low-TF Mice Research Articles

Abstract 609: Heterozygous Tissue Factor Deficiency Protects Against Factor VII Activation And Consumption In A Murine Endotoxemia Model

Background: At least 1 in 25,000 humans carry F3 null alleles predicted to cause heterozygous tissue factor (TF) deficiency. Mice with heterozygous TF deficiency ( F3 +/- ) exhibit increased bleeding times and reduced thrombus formation in some models, and are protected against lethal thrombosis in penetrant thrombophilia. TF supports the activation of factor VII (FVII) in vitro , an effect that is evident in vivo in the extreme case of low-TF mice expressing ~1% of basal TF levels. Aim: To determine the impact of heterozygous TF deficiency on FVII activation in vivo . Methods: Citrated plasma was isolated via inferior vena cava blood draw from F3 +/- and wildtype littermate mice at 10-11 weeks of age. To induce coagulation activation through endotoxemia, mice were challenged with intraperitoneal injection of 8 mg/kg LPS for 6 hours. Plasma TF was determined by ELISA. FVII and FVIIa levels were measured by FVII activity and Staclot VIIa-rTF clotting assays, respectively. n≥10 mice in all experiments. Results: F3 +/- mice have significantly reduced plasma TF antigen levels compared to wildtype littermate controls under basal conditions (6.5 pg/mL vs 11.6 pg/mL, p<0.001), differences that persist following endotoxemic challenge (9.9 pg/mL vs 14.8 pg/mL, p<0.001). At baseline, F3 +/- mice had similar circulating levels of zymogen FVII and FVIIa compared to controls. However, the levels of FVIIa correlated with FVII activity levels in F3 +/- mice (r 2 =0.66, p=0.003), whereas no correlation between FVIIa and FVII activity levels was observed in controls (r 2 =0.17, p=0.23). During endotoxemia, F3 +/- mice maintained significantly higher total FVII (85.6% vs 66.1%, p<0.05) and FVIIa (52.9% vs 35.4%, p<0.05) as compared to controls. Conclusions: FVII and FVIIa levels are unaffected by heterozygous TF deficiency in healthy mice, but FVIIa levels are dependent on the amount of FVII when TF is limiting. When TF expression and coagulation activation are augmented as during endotoxemic challenge, F3 +/- mice are protected against aberrant FVII activation and consumption. These studies suggest that human genetic variation influencing TF dosage could influence FVII activation and disease outcomes.

Cardiac Tissue Factor Regulates Inflammation, Hypertrophy, and Heart Failure in Mouse Model of Type 1 Diabetes.

Patients with diabetes have an increased risk of heart failure (HF). Diabetes is highly prevalent in HF with preserved ejection fraction (HFpEF), which is on the rise worldwide. The role of diabetes in HF is less established, and available treatments for HF are not effective in patients with HFpEF. Tissue factor (TF), a transmembrane receptor, plays an important role in immune cell inflammation and atherothrombosis in diabetes. However, its role in diabetes-induced cardiac inflammation, hypertrophy, and HF has not been studied. In this study, we used wild-type (WT), heterozygous, and low-TF (with 1% human TF) mice to determine the role of TF in type 1 diabetes-induced HF. We found significant upregulation of cardiac TF mRNA and protein levels in diabetic WT hearts compared with nondiabetic controls. WT diabetic hearts also exhibited increased inflammation and cardiac hypertrophy versus controls. However, these changes in cardiac inflammation and hypertrophy were not found in low-TF mice with diabetes compared with their nondiabetic controls. TF deficiency was also associated with improved cardiac function parameters suggestive of HFpEF, which was evident in WT mice with diabetes. The TF regulation of inflammation and cardiac remodeling was further dependent on downstream ERK1/2 and STAT3 pathways. In summary, our study demonstrated an important role of TF in regulating diabetes-induced inflammation, hypertrophy, and remodeling of the heart leading to HFpEF.

Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor

AbstractThe intrinsic tenase complex (FIXa-FVIIIa) of the intrinsic coagulation pathway and, to a lesser extent, thrombin-mediated activation of FXI, are necessary to amplify tissue factor (TF)-FVIIa–initiated thrombin generation. In this study, we determined the contribution of murine FIX and FXI to TF-dependent thrombin generation in vitro. We further investigated TF-dependent FIX activation in mice and the contribution of this pathway to hemostasis. Thrombin generation was decreased in FIX- but not in FXI-deficient mouse plasma. Furthermore, injection of TF increased levels of FIXa-antithrombin complexes in both wild-type and FXI−/− mice. Genetic studies were used to determine the effect of complete deficiencies of either FIX or FXI on the survival of mice expressing low levels of TF. Low-TF;FIX−/y male mice were born at the expected frequency, but none survived to wean. In contrast, low-TF;FXI−/− mice were generated at the expected frequency at wean and had a 6-month survival equivalent to that of low-TF mice. Surprisingly, a deficiency of FXI, but not FIX, exacerbated the size of blood pools in low-TF placentas and led to acute hemorrhage and death of some pregnant dams. Our data indicate that FIX, but not FXI, is essential for survival of low-TF mice after birth. This finding suggests that TF-FVIIa–mediated activation of FIX plays a critical role in murine hemostasis. In contrast, FXI deficiency, but not FIX deficiency, exacerbated blood pooling in low-TF placentas, indicating a tissue-specific requirement for FXI in the murine placenta under conditions of low TF.

A combined deficiency of tissue factor and PAR-4 is associated with fatal pulmonary hemorrhage in mice

IntroductionMice with a complete absence of tissue factor (TF) die during embryonic development whereas mice with low levels of TF (Low-TF mice) survive to adulthood. Low-TF mice exhibit spontaneous hemorrhage in various organs, including the lung. In contrast, mice can survive without protease-activated receptor (PAR)-4, which is the major thrombin receptor on mouse platelets. We determined the effect of combining a deficiency PAR-4 (primary hemostasis) with a deficiency in TF (secondary hemostasis) on embryonic development and survival of adult mice. Materials and methodsLow-TF mice (mTF−/−, hTF+/+) were crossed with PAR-4−/− mice to generate heterozygous mice (mTF+/−, hTF+/−, PAR-4+/−). These mice were intercrossed to generate Low-TF mice lacking PAR-4. Mice surviving to wean were genotyped and survival was monitored for 6months. ResultsWe observed the expected number of Low-TF,PAR-4−/− mice at wean indicating survival in utero and after birth. However, an absence of PAR-4 was associated with premature death of all Low-TF,PAR-4−/− mice in the 6month observational period. This compares with 40% death of the Low-TF,PAR-4+/+ mice (p=0.003). Low-TF,PAR-4+/− mice had an intermediate phenotype with 55% of the mice dying within 6months. The primary cause of mortality of Low-TF,PAR-4−/− mice was pulmonary hemorrhage. ConclusionsLow-TF,PAR-4−/− mice survive into adulthood, but combining a deficiency of primary hemostasis (PAR-4 deficiency) with secondary hemostasis (low levels of TF) leads to premature death primarily due to pulmonary hemorrhage.

Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus‐infected mice

Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection. Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection.

Pharmacological Concentrations of Recombinant Factor VIIa Restores Hemostasis in Acquired Hemophilia Primarily Independent of Tissue Factor

