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Tissue Factor (TF) is the initiator of blood coagulation but also functions as a signal transduction receptor. TF expression in breast cancer is associated with higher tumor grade, metastasis and poor survival. The role of TF signaling on the early phases of metastasis has never been addressed. Here, we show an association between TF expression and metastasis as well as cancer stemness in 574 breast cancer patients. In preclinical models, blockade of TF signaling inhibited metastasis tenfold independent of primary tumor growth. TF blockade caused a reduction in epithelial-to-mesenchymal-transition, cancer stemness and expression of the pro-metastatic markers Slug and SOX9 in several breast cancer cell lines and in ex vivo cultured tumor cells. Mechanistically, TF forms a complex with beta 1-integrin leading to inactivation of beta 1-integrin. Inhibition of TF signaling induces a shift in TF-binding from alpha 3 beta 1-integrin to alpha 6 beta 4 and dictates FAK recruitment, leading to reduced epithelial-to-mesenchymal-transition and tumor cell differentiation. In conclusion, TF signaling inhibition leads to reduced pro-metastatic transcriptional programs, and a subsequent integrin beta 1 and beta 4-dependent reduction in metastasic dissemination.

Simple Summary Alternatively spliced tissue factor (asTF) is a naturally occurring isoform of tissue factor (TF) generated via the omission of exon 5 during the processing of TF’s primary transcript. In human and mouse, asTF protein features a unique C-terminus that lacks a transmembrane domain, rendering it soluble. asTF protein is able to associate with a subset on integrins on cell surfaces, which can trigger outside-in signaling programs in a variety of cell types. In this review, we discuss recent findings on asTF’s proto-oncogenic effects, regulatory mechanisms enabling asTF’s biosynthesis, and asTF’s potential as a biomarker and therapeutic target. Abstract In human and mouse, alternative splicing of tissue factor’s primary transcript yields two mRNA species: one features all six TF exons and encodes full-length tissue factor (flTF), and the other lacks exon 5 and encodes alternatively spliced tissue factor (asTF). flTF, which is oftentimes referred to as “TF”, is an integral membrane glycoprotein due to the presence of an alpha-helical domain in its C-terminus, while asTF is soluble due to the frameshift resulting from the joining of exon 4 directly to exon 6. In this review, we focus on asTF—the more recently discovered isoform of TF that appears to significantly contribute to the pathobiology of several solid malignancies. There is currently a consensus in the field that asTF, while dispensable to normal hemostasis, can activate a subset of integrins on benign and malignant cells and promote outside-in signaling eliciting angiogenesis; cancer cell proliferation, migration, and invasion; and monocyte recruitment. We provide a general overview of the pioneering, as well as more recent, asTF research; discuss the current concepts of how asTF contributes to cancer progression; and open a conversation about the emerging utility of asTF as a biomarker and a therapeutic target.

In 2021, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer deaths in the United States. This is largely due to a lack of symptoms and limited treatment options, which extend survival by only a few weeks. There is thus an urgent need to develop new therapies effective against PDAC. Previously, we have shown that the growth of PDAC cells is suppressed when they are co-implanted with RabMab1, a rabbit monoclonal antibody specific for human alternatively spliced tissue factor (asTF). Here, we report on humanization of RabMab1, evaluation of its binding characteristics, and assessment of its in vivo properties. hRabMab1 binds asTF with a KD in the picomolar range; suppresses the migration of high-grade Pt45.P1 cells in Boyden chamber assays; has a long half-life in circulation (~ 5 weeks); and significantly slows the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice when administered intravenously. Immunohistochemical analysis of tumor tissue demonstrates the suppression of i) PDAC cell proliferation, ii) macrophage infiltration, and iii) neovascularization, whereas RNAseq analysis of tumor tissue reveals the suppression of pathways that promote cell division and focal adhesion. This is the first proof-of-concept study whereby a novel biologic targeting asTF has been investigated as a systemically administered single agent, with encouraging results. Given that hRabMab1 has a favorable PK profile and is able to suppress the growth of human PDAC cells in vivo, it comprises a promising candidate for further clinical development.

Phosphatidylserine (PS) is often externalized in viable pancreatic cancer cells and is therapeutically targetable using PS-selective drugs. One of the first-line treatments for advanced pancreatic cancer disease, gemcitabine (GEM), provides only marginal benefit to patients. We therefore investigated the therapeutic benefits of combining GEM and the PS-targeting drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), for treating pancreatic ductal adenocarcinoma (PDAC). Using cell-cycle analyses and a cell surface PS-based sorting method in vitro, we observed an increase in surface PS as cells progress through the cell cycle from G1 to G2/M. We also observed that GEM treatment preferentially targets G1 phase cells that have low surface PS, resulting in an increased median surface PS level of PDAC cells. Inversely, SapC-DOPS preferentially targets high surface PS cells that are predominantly in the G2/M phase. Finally, combination therapy in subcutaneous and orthotopic PDAC tumors in vivo with SapC-DOPS and GEM or Abraxane (Abr)/GEM (one of the current standards of care) significantly inhibits tumor growth and increases survival compared with individual treatments. Our studies confirm a surface PS and cell cycle-based enhancement of cancer cytotoxicity following SapC-DOPS treatment in combination with GEM or Abr/GEM. Thus, PDAC patients treated with Abr/GEM may benefit from concurrent administration of SapC-DOPS.

Objective Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. Methods We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). Results Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. Conclusions These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.

