreover, employing qPCR analysis and T cell culture, we further demonstrated that TF expression in tumor cells is sufficient to block the synthesis and secretion of T cell-recruiting chemokine CXCL9/10/11. Treatment of TF-high TNBC cells with anti-TF or TF-knockout all stimulated CXCL9/10/11 production, promoted T cell migration and effector function. Thus, we have identified a new mechanism of TF in TNBC tumor progression and therapy resistance.

The Heparan Sulfate Proteoglycan Syndecan-1 Triggers Breast Cancer Cell-Induced Coagulability by Induced Expression of Tissue Factor.

Syndecan-1 (Sdc-1) upregulation is associated with poor prognosis in breast cancer. Sdc-1 knockdown results in reduced angiogenesis and the dysregulation of tissue factor (TF) pathway constituents. Here, we evaluate the regulatory mechanisms and functional consequences of the Sdc-1/TF-axis using Sdc-1 knockdown and overexpression approaches in MCF-7 and MDA-MB-231 breast cancer cells. Gene expression was analyzed by means of qPCR. Thrombin generation and cell migration were detected. Cell-cycle progression and apoptosis were investigated using flow cytometry. In MDA-MB-231 cells, IL6, IL8, VEGF, and IGFR-dependent signaling affected TF pathway expression depending on Sdc-1. Notably, Sdc-1 depletion and TF pathway inhibitor (TFPI) synergistically affected PTEN, MAPK, and STAT3 signaling. At the functional level, the antiproliferative and pro-apoptotic effects of TFPI depended on Sdc-1, whereas Sdc-1's modulation of cell motility was not affected by TFPI. Sdc-1 overexpression in MCF-7 and MDA-MB-231 cells led to increased TF expression, inducing a procoagulative phenotype, as indicated by the activation of human platelets and increased thrombin formation. A novel understanding of the functional interplay between Sdc-1 and the TF pathway may be compatible with the classical co-receptor role of Sdc-1 in cytokine signaling. This opens up the possibility of a new functional understanding, with Sdc-1 fostering coagulation and platelet communication as the key to the hematogenous metastatic spread of breast cancer cells.

Tumor-expressed factor VII is associated with survival and regulates tumor progression in breast cancer.

Cancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer treatment, clinical studies on the effect of such modalities on cancer progression have not yet been successful for unknown reasons. In normal physiology, complex formation between the subendothelial-expressed tissue factor (TF) and the blood-borne liver-derived factor VII (FVII) results in induction of the extrinsic coagulation cascade and intracellular signaling via protease-activated receptors (PARs). In cancer, TF is overexpressed and linked to poor prognosis. Here, we report that increased levels of FVII are also observed in breast cancer specimens and are associated with tumor progression and metastasis to the liver. In breast cancer cell lines, tumor-expressed FVII drives changes reminiscent of epithelial-to-mesenchymal transition (EMT), tumor cell invasion, and expression of the prometastatic genes, SNAI2 and SOX9. In vivo, tumor-expressed FVII enhanced tumor growth and liver metastasis. Surprisingly, liver-derived FVII appeared to inhibit metastasis. Finally, tumor-expressed FVII-induced prometastatic gene expression independent of TF but required a functional endothelial protein C receptor, whereas recombinant activated FVII acting via the canonical TF:PAR2 pathway inhibited prometastatic gene expression. Here, we propose that tumor-expressed FVII and liver-derived FVII have opposing effects on EMT and metastasis.

Tissue factor-induced fibrinogenesis mediates cancer cell clustering and multiclonal peritoneal metastasis.

Peritoneal metastasis is one of the most frequent causes of death in several types of advanced cancers; however, the underlying molecular mechanisms remain largely unknown. In this study, we exploited multicolor fluorescent lineage tracking to investigate the clonality of peritoneal metastasis in mouse xenograft models. When peritoneal metastasis was induced by intraperitoneal or orthotopic injection of multicolored cancer cells, each peritoneally metastasized tumor displayed multicolor fluorescence regardless of metastasis sites, indicating that it consists of multiclonal cancer cell populations. Multicolored cancer cell clusters form within the peritoneal cavity and collectively attach to the peritoneum. In vitro, peritoneal lavage fluid or cleared ascitic fluid derived from cancer patients induces cancer cell clustering, which is inhibited by anticoagulants. Cancer cell clusters formed in vitro and in vivo are associated with fibrin formation. Furthermore, tissue factor knockout in cancer cells abrogates cell clustering, peritoneal attachment, and peritoneal metastasis. Thus, we propose that cancer cells activate the coagulation cascade via tissue factor to form fibrin-mediated cell clusters and promote peritoneal attachment; these factors lead to the development of multiclonal peritoneal metastasis and may be therapeutic targets.