High doses of recombinant FVIIa (rFVIIa) are used successfully to treat hemophilia patients with inhibitory antibodies against FVIII and FIX. Despite its widespread and successful use of treating bleeding disorders for many years, the understanding of the mechanism of rFVIIa action in hemophilia therapy is incomplete. A majority of the studies, mostly in vitro studies, suggested that activation of FX by rFVIIa bound to phospholipids exposed on the activated platelets is responsible for the hemostatic effect of rFVIIa in hemophilia patients. However, other in vitro studies and mathematical modeling indicated that the action of rFVIIa at therapeutic doses is dominated by the TF-dependent pathway, partly because of the need to overcome the inhibitory effect of zymogen FVII, with a minor contribution from phospholipid-dependent mechanism. In the present study, we investigated whether TF plays a role in rFVIIa-mediated hemostasis in hemophilia by employing transgenic mice expressing low levels of human TF (Low TF mice) or normal levels of human TF (HTF mice; breeding pairs for these mice were provided by Nigel Mackman). Mice were administered with FVIII mAb to induce acquired hemophilia. The saphenous vein incision bleeding model was used to evaluate the hemostasis. In this model, saphenous vein was exposed and bleeding was induced by a sharp incision of the vein with a fine scissors. The bleeding from the cut was monitored for 30 min while blood was collected onto a filter paper by capillary action. After each hemostasis incident, the clot was disrupted gently to reinitiate a new bleeding episode, and average time to achieve hemostasis (ATH) was calculated.First, we investigated the role of TF in hemostasis by comparing the bleeding episodes in low TF and HTF mice following the saphenous vein incision. In the saphenous vein bleeding model, control wild-type mice had a median ATH of 60 sec (n=6) and hemophilia A mice failed to achieve hemostasis within 30 min experimental time frame (n=6). HTF mice had essentially very similar ATH compared to that of the wild-type mice, i.e., 64 sec (n=5). In contrast, low TF mice had a substantially longer median ATH, 90 sec (n=6), which is statistically significantly longer than that of ATH of wild-type or HTF mice. Administration of TF antibodies (1 mg/kg) to neutralize remaining traces of TF in Low TF mice further prolonged the bleeding time to a median ATH of 309 sec (n=6). These data clearly illustrates the role of TF in hemostasis.Next, we created acquired hemophilic condition in low TF and HTF mice by injecting FVIII mAb (1 mg/kg). Low TF mice also received TF antibodies. Administration of FVIII mAb markedly prolonged the bleeding time in both groups of mice (between 300 to 1800 sec), with a median ATH of 900 sec. Administration of rFVIIa (4 mg/kg) to HTF hemophilic mice reduced the ATH to a median of 51 sec (mean ± SD, 50 ± 5 sec, n=6). The same concentration of rFVIIa also restored hemostasis in hemophilic low TF mice with a median ATH of 66 sec (mean ±SD, 70 ± 18 sec, n=6). This value is not statistically significantly different from that of ATH of wild-type mice. Although rFVIIa restored the hemostasis in both HTF and low TF mice, it appears that rFVIIa is slightly, but statistically significantly, more effective in HTF mice compared to low TF mice in restoring the hemostasis (p = 0.025). However, we did not observe any significant differences between these two groups in the volume of blood loss following rFVIIa administration. In summary, our data strongly support the hypothesis that high concentrations of rFVIIa restore hemostasis in hemophilia primarily in TF-independent manner. More detailed studies are needed to completely eliminate the role of TF in rFVIIa mediated hemostasis. DisclosuresNo relevant conflicts of interest to declare.

Impaired Hemostasis Caused By Deficit of Tissue Factor-Triggered Blood Coagulation Leads to Alveolar Hemorrhages and Increased Mortality in Influenza A Infection