Tissue Factor, the initiator of the extrinsic pathway of coagulation, and its isoform alternatively spliced Tissue Factor (asTF) are often overexpressed in cancer cells. Previously, we have shown that RabMab1, a rabbit monoclonal antibody specific for human asTF, disrupts the binding of asTF to beta-integrins and thereby inhibits the growth of breast cancer and pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro and in vivo. Here, we report on humanization of RabMab1, assessment of its binding characteristics, and determination of its in vivo properties. The variable regions of the heavy and light chains of hybridoma derived RabMab1 were cloned and used to generate a rabbit/human chimera (cRabMab1). Substitutions of species-specific residues were then carried out to produce humanized RabMab1 (hRabMab1; IgG1 isotype). Antigen binding was assessed via anti-human IgG Fc capture biosensor assays. cRabMab1 was found to have a KD of 4.24 nM whereas hRabMab1 was found to have a KD in the low picomolar range, which could not be precisely measured due to an extremely slow off-rate. The in vivo half-lives of each were determined to be 280 and 908 hours, respectively. Orthotopic co-implantation of cRabMab1 with high-grade, human PDAC cell line Pt45.P1 in athymic nude mice resulted in tumors that were 82% and 92% smaller than tumors in the vehicle and IgG control group groups, respectively, with no statistical difference between vehicle and IgG isotype control groups. Intravenous administration of hRabMab1 slowed the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice by 64% and 61% compared to vehicle and isotype control groups, respectively; again, there was no difference between vehicle and IgG isotype control groups. Immunohistochemical analysis of tumor tissue revealed a statistically significant 68% reduction in neovascularization (CD31), a 58% reduction in M2-polarized macrophages (CD206), and a 24% reduction in proliferating cells (Ki67) in the hRabMab1 cohort. RNAseq analysis of tumor tissue revealed that components of the focal adhesion system were the most affected by hRabMab1 treatment. In addition, a significant suppression of pathways that promote mitosis and the cell cycle was seen in hRabMab1-treated tumors. By qRT-PCR, we validated a greater than 82% reduction in the expression of HBEGF, a gene encoding a cell surface mitogen, and a greater than 68% reduction in the expression of STN1, a gene encoding a component of the CST-complex, a telomere replication and maintenance structure. This is the first proof-of-concept study whereby a novel biologic that inhibits asTF has been used as a systemically administered single agent, with encouraging results. Because hRabMab1 has a favorable pharmacokinetic profile and is able to suppress PDAC tumor cell growth in vivo, it is an attractive candidate for further clinical development. Citation Format: Clayton S. Lewis, Aniruddha Karve, Kateryna Matiash, Timothy Stone, Jingxing Li, Jordan Wang, Henri Versteeg, Bruce Aronow, Syed Ahmad, Pankaj Desai, Vladimir Bogdanov. Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced tissue factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 908.

Background Full length Tissue factor (flTF) is a key player in hemostasis and also likely contributes to venous thromboembolism (VTE), the third most common cardiovascular disease. flTF and its minimally coagulant isoform, alternatively spliced TF (asTF), have been detected in thrombi, suggesting participation of both isoforms in thrombogenesis, but data on participation of asTF in hemostasis is lacking. Therefore, we assessed the role of asTF in flTF cofactor activity modulation, using a co-expression system. Objective To investigate the interplay between flTF and asTF in hemostasis on endothelial cell surface. Methods Immortalized endothelial (ECRF) cells were adenovirally transduced to express asTF and flTF, after which flTF cofactor activity was measured on cells and microvesicles (MVs). To study co-localization of flTF/asTF proteins, confocal microscopy was performed. Finally, intracellular distribution of flTF was studied in the presence or absence of heightened asTF levels. Results Levels of flTF antigen and cofactor activity were not affected by asTF co-expression. asTF and flTF were found to localize in distinct subcellular compartments. Only upon heightened overexpression of asTF, lower flTF protein levels and cofactor activity were observed. Heightened asTF levels also induced a shift of flTF from non-raft to lipid raft plasma membrane fractions, and triggered the expression of ER stress marker BiP. Proteasome inhibition resulted in increased asTF – but not flTF – protein expression. Conclusion At moderate levels, asTF appears to have negligible impact on flTF cofactor activity on endothelial cells and MVs; however, at supra-physiological levels, asTF is able to reduce the levels of flTF protein and cofactor activity.

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.

Tissue Factor (TF) is the initiator of blood coagulation but also functions as a signal transduction receptor. TF expression in breast cancer is associated with higher tumor grade, metastasis and poor survival. The role of TF signaling on the early phases of metastasis has never been addressed. Here, we show an association between TF expression and metastasis as well as cancer stemness in 574 breast cancer patients. In preclinical models, blockade of TF signaling inhibited metastasis tenfold independent of primary tumor growth. TF blockade caused a reduction in epithelial-to-mesenchymal-transition, cancer stemness and expression of the pro-metastatic markers Slug and SOX9 in several breast cancer cell lines and in ex vivo cultured tumor cells. Mechanistically, TF forms a complex with β1-integrin leading to inactivation of β1-integrin. Inhibition of TF signaling induces a shift in TF-binding from α3β1-integrin to α6β4 and dictates FAK recruitment, leading to reduced epithelial-to-mesenchymal-transition and tumor cell differentiation. In conclusion, TF signaling inhibition leads to reduced pro-metastatic transcriptional programs, and a subsequent integrin β1 and β4-dependent reduction in metastasic dissemination.

OBJECTIVE: Mutations in Krüppel like factor-11 (KLF11), a gene also known as maturity-onset diabetes of the young type 7, contribute to the development of diabetes. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell (VSMC) biology and arterial thrombosis. APPROACH AND RESULTS: Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout (Klf11 KO) mice while bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that VSMC specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (TF, encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 KO mice, as determined by real-time quantitative PCR and immunofluorescence. Furthermore, VSMCs isolated from Klf11 KO mouse aortas showed increased TF expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, siRNA-mediated knockdown of KLF11 increased TF expression. Consistent results were observed upon adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. CONCLUSIONS: Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in VSMCs, constituting a potential molecular target for inhibition of arterial thrombosis.

Essentials Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET). TF knockdown inhibits proliferation of human pNET cells in vitro. mTOR kinase inhibitor sapanisertib/MLN0128 suppresses TF expression in human pNET cells. Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo. SUMMARY: Background Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs). Objective To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg-1 , oral gavage) for 34 days. Results Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice. Conclusions TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.

Tissue Factor, the initiator of the extrinsic pathway of coagulation, and its isoform alternatively spliced Tissue Factor (asTF) are often overexpressed in cancer cells. While asTF is only minimally procoagulant, it activates both the extracellular mechanical and intracellular signaling functions of beta-integrins. This leads to activation of multiple MAPK pathways thereby promoting increases in proliferation, migration, and angiogenesis. Previously, we have shown that RabMab1, a rabbit monoclonal antibody specific for human asTF, disrupts the binding of asTF to beta-integrins and thereby inhibits the growth of breast cancer and pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro and in vivo. Here, we report on humanization of RabMab1, assessment of its binding characteristics, and determination of its in vivo properties. The variable regions of the heavy and light chains of hybridoma derived RabMab1 were cloned and used to generate a rabbit/human chimera (cRabMab1). Substitutions of species-specific residues were then carried out to produce humanized RabMab1 (hRabMab1; IgG1 isotype). Antigen binding was assessed via anti-human IgG Fc capture biosensor assays. cRabMab1 was found to have a KD of 4.24 nM whereas hRabMab1 was found to have a KD in the low picomolar range, which could not be precisely measured due to an extremely slow off-rate. The in vivo half-lives of each were determined to be 280 and 908 hours, respectively. Orthotopic co-implantation of cRabMab1 with high-grade, human PDAC cell line Pt45.P1 in athymic nude mice resulted in tumors that were 82% and 92% smaller than tumors in the vehicle and IgG control group groups, respectively, with no statistical difference between vehicle and IgG isotype control groups. Intravenous administration of hRabMab1 slowed the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice by 64% and 61% compared to vehicle and isotype control groups, respectively; again, there was no difference between vehicle and IgG isotype control groups. Immunohistochemical analysis of tumor tissue revealed a statistically significant 68% reduction in neovascularization (CD31), a 58% reduction in M2-polarized macrophages (CD206), and a 24% reduction in proliferating cells (Ki67) in the hRabMab1 cohort. RNAseq analysis of tumor tissue revealed a significant suppression of pathways that promote mitosis and the cell cycle. By qRT-PCR, we validated a greater than 82% reduction in the expression of HBEGF, a gene encoding a cell surface mitogen, and a greater than 68% reduction in the expression of STN1, a gene encoding a component of the CST-complex, a telomere replication and maintenance structure. This is the first proof-of-concept study whereby a novel biologic that targets asTF has been used as a systemically administered single agent, with encouraging results. Because hRabMab1 has a favorable pharmacokinetic profile and is able to suppress PDAC tumor growth in vivo, it comprises an attractive candidate for further clinical development. Citation Format: Clayton S. Lewis, Aniruddha Karve, Kateryna Matiash, Timothy Stone, Pankaj Desai, Vladimir Y. Bogdanov. Preclinical in vivo characterization of a first-in-class humanized antibody targeting alternatively spliced Tissue Factor [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-041.