Inhibition of protein disulfide isomerase with PACMA-31 regulates monocyte tissue factor through transcriptional and posttranscriptional mechanisms.

INTRODUCTION: Protein disulfide isomerase (PDI) contributes to tissue factor (TF) regulation in monocytes. While bacitracin and quercetin-3-rutinoside mitigate myeloid TF production, the effect of PACMA-31, a more specific PDI inhibitor with distinct pharmacologic properties, remains unclear. MATERIALS AND METHODS: Lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMCs) or citrate-anticoagulated whole blood was carried out in the presence of PACMA-31 or DMSO vehicle before monocytes were analyzed for TF expression, including antigen, procoagulant activity (PCA) and mRNA, release of IL-6 and TNFα, and LPS-induced signaling pathways. RESULTS: While PACMA-31 alone had no effect, coincubation with LPS and PACMA-31 (25 µM) enhanced LPS-induced monocyte TF production in whole blood. The effect was at least partially regulated on the transcriptional level and could not be explained by increased phosphatidylserine membrane exposure. In contrast, the same PACMA-31 concentrations were cytotoxic in isolated PBMCs. A lower dose of PACMA-31, however, restored the stimulating effect by enhancing IκB-NFκB signaling that also increased the release of IL-6 and TNFα. The protease-activated receptor 2 (PAR2) inhibitor ENMD547 but not TF antibody 10H10 or factor Xa inhibitor rivaroxaban prevented the stimulatory effect of PACMA-31 on inflammatory monocytes. In sharp contrast, short time incubation of LPS-stimulated PBMCs with 25 µM PACMA-31 was non-cytotoxic and significantly inhibited cellular TF PCA but not surface antigen expression. CONCLUSIONS: PACMA-31 regulates monocyte TF in a concentration-dependent manner by opposing transcriptional and posttranscriptional mechanisms. While low concentrations of PACMA-31 augment monocyte TF production by amplifying LPS-dependent PAR2 signaling, high concentrations convert monocyte TF into its non-coagulant state.

Evaluation of the mechanisms of heme-induced tissue factor activation: Contribution of innate immune pathways.

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to in vivo hemostasis. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of activated factor X (FXa). Heme was capable of inducing both TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Our results provide additional details on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

Metformin Inhibits Multiple Myeloma Serum-induced Endothelial Cell Thrombosis by Down-Regulating miR-532.

BACKGROUND: Thrombotic complications in multiple myeloma (MM) impairs the quality of life in patients. Metformin has a certain effect on anti-thrombosis, but its role and mechanism in MM-induced thrombosis are still uncovered. Therefore, this study evaluated the effect of metformin on MM-induced thrombosis. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to normal serum (15%), MM serum (15%), metformin (0.01 mmol/L), or MM serum, and metformin simultaneously. The expression of tissue factor (TF) in HUVECs was detected by flow cytometry and quantitative real-time polymerase chain reaction PCR (qRT-PCR). QRT-PCR was also used to determine the expressions of endothelial protein C receptor (EPCR) and miR-532. The generation of thrombin and activated protein C was measured by thrombin generation and protein C activation assays. EPCR, extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathway related protein expressions were detected by western blot. RESULTS: MM serum increased the expressions of TF and miR-532, induced thrombin generation, inhibited EPCR and protein C activation in HUVECs. And metformin could reverse the effects of MM serum on the expressions of TF, EPCR and miR-532, thrombin generation, protein C activation in HUVECs. However, miR-532 mimic reversed the effects of metformin and promoted the levels of thrombosis-related indicators in HUVECs. Moreover, metformin activated the ERK 1/2, p38 MAPK, and NF-κB pathways but miR-532 mimic suppressed the pathway activation. CONCLUSIONS: Metformin played an inhibitory effect on MM serum-induced HUVEC thrombosis, suggesting that metformin could serve as a novel antithrombotic approach for MM patients.

Tanshinone IIA Inhibits Tissue Factor Expression Induced by Thrombin in Human Umbilical Vein Endothelial Cells via PAR-1 and p38 MAPK Signaling Pathway.