Objective: Viral infections lead to activation of coagulation which enhances inflammatory responses. For instance, influenza A is associated with activation of coagulation and the increased risk for thrombotic events, such as myocardial infarction. Here we investigated the role of the coagulation cascade and platelets in influenza A virus (IAV) infection in mice.Methods: To investigate the role of the coagulation cascade in influenza infection, we examined the effect of either low levels of tissue factor or wild-type mice that were administered with 4 μg/mL of warfarin via drinking water. To study the role of platelets in influenza infection, we examined protease-activated receptor 4 (PAR-4, the main functional receptor on mouse platelets) deficient mice or wild-type mice treated with 0.15 mg/mL of clopidogrel or 0.4 mg/mL of aspirin via drinking water. All mice were infected intranasally with mouse adapted influenza A H1N1/PR8 (IAV). Changes in body weights and survival were analyzed up to 14 days after infection. In addition, bronchoalveolar lavage fluid (BALF) or lungs were collected and analyzed 7 or 14 days after virus challenge.Results: LowTF mice exhibited higher mortality rate and increased loss of body weights compared to littermate control mice after IAV infection (P<0.01). At day 7, BALF of LowTF was red and had increased red blood cell numbers and hemoglobin levels compared to infected control mice (P<0.001). Thrombin-antithrombin complex (TAT) levels in the BALF of infected LowTF mice were lower than that of control mice. In addition, lung histology revealed that LowTF mice had extensive hemorrhages. Similarly, mice treated with warfarin and infected with IAV showed higher mortality compared with the warfarin-treated mice without infection or IAV-infected mice without drugs. Warfarin treated mice also had red BALF, higher red blood cell number and hemoglobin levels in the BALF, and lung bleeding. PAR-4 deficient mice exhibited visible lung hemorrhages with increased hemoglobin levels in the BAL (P<0.01) and increased mortality compared to controls (P<0.05), although the bleeding was less than LowTF mice. Treatment of mice with clopidogrel or aspirin did not increased lung bleeding or mortality.Conclusion: Pulmonary hemostasis requires both TF-mediated activation of blood coagulation and thrombin activation of platelets during IAVinfection. Use of antithrombotics during IAV infection may increase the risk of lung bleeding. DisclosuresNo relevant conflicts of interest to declare.

Impaired Hemostasis Leads To Alveolar Hemorrhages and Increased Mortality In Influenza A Infection

ObjectiveViral infections lead to activation of coagulation which enhances inflammatory responses. For instance, influenza A is associated with activation of coagulation and the increased risk for thrombotic events, such as myocardial infarction. Protease-activated receptor 4 (PAR-4) is the main functional receptor on mouse platelets. Here, we investigated the role of tissue factor (TF) and platelets in influenza A infection in mice. MethodsWe used male LowTF mice, which express 1% of normal TF levels, PAR-4 deficient mice and respective control mice. All mice were infected intranasal with influenza A H1N1/PR8. Changes in body weights and survival were analyzed up to 14 days after infection. In addition, bronchoalveolar lavage (BAL) was collected and analyzed 7 days after virus challenge. ResultsLowTF mice exhibited increased loss of body weights compared to littermate control mice between day 4 and 6 after infection (P<0.05). At day 7, BAL of LowTF had increased levels of free hemoglobin compared to infected control mice (2.60±0.39 g/dL vs. 0.05±0.02 g/dL, P<0.05). In addition, LowTF lungs exhibited increased protein levels in the BAL compared to controls (1.52±0.18 mg/mL vs. 0.70±0.26 mg/mL, P<0.05). Lung histology revealed that LowTF mice had extensive hemorrhages. The impaired hemostasis resulted in increased mortality in LowTF mice compared to control mice (90% vs 30%, P<0.004, N=9-10, Figure). Similarly, mice with defective thrombin signaling in platelets (PAR-4-/-) exhibited visible lung hemorrhages with increased hemoglobin levels in the BAL (P<0.05) at day 7 and increased mortality compared to controls (57% vs 10%, P<0.03, N=9-14, Figure). [Display omitted] ConclusionsTF mediated activation of coagulation and PAR-4 dependent platelet activation are essential for pulmonary hemostasis during influenza A infection. Disclosures:No relevant conflicts of interest to declare.

Abstract 1091: Procoagulant receptor tissue factor provokes the escape from brain tumor dormancy.