Published version. Source at http://doi.org/10.18632/oncotarget.7955. License CC BY 3.0. Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to β1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread.

Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC –/– mice. In RBCs from HFD-fed wild-type and DARC –/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

OBJECTIVES In patients with chronic liver diseases, hypercoagulability can contribute to the progression of fibrosis and complications of cirrhosis. Tissue factor (TF) is a transmembrane glycoprotein that initiates the extrinsic pathway of blood coagulation. Recent investigations have established that TF is elevated in patients with pancreatic cancer, blood disorders, diabetes, and cardiovascular disease. Alternatively spliced tissue factor (asTF), a secreted form of TF, induces angiogenesis and exhibits low-level procoagulant activity. The aim of this study was to investigate whether the circulating levels of asTF are elevated in the plasma of patients with liver disease. MATERIALS AND METHODS In a single-center study, we retrospectively analyzed asTF plasma levels in healthy participants and patients having stage F0-F3 liver fibrosis, liver cirrhosis, as well as hepatocellular carcinoma (HCC). AsTF plasma levels were measured using a sandwich enzyme-linked immunosorbent assay. Values were expressed as median with interquartile range (IQR). RESULTS The lowest median plasma asTF concentration (94 pg/ml, IQR: 33-275) was found in the healthy control group. The patients with low-grade liver fibrosis (F0-F1 group) displayed the highest median asTF concentration (404 pg/ml, IQR: 277-789). Significant differences between the asTF levels in the plasma of healthy participants and those in patients with grade F0-F1 fibrosis (P

Summary Background We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation. Methods Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells. Results In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC. Conclusions Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity.

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Background: Hepatocytes produce a variety of clotting and fibrinolytic factors. In chronic liver disease (CLD), biosynthesis of these factors is often altered leading to hemostatic aberrations. Recently, the usefulness of PT/INR - one of the five parameters used in the Child-Pugh (CP) scoring system - has been questioned. In this retrospective study, we explored the patterns of plasma alterations relative to CP scores to establish a plasma-based panel that can help evaluate the extent of CLD severity without the use of PT/INR and/or such parameters as the extent of ascites and hepatic encephalopathy. Methods: Blood was collected via venipuncture (0.129 mol/l sodium citrate) from CLD patients suffering from cirrhosis due to untreated HCV and/or excessive alcohol consumption, that were previously assigned a CP score: CP-A, n=12; CP-B, n=19; CP-C, n=11. Healthy control subjects (n=30) were age and sex matched; all subjects gave informed consent. Within 2 h post-collection, specimens were centrifuged for 15 min at 1500 x g, and then 13000 x g for 2 min to obtain plasma that was immediately frozen at -80°C until used. The following was measured: pro-coagulant phospholipid, fXa, D-dimer, fVIII, sP-selectin, thrombomodulin, protein C, protein S, alternatively spliced Tissue Factor (asTF), free TFPI, TF/TFPI ratio, and TFPI/protein S ratio. Thrombin generation tests (TGT) with and without thrombomodulin were performed. To interrogate the biology of CLD, patient plasma was also analyzed for the levels of microvesicles (MVs) of various origin: endothelium (CD31+/CD41-), monocytes (CD14+), leukocytes (CD11b+), neutrophils (CD16+), platelets (CD41a+, CD41a+/CD62P+, CD62P+), and exosomes (CD63+). Platelet counts and the levels of Annexin V+ MVs were also evaluated. Results were analyzed using GraphPad Prism v.6.03, and the parameters that were significantly different between control and CLD plasma were further evaluated. A system was developed with specified cutoffs for each parameter, derived using clinically normal ranges as well as the data generated in this study, to compile a composite score for CLD severity. Results: CLD plasma had endogenous thrombin potential comparable to that of control plasma; however, the rate of thrombin generation and peak thrombin generation diminished with increased CLD severity (both p < 0.0001), prolonging the time until peak (p < 0.01) as assessed by TGT. Of the 14 remaining parameters investigated in CLD and control plasma, the levels of fVIII, D-dimer, and asTF increased with CLD severity while protein C, fV, protein S, and sP-selectin levels decreased (Table 1) and in combination, these 7 parameters were able to predict the previously assigned CP scores with high accuracy. Our scoring system assigns a numeric value (0, 1, or 2) for each parameter; a cumulative score of Conclusions: Here we report a novel plasma panel with potential utility in assessing CLD severity in a point-of-care setting. Our scoring system exhibits 97.2% accuracy and does not require clinical assessment beyond plasma analysis. asTF levels positively correlate with those of CD31+/CD41- MVs and both increase with CLD severity; this is in agreement with earlier reports of inflammatory cytokines eliciting asTF secretion from endothelial cells. Larger-scale prospective studies are warranted to validate our findings. Disclosures No relevant conflicts of interest to declare.