BACKGROUND: The potential signaling pathway of TSA suppressing TF expression induced by thrombin was unknown. Thus, the transcription of TF in HUVECs and the expressions of DCF, phospho-p38 MAPK, NADPH oxidase 4, PAR-1, and NF-κB were detected in our study. METHODS: HUVECs were randomly divided into control group, thrombin-treated group (with 5 U/mL of thrombin), and 4 TSA-treated groups (with 5 U/mL of thrombin plus TSA with 4 different concentrations of 1 µg/mL, 10 µg/mL, 100 µg/mL, and 1 mg/mL, respectively). RESULTS: After incubation with thrombin for 6 h at 37°C, the results showed increased TF mRNA, TF procoagulant activity, and antigen of TF in HUVECs of thrombin-treated group (p < 0.01); however, they were restored by TSA in a dose-dependent manner (p < 0.01). In addition, reactive oxygen species (ROS), phospho-p38 MAPK, NADPH oxidase 4, NF-κB, and PAR-1 expressed more intensively, and phosphorylated Akt decreased obviously in HUVECs after thrombin stimulation (p < 0.01); however, they were reversed to different extents by TSA in a dose-dependent manner (p < 0.01). CONCLUSIONS: Study suggests that TSA inhibits TF expression induced by thrombin in cultured HUVECs, and the potential signaling pathway of which is TSA interrupts the activation of PAR-1 and NADPH oxidase as well as derivative ROS generation, thereafter suppresses the activation of NF-κB, the upstream signal molecule of TF, via hampering phosphorylation of p38 MAPK and dephosphorylation of Akt, and finally inhibits thrombin-induced TF overexpression.

Expression of tissue factor and TF-mediated integrin regulation in HTR-8/SVneo trophoblast cells.

Placenta is a crucial source of Tissue Factor (TF) to initiate coagulation. As far as the TF is concern, aberrant expression of TF has been reported to have a significant role in thrombosis, inflammation, cancer metastasis and atherosclerosis. It is evident that TF and TF-FVIIa complex has major roles in the disease process beyond hemostasis and thrombosis. On the other hand, TF-FVII-dependent signaling primarily activates PAR2 and inducing pro-angiogenic and immune-modulating cytokines in tumor environment. However, the role of TF has not been delineated in placental functions. Integrin typically binds to the extracellular matrix which in turn mediate cell-cell adhesion and cell behavior for migration. Dysregulation of integrin expression affects cell interaction, proliferation, and migration. Therefore, this study aims to ascertain the expression of TF in HTR-8/SVneo trophoblast cell line and its role in signal transduction of integrin (ITGα1, ITGα2, ITGß1) regulation concerning the invasion of trophoblasts. We have used RT-PCR and Western blot for the gene and protein expression analysis respectively. In addition, cell migration assays, MTT, and DAPI were performed to examine migration, cytotoxicity and apoptosis effect of FVIIa. The results suggest that the gene and protein level expressions of TF were predominant in HTR-8/SVneo cell line. Further, the cytotoxicity and apoptosis in HTR-8/SVneo cells were not observed when treated with FVIIa. The cells treated with FVIIa shown a dose-dependent up-regulation of integrin(s) (**p < 0.01, *p < 0.05) when compared to control. Migration of the HTR-8/SVneo cells was observed without any apoptosis in FVIIa-treated cells when compared to that of control. On the whole, these observations delineated the TF-FVIIa interaction in modulating the TF-dependent integrin signal transduction in HTR-8/SVneo trophoblast cell line.

KLF11 Protects against Venous Thrombosis via Suppressing Tissue Factor Expression.

Krüppel-like factors (KLFs) play essential roles in multiple biological functions, including maintaining vascular homeostasis. KLF11, a causative gene for maturity-onset diabetes of the young type 7, inhibits endothelial activation and protects against stroke. However, the role of KLF11 in venous thrombosis remains to be explored. Utilizing stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs), we identified an increase of KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-α (TNF-α)-induced tissue factor (TF) gene transcription. Using reporter gene assay, chromatin immunoprecipitation assay, and co-immunoprecipitation, we revealed that KLF11 could reduce TNF-α-induced binding of early growth response 1 (EGR1) to TF gene promoter in ECs. In addition, we demonstrated that conventional Klf11 knockout mice were more susceptible to developing stasis-induced DVT. These results suggest that under prothrombotic conditions, KLF11 downregulates TF gene transcription via inhibition of EGR1 in ECs. In conclusion, KLF11 protects against venous thrombosis, constituting a potential molecular target for treating thrombosis.

Role of tissue factor in delayed bone repair induced by diabetic state in mice.