Abstract Occult persistence of transformed cells is known to precede the onset of clinically apparent malignancies, accompany disease remission and micrometastasis. While the mechanisms governing this dormant state remain poorly understood, tissue injury and wound healing responses have often been implicated in its cessation, but the surrounding circumstances remain largely obscure. Notably, the activation of hemostasis represents the first response to the exogenous and endogenous tissue injury, and is linked to inflammation, angiogenesis and growth promoting microenvironment. Tissue factor (TF) acts as the main trigger of the coagulation cascade in this setting, and is known to be overexpressed in cancer cells, including in high grade brain tumors such as glioblastoma (GBM). Here we report that in a model of GBM, the TF-mediated activation of the coagulation system may act as the sufficient initial trigger in the cessation of tumor dormancy, and the onset of inflammation and angiogenesis. We also document that while TF may play a role in the subsequent tumor progression, its expression by cancer and host cells may no longer be absolutely required in established tumors. Thus, we observed that indolent human glioma cells (U373) remain viable, but dormant for over 250 days post subcutaneous and orthotopic inoculation, with no evidence of tumor growth, as detected by bioluminescent monitoring. The enforced expression of TF is sufficient to trigger tumor formation by these cells, albeit after long latency (30-90 days). During this latent phase TF-expressing glioma cells, but not their TF-negative counterparts, are able to recruit rich stroma, containing CD11b+ myeloid cells and CD105+ blood vessels. These cells also evolve to become rapidly tumorigenic and gain a dramatic change to their transcriptome. Similar aggressiveness can also be induced in U373 cells by the expression of the oncogenic epidermal growth factor receptor variant (EGFRvIII), which also provokes upregulation of TF and onset of the procoagulant phenotype (U373vIII cells). Targeting TF in U373vIII tumors, as well as their implantation into TF-hypomorphic recipients (low-TF mice) delays, but does not abrogate experimental GBM progression. We generated spontaneous GBM tumors by intracranial injection of the oncogenic cDNA (SV40, N-ras), which becomes expressed in brain astrocytes under the control of the Sleeping Beauty (SB) transposase. Disruption of the TF gene in the astrocytic lineage of such mice (Cre/nestinTF-/-) resulted in a partial tumor growth inhibition. We postulate that activation of the coagulation system (via TF) may play a role in disruption of the dormant behavior of brain tumor cells and can serve as targets in prevention of disease onset. However, targeting TF alone is only partially effective as a measure to control the growth of established astrocytic tumors. Acknowledgments: We thank Dr. John Ohlfest for providing us with the SB transposon system. Citation Format: Nathalie Magnus, Delphine Garnier, Brian Meehan, Maryam Hashemi, Tae-Hoon Lee, Chloe Milsom, Nada Jabado, Rafal Pawlinski, Nigel Mackman, Janusz Rak. Procoagulant receptor tissue factor provokes the escape from brain tumor dormancy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1091. doi:10.1158/1538-7445.AM2013-1091

Role of the tissue factor pathway in the biology of tumor initiating cells

Oncogenic transformation and aberrant cellular differentiation are regarded as key processes leading to malignancy. They produce heterogenous cellular populations including subsets of tumour initiating cells (TICs), also known as cancer stem cells (CSCs). Intracellular events involved in these changes profoundly impact the extracellular and systemic constituents of cancer progression, including those dependent on the vascular system. This includes angiogenesis, vasculogenesis, activation of the coagulation system and formation of CSC-related and premetastatic niches. Tissue factor (TF) is a unique cell-associated receptor for coagulation factor VIIa, initiator of blood coagulation, and mediator of cellular signalling, all of which influence vascular homeostasis. Our studies established a link between oncogenic events, angiogenesis and the elevated expression of TF in several types of cancer cells. The latter suggests that cancer coagulopathy and cellular events attributed to the coagulation system may have cancer-specific and genetic causes. Indeed, in human glioma cells, a transforming mutant of the epidermal growth factor receptor (EGFRvIII) triggers not only the expression of TF, but also of its ligand (factor VII) and protease activated receptors (PAR-1 and PAR-2). Consequently, tumour cells expressing EGFRvIII become hypersensitive to contact with blood borne proteases (VIIa, thrombin), which upregulate their production of angiogenic factors (VEGF and IL-8), and contribute to formation of the growth promoting microenvironment (niche). Moreover, TF overexpression accompanies features of cellular aggressiveness such as markers of CSCs (CD133), epithelial-to-mesenchymal transition (EMT) and expression of the angiogenic and prometastatic phenotype. Conversely, TF blocking antibodies inhibit tumour growth, angiogenesis, and especially tumour initiation upon injection of threshold numbers of tumourigenic cells. Likewise, TF depletion in the host compartment (e.g. in low-TF mice) perturbs tumour initiation. These observations suggest that both cancer cells and their adjacent host stroma contribute TF activity to the tumour microenvironment. We postulate that the TF pathway may play an important role in formation of the vascular niche for tumour initiating CSCs, through its procoagulant and signalling effects. Therapeutic blockade of these mechanisms could hamper tumour initiation processes, which are dependent on CSCs and participate in tumour onset, recurrence, drug resistance and metastasis.