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

Upregulation of tissue factor (TF) expression on activated donor endothelial cells (ECs) triggered by the immune response (IR) has been considered the main initiator of consumptive coagulopathy (CC). In this study, we aimed to identify potential factors in the development of thrombocytopenia and CC after genetically engineered pig liver transplantation in baboons. Baboons received a liver from either an α1,3-galactosyltransferase gene-knockout (GTKO) pig (n = 1) or a GTKO pig transgenic for CD46 (n = 5) with immunosuppressive therapy. TF exposure on recipient platelets and peripheral blood mononuclear cell (PBMCs), activation of donor ECs, platelet and EC microparticles, and the IR were monitored. Profound thrombocytopenia and thrombin formation occurred within minutes of liver reperfusion. Within 2 h, circulating platelets and PBMCs expressed functional TF, with evidence of aggregation in the graft. Porcine ECs were negative for expression of P- and E-selectin, CD106, and TF. The measurable IR was minimal, and the severity and rapidity of thrombocytopenia were not alleviated by prior manipulation of the IR. We suggest that the development of thrombocytopenia/CC may be associated with TF exposure on recipient platelets and PBMCs (but possibly not with activation of donor ECs). Recipient TF appears to initiate thrombocytopenia/CC by a mechanism that may be independent of the IR.

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.

The initiator of coagulation, full-length tissue factor (flTF), in complex with factor VIIa, influences angiogenesis through PAR-2. Recently, an alternatively spliced variant of TF (asTF) was discovered, in which part of the TF extracellular domain, the transmembrane, and cytoplasmic domains are replaced by a unique C terminus. Subcutaneous tumors produced by asTF-secreting cells revealed increased angiogenesis, but it remained unclear if and how angiogenesis is regulated by asTF. Here, we show that asTF enhances angiogenesis in matrigel plugs in mice, whereas a soluble form of flTF only modestly enhances angiogenesis. asTF dose-dependently upregulates angiogenesis ex vivo independent of either PAR-2 or VIIa. Rather, asTF was found to ligate integrins, resulting in downstream signaling. asTF-alpha V beta 3 integrin interaction induces endothelial cell migration, whereas asTF-dependent formation of capillaries in vitro is dependent on alpha 6 beta 1 integrin. Finally, asTF-dependent aortic sprouting is sensitive to beta 1 and beta 3 integrin blockade and a TF-antibody that disrupts asTF-integrin interaction. We conclude that asTF, unlike flTF, does not affect angiogenesis via PAR-dependent pathways but relies on integrin ligation. These findings indicate that asTF may serve as a target to prevent pathological angiogenesis

Tumor necrosis factor (TNF)-α–stimulated human umbilical vein endothelial cells express 2 naturally occurring forms of tissue factor (TF), the primary initiator of blood coagulation: the soluble alternatively spliced isoform and the full-length TF isoform. The regulatory pathways enabling this phenomenon are completely unknown. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing via phosphorylation of serine/arginine-rich proteins. In this study, we examined effects of serine/arginine-rich protein kinases on TF splicing following stimulation with TNF-α. Human endothelial cells were pretreated with specific inhibitors or small interfering RNAs against Cdc2-like kinases and DNA topoisomerase I before stimulation with TNF-α. TF levels were determined by semiquantitative RT-PCR, real-time PCR, and Western blotting. Cellular procoagulant activity was analyzed in a chromogenic TF activity assay. All 4 known Cdc2-like kinases forms were expressed in human endothelial cells. Selective inhibition of Cdc2-like kinases and DNA topoisomerase I elicited distinct changes in TF biosynthesis in TNF-α–stimulated endothelial cells, which impacted endothelial procoagulant activity. This study is the first to demonstrate that serine/arginine-rich protein kinases modulate splicing of TF pre-mRNA in human endothelial cells and, consequently, endothelial procoagulant activity under inflammatory conditions.

Objectives: The aim of this study was to investigate the effect of the antioxidants pyrrolidine dithiocarbamate (PDTC) and N -acetylcysteine (NAC) on the ionizing radiation (IR)- and tumor necrosis factor-α (TNF-α) induced tissue factor (TF) expression and its release from human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were irradiated with a single dose of either 5 Gy or 10 Gy and stimulated with TNF-α (10 ng/mL) in the presence or absence of PDTC and NAC, respectively. Quantitative real-time PCR, ELISA, and TF activity measurements were performed, including TF activity in the supernatant. Apoptosis was detected by flow cytometric active caspase-3 measurement and formation of reactive oxygen species (ROS) by chemiluminescence. Results: We demonstrated a thus far uninvestigated persistent induction of TF expression in HUVECs after treatment with IR and TNF-α. Combined stimulation with IR and TNF-α led to an immense shedding of microparticle-associated TF which was positively correlated with apoptosis and ROS formation. Antioxidative pre-treatment reduced not only apoptosis and ROS formation, but also the release of thrombogenic microparticles. Conclusions: Antioxidative treatment inhibited apoptosis and shedding of microparticles, thereby reducing thrombogenicity. Thus, antioxidants may help to prevent late thrombosis after antiproliferative treatment when used in combination with anticoagulants.

Tissue factor (TF), the main trigger of blood coagulation, is essential for normal hemostasis. Over the past 20 years, heightened intravascular levels and activity of TF have been increasingly perceived as an entity that significantly contributes to venous as well as arterial thrombosis. Various forms of the TF protein in the circulation have been described and proposed to be thrombogenic. Aside from cell and vessel wall-associated TF, several forms of non-cell-associated TF circulate in plasma and may serve as a causative factor in thrombosis. At the present time, no firm consensus exists regarding the extent, the vascular setting(s), and/or the mechanisms by which such TF forms contribute to thrombus initiation and propagation. Here, we summarize the existing paradigms and recent, sometimes paradigm-shifting findings elucidating the structural, mechanistic, and pathophysiological characteristics of plasma-borne TF.

Purpose: In order to elucidate anticancer effects of monocarbonyl analogs of curcumin (MACs), we have undertaken the present study to obtain information regarding drug targets by using a microarray approach, and to study the cellular localization of EF24 and the activity of two key transcription factors, AP-1 and NF-κB, involved in complex cellular responses of cell survival and death. Methods: Cytotoxic activity of various drugs was evaluated using a Neutral Red Dye assay. Cellular localization of biotinylated EF24 (active) and reduced EF24 (inactive) was determined using light and confocal microscopy. Measurement of transcription factor binding was carried out using Transfactor ELISA kits (BD Clontech, Palo Alto, CA). Gene microarray processing was performed at Expression Analysis, Inc (Durham, NC) using Affymetrix Human U133A Gene Chips.Results: In this study, we demonstrated that EF24 and UBS109 exhibit much more potent cytotoxic activity against pancreatic cancer than the current standard chemotherapeutic agent gemcitabine. EF24, rapidly localizes to the cell nucleus. The compound modulates the DNA binding activity of NF-κB and AP-1 in MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells. Immunohistochemical studies utilizing biotinylated-EF24 and chemically-reduced EF24 show that the unsaturated compound and biotinylated EF24, but not reduced EF24, translocates to the nucleus within 30 minutes after the addition of drug. Through a gene microarray study, EF24 is shown to affect genes directly involved in cytoprotection, tumor growth, angiogenesis, metastasis and apoptosis. Conclusion: EF24 and UBS109 warrant further investigation for development of pancreatic cancer therapy. The dualistic modulations of gene expression may be a manifestation of the cell responses for survival against oxidative stress by EF24. However, the cytotoxic action of EF24 ultimately prevails to kill the cells.