BACKGROUND: Tissue factor (TF) is the primary activator of the extrinsic coagulation protease cascade. Although TF plays roles in various pathological states, such as thrombosis, inflammatory diseases, cancer, and atherosclerosis, its involvement in bone metabolism remains unknown. MATERIALS AND METHODS: The present study examined the roles of TF in delayed bone repair induced by a diabetic state in mice using wild-type (WT) and low TF-expressing (LTF) male mice. A diabetic state was induced by intraperitoneal injections of streptozotocin (STZ). RESULTS: A prolonged diabetic state significantly reduced total and trabecular bone mineral densities (BMD) as well as cortical bone thickness in WT and LTF mice; these BMD parameters were similar between WT and LTF mice treated with or without STZ. The diabetic state induced in WT mice delayed the repair of the femur following injury. The diabetic state induced in LTF mice was associated with further delays in bone repair. In in vitro experiments, TF significantly decreased receptor activator of nuclear factor-κB ligand-induced osteoclast formation and osteoclastogenic gene expression in RAW264.7 cells. However, it did not affect the gene expression levels of runt-related transcription factor 2 and osterix as well as alkaline phosphatase activity in mouse primary osteoblasts. CONCLUSION: Low TF state was associated with enhanced bone repair delay induced by diabetic state in mice. The TF-induced suppression of bone remodeling may be a contributing factor to the protective effects of TF against delayed bone repair in a diabetic state.

Synthesis and analysis of small molecules to restrain the function of tissue factor within tumour cells.

Introduction: The restriction of prolyl-protein cis/trans isomerase 1 (Pin1) activity has been shown to prevent the release of tissue factor (TF) leading to the accumulation of the latter protein within the cell. This study tested the ability of novel small molecules to inhibit Pin1, suppress TF activity and release, and induce cellular apoptosis. Methods: Four compounds were designed and synthesised based on modification of 5-(p-methoxyphenyl)-2-methylfuran-3-carbonyl amide and the outcome on MDA-MB-231 and primary cells examined. These compounds contained 3-(2-naphthyl)-D-alanine (4a), D-tryptophan (4b), D-phenylalanine (4c), and D-tyrosine (4d) at the amino-termini. Results: Treatment of cells with compound 4b and 4d reduced the cell-surface TF activity after 60 min on MDA-MB-231 cells. Incubation with compound 4d also reduced TF antigen on the cell surface and its incorporation into microvesicles, while compounds 4a and 4b significantly increased TF release. None of the four compounds significantly altered the total amount of TF antigen or TF mRNA expression. Compound 4b and 4d also suppressed the binding of Pin1 to TF-cytoplasmic domain peptide. However, compound 4d reduced while compound 4b increased the Pin1 isomerase activity. Finally, treatment with compound 4b and 4d reduced the cell numbers, increased nuclear localisation of p53, Bax protein and bax mRNA expression and induced cellular apoptosis in MDA-MB-231 but not primary endothelial cells. Conclusions: In conclusion, we have identified small molecules to regulate the function of TF within cells. Two of these compounds may prove to be beneficial in moderating TF function specifically and restrain TF-mediated tumour growth without detrimental outcomes on normal vascular cells.

Multi-omics analysis reveals the interaction between the complement system and the coagulation cascade in the development of endometriosis.

Endometriosis (EMS) is a disease that shows immune dysfunction and chronic inflammation characteristics, suggesting a role of complement system in its pathophysiology. To find out the hub genes and pathways involved in the pathogenesis of EMs, three raw microarray datasets were recruited from the Gene Expression Omnibus database (GEO). Then, a series of bioinformatics technologies including gene ontology (GO), Hallmark pathway enrichment, protein-protein interaction (PPI) network and gene co-expression correlation analysis were performed to identify hub genes. The hub genes were further verified by the Real-time quantitative polymerase chain reaction (RT-PCR) and Western Blot (WB). We identified 129 differentially expressed genes (DEGs) in EMs, of which 78 were up-regulated and 51 were down-regulated. Through GO functional enrichment analysis, we found that the DEGs are mainly enriched in cell adhesion, extracellular matrix remodeling, chemokine regulation, angiogenesis regulation, epithelial cell proliferation, et al. In Hallmark pathway enrichment analysis, coagulation pathway showed great significance and the terms in which included the central complement factors. Moreover, the genes were dominating in PPI network. Combined co-expression analysis with experimental verification, we found that the up-regulated expression of complement (C1S, C1QA, C1R, and C3) was positively related to tissue factor (TF) in EMs. In this study, we discovered the over expression complement and the positive correlation between complement and TF in EMs, which suggested that interaction of complement and coagulation system may play a role within the pathophysiology of EMS.