Tissue factor-dependent coagulation contributes to α-naphthylisothiocyanate-induced cholestatic liver injury in mice

Separation of concentrated bile acids from hepatic parenchymal cells is a key function of the bile duct epithelial cells (BDECs) that form intrahepatic bile ducts. Using coimmunostaining, we found that tissue factor (TF), the principal activator of coagulation, colocalized with cytokeratin 19, a marker of BDECs in the adult mouse liver. BDEC injury induced by xenobiotics such as alpha-naphthylisothiocyanate (ANIT) causes cholestasis, inflammation, and hepatocellular injury. We tested the hypothesis that acute ANIT-induced cholestatic hepatitis is associated with TF-dependent activation of coagulation and determined the role of TF in ANIT hepatotoxicity. Treatment of mice with ANIT (60 mg/kg) caused multifocal hepatic necrosis and significantly increased serum biomarkers of cholestasis and hepatic parenchymal cell injury. ANIT treatment also significantly increased liver TF expression and activity. ANIT-induced activation of the coagulation cascade was shown by increased plasma thrombin-antithrombin levels and significant deposition of fibrin within the necrotic foci. ANIT-induced coagulation and liver injury were reduced in low-TF mice, which express 1% of normal TF levels. The results indicate that ANIT-induced liver injury is accompanied by TF-dependent activation of the coagulation cascade and that TF contributes to the progression of injury during acute cholestatic hepatitis.

Contribution of Host-Derived Tissue Factor to Tumor Neovascularization

The role of host-derived tissue factor (TF) in tumor growth, angiogenesis, and metastasis has hitherto been unclear and was investigated in this study. We compared tumor growth, vascularity, and responses to cyclophosphamide (CTX) of tumors in wild-type (wt) mice, or in animals with TF levels reduced by 99% (low-TF mice). Global growth rate of 3 different types of transplantable tumors (LLC, B16F1, and ES teratoma) or metastasis were unchanged in low-TF mice. However, several unexpected tumor/context-specific alterations were observed in these mice, including: (1) reduced tumor blood vessel size in B16F1 tumors; (2) larger spleen size and greater tolerance to CTX toxicity in the LLC model; (3) aborted tumor growth after inoculation of TF-deficient tumor cells (ES TF(-/-)) in low-TF mice. TF-deficient tumor cells grew readily in mice with normal TF levels and attracted exclusively host-related blood vessels (without vasculogenic mimicry). We postulate that this complementarity may result from tumor-vascular transfer of TF-containing microvesicles, as we observed such transfer using human cancer cells (A431) and mouse endothelial cells, both in vitro and in vivo. Our study points to an important but context-dependent role of host TF in tumor formation, angiogenesis and therapy.