ObjectivesWe investigated the myocardial localization and expression of tissue factor (TF) and alternatively spliced human tissue factor (asHTF) in patients with dilated cardiomyopathy (DCM).BackgroundTissue factor is expressed in cardiac muscle and may play a role in maintaining myocardial structure.MethodsMyocardial biopsies were obtained from patients with a normal or mildly impaired ejection fraction (EF) (≥50%) and moderate to severely reduced EF (

61 MicroRNA-216a: a cardiac-specific post-transcriptional regulator of capillary rarefaction associated with heart failure {#article-title-2} Background: While cardiomyocytes have been the main subject of extensive research in the past, the role of other cardiac cell types in the pathogenesis of heart failure received considerable less attention. Multiple other cell types, including fibroblasts, endothelial cells and vascular smooth muscle cells make up the heart and increasing evidence suggests that these cell types contribute to myocardial health and disease. Angiogenesis is an important adaptive mechanism of the myocardium to conditions of sustained increased demand. A select number of microRNAs were found to participate in post-myocardial angiogenesis but to date, it is unclear, however, if microRNAs regulate cardiac angiogenesis in the non-ischemic failing myocardium, which represents a clinically more relevant question. Purpose: Establish that non-coding RNAs are main orchestrators of vascular integrity and hemostasis in the heart and changes in expression of the non-coding RNA content in different cardiac cell types underlie defects in neovascularization and progression to heart failure. Results: To identify cardiac angiomiRs we recently completed a functional, genome-wide microRNA knockdown screen in cultured mouse cardiac microvascular endothelial cells. In this setup, we performed the scratch assay by fully automated scratch induction and image capturing and identified several candidate microRNAs. miR-216a, one of those candidates affecting endothelial cell migration, is consistently downregulated in a mouse model of cardiac pressure overload induced by transverse aortic constriction. We generated mice with a somatic gene deletion of miR-216a (miR-216-/-). Ablation of miR-216a induced dramatic effects on cardiac morphology and function. In addition to spontaneous development of pathological cardiac remodeling at baseline, miR-216-/- mice are also extremely sensitive to cardiac stress and die 1-2 weeks after pressure overload or develop severe cardiac phenotypes 3-4 weeks after myocardial infarction. Interestingly, the defects observed correlate with vascular defects including capillary rarefaction and reduced angiogen­­­­ic activity. By combining photoacoustics with high-frequency ultrasound we observed different levels of hypoxia throughout the cardiac tissue confirming lower oxygenation of the miR-216a-/- hearts. The observed effects of miR-216 modulation seem to be related to altered autophagic activity in cardiac endothelial cells. Conclusions: We identified a new angiogenesis-related gene regulatory network where miR-216a underlies the vascular changes associated with pathological cardiac remodeling and/or heart failure. # 62 Divergent effects of pre- and post-conditioning on microvascular function {#article-title-3} Background: A short period of ischemia/reperfusion (I/R) that occurs before a prolonged ischemia (preconditioning; preC) or at reflow (postconditioning; postC) reduces tissue necrosis through activation of regulatory kinases cascades, also reciprocally related, including the GPCR-PKG-PKC cascade and the RISK pathway. In this setting, an important role is played by nitric oxide (NO), which is also the main mediator of endothelium-dependent vasodilation. PreC may also protect microcirculation against I/R injury, while the impact of postC is less clear, and mechanism underlying these effects are not completely elucidated. Purpose: To study the influence of preC and postC on postichemic microcirculation, by specifically evaluating leukocyte recruitment and vasodilating reserve, and to correlate these effects with activation of regulatory kinases. Methods: In rats, cremaster muscle was prepared for in vivo videomicroscopy leaving vascular and neural connections intact. Animals were divided in 4 groups: 1- controls, in which muscle was subjected to 90 min of I and 90 min of R; 2- preC, in which preC was induced before I by 5 min of I and 10 min of R; 3- postC, in which postC was induced at R with 5 cycles of 10 seconds R and 5 seconds I; 4- sham, observed after surgery for an equivalent period of time. Leukocyte recruitment was monitored during I/R; at the end of R, vasodilating endothelium-dependent and -independent reserve was assessed by local superfusion with 10-4 µM acetylcholine and 10-5 µM sodium nitroprusside. Phosphorylation of ERK-1/2, Akt, eNOS, PKCe and GSK3β was measured by Western Blotting on muscle homogenates obtained at the end of experiment in a second set of experiments in which, to avoid possible confounding effects of acetylcholine and sodium nitroprusside on kinase activation, rats were divided in 4 groups as described above but vasodilating challenge was not performed. Results: I/R activated leukocyte recruitment (data not shown) and reduced vasodilating reserve, both endothelium-dependent and independent. Both preC and postC activated kinases and eNOS in a similar degree, and reduced leukocyte recruitment. However, only preC preserved vasodilating reserve (both endothelium-dependent and independent) (*=p

Tissue factor (TF) is an essential enzyme activator that forms a catalytic complex with FVII(a) and initiates coagulation by activating FIX and FX, ultimately resulting in thrombin formation. TF is found in adventitia of blood vessels and the lipid core of atherosclerotic plaques. In unstable coronary syndromes, plaque rupture initiates coagulation by exposing TF to blood. Biologically active TF has been detected in vessel walls and circulating blood. Elevated intravascular TF has been reported in diverse pro-thrombotic syndromes such as myocardial infarction, sepsis, anti-phospholipid syndrome and sickle-cell disease. It is unclear how TF circulates, although it may be present in pro-coagulant microparticles. We now report identification of a form of human TF generated by alternative splicing. Our studies indicate that alternatively spliced human tissue factor (asHTF) contains most of the extracellular domain of TF but lacks a transmembrane domain and terminates with a unique peptide sequence. asHTF is soluble, circulates in blood, exhibits pro-coagulant activity when exposed to phospholipids, and is incorporated into thrombi. We propose that binding of asHTF to the edge of thrombi contributes to thrombus growth by creating a surface that both initiates and propagates coagulation.

Introduction: Recruitment of pericytes (PC), critical to microvascular maturation, requires deposition of basement membrane proteins such as von Willebrand factor (vWF). Loss of PCs ultimately leads to vessel regression and rarefaction. Hypothesis: Splice variants of tissue factor (TF) stabilize the vasculature by supporting EC-PC interactions. Results: We determined that alternatively spliced TF (asTF) resides in Weibel Palade bodies (WPB) of ECs. Overexpression of KLF-2 enhanced asTF protein levels in WPBs. In confluent EC monolayers, asTF was deposited into the extracellular matrix (ECM). In assays employing vascular plexus remodelling on 3D-basement membrane and purified recombinant asTF or full-length TF (flTF), only asTF improved EC-PC interactions. At sites of low KLF-2 expression (low or turbulent flow), total TF mRNA was diminished, leading to low asTF expression. However, upon EC activation by TNFα, total TF mRNA levels rapidly increased and TF protein expression shifted from the ECM-deposited asTF to luminally expressed flTF. Interestingly, flTF-driven EC conversion to a procoagulant state was associated with the release of vWF and asTF from WPBs, depleting the intracellular depots from these proteins. Immunohistochemical staining of kidney specimens of human living donors confirmed that asTF was present at high levels in WPBs of microvascular endothelium. Moreover, after 45 minutes of reperfusion of the transplanted kidney, asTF- and vWF-staining was markedly reduced, consistent with their release from WPB following an inflammatory insult. Conclusion: We show for the first time that quiescent, KLF-2 expressing ECs predominantly generate non-coagulant asTF, which is stored in WPBs and serves to stabilize the microvasculature by supporting EC-PC interactions. In contrast, in low-flow zones or sites with disturbed flow (e.g. at bifurcations), KLF-2 is downregulated, leading to decreased production of asTF and impaired stabilization of the vasculature. During inflammation, acute perturbation of ECs (e.g. exposure to TNFα) is associated with downregulation of KLF-2 and a post-transcriptionally regulated increase in the production of flTF, resulting in a pro-coagulant phenotype and loss of EC-PC interactions.

Background: Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment to successful hematogenous tumor cell metastasis. Objective: Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TMPromice). Methods: Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis. Results: TF-dependent, but contact pathway-independent, syngeneic breast cancer metastasis was associated with marked platelet hyperreactivity and formation of leukocyte-platelet aggregates in immune-competent TMPromice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TMPromice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TMPromice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TMPromice. Conclusions: Metastasis in the hyperthrombotic TMPromouse model is mediated by platelet hyperreactivity and contributions of PAR1 signaling on tumor and host cells. © 2013 International Society on Thrombosis and Haemostasis.

Background: Pancreatic cancer remains extremely difficult to treat, resulting in an 80% 1-year mortality rate. Tissue Factor (TF) serves as a co-factor for serine protease factor VIIa and the full-length (fl)TF/VIIa complex initiates blood coagulation. Alternatively spliced isoform of TF (asTF) occurs naturally, is minimally coagulant and, in contrast to flTF, dispensable for normal hemostasis. flTF is overexpressed in many forms of cancer and contributes to malignant growth; however, targeting flTF is risky due to the possibility of bleeding complications. We previously described that asTF is highly expressed in pancreatic ductal adenocarcinoma (PDAC) which account for ~75% of pancreatic cancer cases. Through non-canonical interactions with β1 integrins, asTF promotes activation of Akt and p42/44 MAPK, thus potentiating cancer cell migration and proliferation. We recently developed asTF-specific monoclonal antibody that inhibited growth and spread of PDAC in vivo in an orthotopic setting (Unruh et al, Oncotarget, in press). At this time it is not known whether, aside from PDAC, asTF also contributes to pathobiology of other pancreatic tumors such as pancreatic neuroendocrine tumors (pNET). Although pNETs comprise a minority subset of pancreatic cancer cases, pNET incidence is rapidly increasing and new therapies are thus needed for patients who ultimately progress on second line therapies which include mTOR inhibitors. Objective: To evaluate i) expression of asTF in pNET, and ii) potential utility of targeting intracellular pathways engaged by asTF in pNET. Methods: A tissue microarray containing 6 human pNET specimens was immunostained for asTF. Two human pNET cell lines, QGP1 and BON (a kind gift of Dr. Courtney Townsend, UTMB), were evaluated for TF isoform expression and their responsiveness to mTOR inhibition using the dual PI3K/mTOR ATP-site competitive inhibitor BEZ235. Results: All 6 pNET tissue specimens stained positive for asTF: 4 samples were highly positive and the remaining 2 moderately positive; normal pancreatic tissue specimens in the microarray exhibited minimal staining for asTF. qRT-PCR and western blotting analyses revealed that BON and QGP1 express flTF as well as asTF mRNA and protein, respectively. Compared to QGP1 cells, BON cells expressed ~3 fold more asTF and ~10 fold more flTF. Western blotting revealed that, compared to QGP1 cells, BON cells express significantly higher levels of β1 integrin. Treatment of QGP1 and BON cells with BEZ235 (400nM, 48 hours) markedly suppressed phosphorylation of mTOR targets including Akt, S6K1, 4E-BP1, and ULK1. Conclusion: To our knowledge, this is the first study to report that flTF and asTF are expressed in pNET tumor tissue and cultured cells. In pNET cell lines QGP1 and BON, BEZ235 effectively reduced phosphorylation of Akt, a downstream target of the pathways activated by asTF:β1 interactions. Further studies are warranted to evaluate asTF as a potential target in pNET, specifically strategies aimed at concurrent inhibition of asTF:β1 interactions and Akt/S6K1 activity. Citation Format: Clayton S. Lewis, Hala Elnakat Thomas, Fred V. Lucas, Vladimir Y. Bogdanov.{Authors}. Expression of alternatively spliced tissue factor in pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A39.

Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells.

Upregulation of tissue factor (TF) expression on activated donor endothelial cells (ECs) triggered by the immune response (IR) has been considered the main initiator of consumptive coagulopathy (CC). In this study, we aimed to identify potential factors in the development of thrombocytopenia and CC after genetically engineered pig liver transplantation in baboons. Baboons received a liver from either an α1,3-galactosyltransferase gene-knockout (GTKO) pig (n = 1) or a GTKO pig transgenic for CD46 (n = 5) with immunosuppressive therapy. TF exposure on recipient platelets and peripheral blood mononuclear cell (PBMCs), activation of donor ECs, platelet and EC microparticles, and the IR were monitored. Profound thrombocytopenia and thrombin formation occurred within minutes of liver reperfusion. Within 2 h, circulating platelets and PBMCs expressed functional TF, with evidence of aggregation in the graft. Porcine ECs were negative for expression of P- and E-selectin, CD106, and TF. The measurable IR was minimal, and the severity and rapidity of thrombocytopenia were not alleviated by prior manipulation of the IR. We suggest that the development of thrombocytopenia/CC may be associated with TF exposure on recipient platelets and PBMCs (but possibly not with activation of donor ECs). Recipient TF appears to initiate thrombocytopenia/CC by a mechanism that may be independent of the IR.