CARDIAC TISSUE FACTOR: ROLES IN PHYSIOLOGY AND FIBROSIS

1. The aim of the present review is to discuss: (i) the role of tissue factor (TF) in the heart and focus on its potential role in maintaining normal extracellular cardiac homeostasis; (ii) the means by which TF may be contributing to extracellular matrix regulation and vascular maintenance; and (iii) potential mechanisms whereby reduced TF expression leads to cardiac fibrosis. 2. Tissue factor is the main initiator of the coagulation cascade in response to tissue injury, but it is also involved in numerous other biological processes, including angiogenesis, cell migration, apoptosis, metastasis and inflammation. 3. Tissue factor is implicated in cardiovascular disease and its localization and level of expression in cardiomyocytes suggests a unique role in maintaining the structure and function of cardiac muscle. 4. It has been shown that TF(-/-) mice die in utero as a result of disrupted yolk sac vasculature. Low-TF mice, which have transgenic expression of TF at less than 1% of normal levels, are rescued from lethality. 5. Low-TF mice develop cardiac fibrosis in a gender-dependent manner that may be dependent on differential expression of urokinase plasminogen activator. 6. Intracardiac bleeding in low-TF mice may occur as a result of a primary haemostasis defect and/or as a result of disrupted vascular maintenance. The mechanism, when elucidated, will have important therapeutic implications and may provide novel strategies for the treatment of cardiac fibrosis.

The development of cardiac fibrosis in low tissue factor mice is gender-dependent and is associated with differential regulation of urokinase plasminogen activator

Tissue factor (TF) initiates the protease coagulation cascade in response to tissue injury. Homozygous deficiency of murine TF results in embryonic lethality, which is rescued by low-level expression of human TF. These low-TF mice have been shown to develop cardiac fibrosis. We tested the hypothesis that the development of cardiac fibrosis in low-TF mice results from dysregulated protease expression and is affected by gender. Mice were divided into the age groups 2–5, 6–12, 13–18 and 19+ weeks. Fibrosis was assessed by trichrome staining. Protease expression was measured in male and female mice by RT-PCR for mRNA and zymography, ELISA or immunoblot for protein. Urokinase plasminogen activator (uPA) activity was determined by zymography and chromogenic substrate assay. A marked gender effect was noted for the development of fibrosis, with interstitial collagen deposition occurring from 9 weeks in male low-TF mice, but not until 19 weeks in low-TF females. This delayed onset in females was accompanied by delayed up-regulation of molecular markers of injury. Matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 expression were up-regulated in the hearts of male low-TF mice from 6 to 12 weeks and in females from 19 weeks. MMP/TIMP dysregulation was not seen prior to cardiac fibrosis and did not appear to explain the gender differences. However, uPA expression and activity were down-regulated prior to cardiac fibrosis in low-TF females, but were up-regulated in age-matched males. This suggests that the down-regulation of uPA in female low-TF mice protects them from more severe cardiac fibrosis.

The role of tumor-and host-related tissue factor pools in oncogene-driven tumor progression

Oncogenic events play an important role in cancer-related coagulopathy (Trousseau syndrome), angiogenesis and disease progression. This can, in part, be attributed to the up-regulation of tissue factor (TF) and release of TF-containing microvesicles into the pericellular milieu and the circulation. In addition, certain types of host cells (stromal cells, inflammatory cells, activated endothelium) may also express TF. At present, the relative contribution of host- vs tumor-related TF to tumor progression is not known. Our recent studies have indicated that the role of TF in tumor formation is complex and context-dependent. Genetic or pharmacological disruption of TF expression/activity in cancer cells leads to tumor growth inhibition in immunodeficient mice. This occurred even in the case of xenotransplants of human cancer cells, in which TF overexpression is driven by potent oncogenes (K-ras or EGFR). Interestingly, the expression of TF in vivo is not uniform and appears to be influenced by many factors, including the level of oncogenic transformation, tumor microenvironment, adhesion and the coexpression of markers of cancer stem cells (CSCs). Thus, minimally transformed, but tumorigenic embryonic stem (ES) cells were able to form malignant and angiogenic outgrowths in the absence of TF. However, these tumors were growth inhibited in hosts (mice) with dramatically reduced TF expression (low-TF mice). Depletion of host TF also resulted in changes aff5cting vascular patterning of some, but not all types of tumors. These observations suggest that TF may play different roles growth and angiogenesis of different tumors. Moreover, both tumor cell and host cell compartments may, in some circumstances, contribute to the functional TF pool. We postulate that activation of the coagulation system and TF signaling, may deliver growth-promoting stimuli (e.g. fibrin, thrombin, platelets) to dormant cancer stem cells (CSCs). Functionally, these influences may be tantamount to formation of a provisional ( TF-dependent) cancer stem cell niche. As such these changes may contribute to the involvement of CSCs in tumor growth, angiogenesis and metastasis.