Rationale: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. Methods and Results: Cell-specific knockout mice were generated by Cre/ loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout ( myeKlf2 −/− ) mice were similar to myeKlf2 +/+ macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2 +/+ macrophages, myeKlf2 −/− macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2 −/− mice also adhered more robustly to endothelial cells, and fewer myeKlf2 −/− neutrophils survived in culture over a 24-hour period in comparison with myeKlf2 +/+ neutrophils. When myeKlf2 −/− mice were mated to Ldlr −/− mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2 −/− Ldlr −/− mice compared with myeKlf2 +/+ Ldlr −/− littermates. The increased atherosclerosis in myeKlf2 −/− Ldlr −/− mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2 +/+ Ldlr −/− mice. Conclusions: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.

Summary. Background: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. Objective: To examine the role of integrin signaling in TF-dependent recruitment of monocytes by endothelial cells. Methods: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ-TF) and recombinant asTF were assessed. Results: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid-rich plaques. Tumor lesions, as well as stromal CD68+ monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ-TF, and this was completely blocked by anti-β1 integrin antibody. asTF- and LZ-TF-treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF-elicited adhesion was more pronounced than that elicited by LZ-TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E-selectin, ICAM-1 and VCAM-1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ∼3-fold under MCP-1 gradient. Conclusions: TF splice variants ligate β1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non-proteolytic, integrin-mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.

Tissue factor profile of human monocytic cells, afforded by alternative splicing, can be greatly influenced by the activity of SR proteins. Following recruitment to solid tissues, peripheral blood monocytes express a number of proinflammatory molecules including TF, a trigger of coagulation that also promotes cell–cell interactions and tissue remodeling. Monocytes express two forms of TF: flTF, a highly coagulant transmembrane form, and asTF, a highly proangiogenic, soluble TF form. Biosynthesis of the two TF forms occurs via alternative processing of exon 5 during pre-mRNA splicing. Its inclusion results in flTF mRNA and its exclusion, asTF mRNA. We developed a splicing reporter system recently and determined that two spliceosomal constituents, SR proteins ASF/SF2 and SRp55, play a pivotal role in exon 5 inclusion. In this report, we show for the first time that two other SR proteins expressed in human monocytes, SRp40 and SC35, antagonize ASF/SF2 and SRp55 by competing for binding to certain sites in exon 5, thereby promoting TF exon 5 exclusion, an event unique to asTF biosynthesis. We also show that the intron preceding TF exon 5 possesses characteristics rarely found in U2 introns. Our findings indicate that modulation of TF pre-mRNA splicing can be accomplished via modification of SR proteins’ activity, facilitating development of novel therapeutic strategies to modulate the “TF profile” of monocytes/macrophages.

Summary. Background: Human monocytes express two naturally occurring forms of circulating tissue factor (TF) – full-length TF, a membrane-spanning protein, and alternatively spliced TF, a soluble molecule. Presence of the variable exon 5 in TF mRNA determines whether the encoded TF protein is transmembrane, or soluble. Recently, an essential SR protein ASF/SF2 was implicated in TF pre-mRNA processing in human platelets. Objective: To examine molecular mechanisms governing regulated processing of TF pre-mRNA in human monocytic cells. Methods and results: In silico analysis of the human TF exon 5, present only in full-length TF mRNA, revealed putative binding motifs termed exonic splicing enhancers (ESE) for the SR proteins ASF/SF2 and SRp55, which were found to be abundantly expressed in monocytic cell lines THP-1 and SC, as well as monocyte-enriched peripheral blood mononuclear cells (PBMC). Using a splice competent mini-gene reporter system transiently expressed in monocytic cells, it was determined that weakening of either five closely positioned ASF/SF2 ESE (bases 87–117) or a single conserved SRp55 ESE (base 39) results in severe skipping of exon 5. ASF/SF2 and SRp55 were found to physically associate with the identified ESE. Conclusions: SR proteins ASF/SF2 and SRp55 appear to interact with the variable TF exon 5 through ESE at bases 39 and 87–117. Weakening of the above ESE modulates splicing of TF exon 5. This study is the first to identify and experimentally characterize cis-acting splicing elements involved in regulated biosynthesis of human TF.

Purpose: Here we study the mechanisms by which alternatively spliced Tissue Factor (asTF) contributes to pancreatic cancer progression in an advanced tumor micro-environment. Background: There is a well-established association of the hemostatic system with cancer. Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main trigger of coagulation, undergoes alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF does not have a physiologic function in hemostasis, but its expression positively correlates with advanced tumor stages, including pancreatic adenocarcinoma; asTF acts as a cell agonist by binding beta-1 intregrins. The hypoxia-inducible enzyme carbonic anhydrase IX (CAIX) may be overexpressed by cancer cells as a result of increased glycolysis and acidic pH. While it increases cancer cell survival by maintaining intracellular pH, carbonic anhydrase IX decreases the extracellular pH, making the extracellular space more acidic, which can increase cancer cell motility. Methods: asTF-overexpressing pancreatic ductal adenocarcinoma cell line Pt45P1/asTF+ and the parent cell line Pt45P1 were tested for growth and mobility under normoxic conditions (5% CO2, ambient O2, 1000 mg/ml glucose) that model early stage tumors, and in the hypoxic environment of advanced-stage cancers (20% CO2, 1% O2 and 5 mM lactate). Results: asTF overexpression in Pt45P1 cells conveyed higher proliferative ability. According to propidium iodide staining and flow cytometry, the major fraction of Pt45P1/asTF+ cells resides in the dividing G2/M phase of the cell cycle, while control Pt45P1 cells are mostly confined to the quiescent G0/G1 phase. asTF overexpression is also associated with significantly higher cell mobility. These observations were similar for cells plated under both normoxia and hypoxia. While normoxic cell culture conditions have been traditionally designed to maximize cell growth, an aggressive phenotype that may arise in an advanced-stage micro-environment prompted us to study the mechanisms that might promote invasive behavior under hypoxic tumor conditions. In a hypoxic environment, we observed an upregulation of carbonic anhydrase IX which was more pronounced in Pt45P1/asTF+ cells. Inhibition of carbonic anhydrase IX by the compound U-104 significantly decreased cell growth and mobility of Pt45P1/asTF+ cells in hypoxia, but not in normoxia. Conclusion: Carbonic anhydrase IX is a novel downstream mediator of the asTF's effects in pancreatic cancer progression under hypoxic conditions that model advanced stage tumor micro-environment. Citation Format: Divya Ramchandani, Dusten Unruh, Vladimir Y. Bogdanov, Georg F. Weber. Alternatively spliced tissue factor promotes pancreatic cancer progression via carbonic anhydrase IX. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3001. doi:10.1158/1538-7445.AM2015-3001

Summary. Tissue factor (TF) is a transmembrane glycoprotein that initiates coagulation and plays a critical role in regulating hemostasis and thrombosis. We have recently reported a naturally occurring, soluble form of human tissue factor (asTF) generated by alternative splicing. This splice variant has a novel C-terminus with no homology to that of the full-length TF (flTF), lacks a transmembrane domain, and is active in the presence of phospholipids. Mouse models offer unique opportunities to examine the relative importance of flTF and asTF in mediating thrombosis, the response to arterial injury, and ischemic damage. To that end, we have identified and characterized murine asTF (masTF). Like the human splice variant, masTF lacks a transmembrane domain and has a unique C-terminus. We have generated antibodies specific to masTF and murine flTF (mflTF) to examine the expression of both forms of TF. masTF antigen is widely and abundantly expressed, with a pattern similar to that of mflTF, in adult tissues, in experimentally induced thrombi, and during development. These studies demonstrate that masTF contributes to the pool of total TF and may thus play an important role in mediating TF-dependent processes.

Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF). Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. asTF is abundant in PDAC lesions and its constitutive overexpression promotes tumor growth, metastatic spread, monocyte recruitment, and elevated procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we determined if 1) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; 2) asTF contributes to PDAC cell migration; and 3) monoclonal antibody-based targeting of tumor-derived asTF slows PDAC progression and/or asTF release into circulation in an orthotopic setting. Nude mice were orthotopically transplanted with human PDAC cells harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi); mice received Dox (2µg/mL) in sucrose at day 1 (“Dox”), day 25 (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo over 7 weeks via SapC-DOPS imaging. Migration of Pt45P1/asTFi cells toward serum was assessed using laminin-coated transwell inserts. Plasma of PDAC tumor-bearing mice was assayed for asTF using a custom ELISA. Pt45P1 cells were resuspended in PBS containing anti-asTF rabbit monoclonal antibody Rb1 and implanted in a) nude mice; b) SCID mice, and c) SCID mice with TF levels reduced by 99% (SCID/TF-low) and tumor progression was monitored as above over 5 weeks. Paraffin-embedded tumor specimens were assessed for vessel density (isolectin B4 and/or anti-CD31 staining) and monocyte/macrophage infiltration (anti-F4/80 and/or anti-CD206 staining). “Late Dox” mice developed tumors comparable in size and vessel density to those in “Dox” mice, yet less infiltrated with macrophages (Dox: 70±22 cells/LPF, late Dox: 51±17 cells/LPF, p=0.009); tumor spread was significantly reduced in "late Dox" mice compared to "Dox" mice (7.52e+7 vs 1.22e+8 P/s/mm2, p=0.01). "Dox" mice also had high levels of asTF in circulation (~1 ng/mL), which did not differ significantly from those in “late Dox” mice. The PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p We here report that asTF can promote PDAC progression during early and late stages of the disease. asTF-β1 integrin interaction renders PDAC cells more motile, while Rb1 fully inhibits asTF-potentiated PDAC cell migration. Host-derived TF has no impact on the size of primary PDAC tumor, yet it contributes to tumor vascularization and, consequently, metastatic spread. In the presence of Rb1 in nude and/or SCID mice, Pt45P1 cells grow significantly smaller tumors with fewer monocytes and blood vessels that release less asTF in the circulation. Thus, antibody-based targeting of asTF may comprise a novel strategy to stem PDAC progression. Disclosures No relevant conflicts of interest to declare.

Tissue Factor (TF) present in blood cells and plasma is referred to as blood-borne or circulating TF. TF has been implicated in the pathogenesis of several chronic disease states, most notably cardiovascular disease/thrombosis, diabetes, and cancer. Full-length TF is an integral membrane protein while alternatively spliced TF can be secreted in a free form and features a unique C-terminal domain enabling its selective detection in bio-specimens. Recently, asTF was shown to circulate in the blood of metastatic breast cancer patients at concentrations exceeding 1 ng/mL (Kocaturk et al, PNAS 2013), and it promoted tumor growth and spread in an orthotopic model of pancreatic ductal adenocarcinoma (PDAC, Unruh et al, Int J Cancer, 2014). asTF protein acts as a cell agonist driving angiogenesis, cancer cell proliferation, and monocyte recruitment via integrin binding. It is not known whether circulating asTF may contribute to or serve as a biomarker in patients suffering from cardiovascular disease, diabetes, and/or solid cancers including PDAC. We evaluated circulating asTF in healthy subjects and individuals with ongoing acute coronary syndrome (ACS); diabetes mellitus (DM); ongoing ACS+DM; and PDAC. Samples of platelet poor plasma from 204 subjects were obtained from University of Cincinnati Cancer Institute’s Tumor Bank and Diagnostica Stago collections, blood specimens drawn from emergency room visitors at four medical centers in the US, and George King Bio-Medical, Inc. ACS was defined by positive troponin levels; DM was self-identified. Blood was drawn into tubes containing heparin (ASC, DM, ACS+DM), acid citrate dextrose (PDAC), or sodium citrate (healthy subjects), centrifuged at 3000 rpm for 15 min at 4°C, and stored at -80°C until use. Blinded asTF ELISA was performed on plasma samples as per the prototype-tailored procedure (Diagnostica Stago). Samples with asTF concentrations ≥0.2 ng/mL were deemed positive. asTF concentrations are presented as mean±SD. Kruskal-Wallis one-way analysis of variance was used to compare differences in concentration levels between the cohorts; Chi-Square and/or Fisher’s exact test were used to compare proportions. asTF protein was detectable in the plasma of 3/19 (15.8%) subjects in the healthy cohort (CORE Set 50, George King Bio-Medical); 7/38 (18.4%) in the no ACS/no DM cohort (emergency room visitors’ control group); 2/40 (5%) in the DM cohort; 5/39 (12.8%) in the ACS cohort; 4/25 (16.0%) in the ACS/DM cohort; and 20/43 (46.5%) in the PDAC cohort; the proportion of PDAC patients positive for asTF was significantly higher compared to that in all other cohorts (p We here report that asTF at levels ≥0.2 ng/mL occurs more frequently in the plasma of patients with PDAC compared to healthy subjects and/or individuals with ACS, DM, and ACS/DM. Further, PDAC patients whose plasma asTF levels were equal to or exceeded 0.2 ng/mL had a significantly lower chance to qualify for tumor resection, irrespective of initial pre-surgical diagnostic evaluation. asTF may thus comprise a novel marker of aggressive PDAC phenotype with potential utility in patient stratification, warranting prospective evaluation of larger PDAC patient cohorts. Disclosures No relevant conflicts of interest to declare.

Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.