A balance between tissue factor and tissue factor pathway inhibitor is required for embryonic development and hemostasis in adult mice

AbstractInactivation of the murine tissue factor (TF) gene or tissue factor pathway inhibitor 1 (TFPI) gene results in embryonic lethality, indicating that both are required for embryonic development. We have shown that expression of low levels of TF from a transgene (hTF) rescues TF-null embryos. However, low-TF mice (mTF–/–/hTF+) have hemostatic defects in the uterus, placenta, heart, and lung. In this study, we hypothesized that the death of TFPI–/– embryos was due to unregulated TF/FVIIa activity and that the hemostatic defects in low-TF mice were due to insufficient TF expression. Therefore, we attempted to rescue TFPI–/– embryos by reducing TF expression, and to restore hemostasis in low-TF mice by abolishing TFPI expression. Intercrossing TFPI+/–/mTF+/–/hTF+/– mice generated close to the expected number of TFPI–/–/low-TF mice at weaning age from 128 offspring, indicating rescue of TFPI–/– embryos from embryonic lethality. Conversely, a decrease in TFPI levels dose-dependently prolonged the survival of low-TF mice and rescued the hemorrhagic defects in the lung and placenta but not in the heart or uterus. These results indicate that the correct balance between TF and TFPI in different organs is required to maintain hemostasis during embryonic development and in adult mice.

Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall

AbstractLeukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF–/–, hTF-Tg+, or “low-TF”) demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation

AbstractTissue factor (TF) is expressed on nonvascular cells and cells within the vessel wall and circulates in blood associated with microparticles. Although blood-borne TF accumulates into the developing thrombus during thrombus formation, the contribution of blood-borne TF and vessel wall TF to thrombin generation in vivo following vessel injury is unknown. To determine the source and role of blood-borne microparticle TF, we studied arterial thrombus formation in a living mouse using intravital microscopy. Platelet, TF, and fibrin accumulation in the developing thrombus was compared in wild-type and low TF mice. Compared to wild-type mice, low TF mice formed very small platelet thrombi lacking TF or fibrin. Wild-type and low TF mice received transplants of bone marrow from wild-type and low TF mice. Arterial thrombi in low TF bone marrow/wild-type chimeric mice had decreased size and decreased TF and fibrin levels. Arterial thrombi in wild-type bone marrow/low TF chimeric mice showed decreased platelet thrombus size but normal TF and fibrin levels. This demonstrates that blood-borne TF associated with hematopoietic cell-derived microparticles contributes to thrombus propagation.

Role of tissue factor in haemostasis, thrombosis, angiogenesis and inflammation: lessons from low tissue factor mice.

Tissue factor (TF) contributes to a variety of biological processes by generating coagulation proteases and by the activation of protease activated receptors (PARs). Studies, using low TF mice, have provided us with novel insights into the role of TF in tissue-specific haemostasis. Low TF mice exhibit defects in the uterus, placenta, heart and lung, which are all tissues that normally express high levels of TF. We propose that these observed defects are primarily due to a reduction in the level of TF to below a critical threshold that is required to maintain adequate haemostasis. Nevertheless, a reduction in TF:FVIIa-dependent signaling may also reduce cell survival and/or compromise the integrity of the vasculature in these organs. Low TF mice are also a useful tool to study the role of TF and the coagulation cascade in other processes, such as thrombosis, inflammation and angiogenesis.

Mice deficient in tissue factor demonstrate attenuated intimal hyperplasia in response to vascular injury and decreased smooth muscle cell migration.

Tissue factor (TF) is the primary initiator of the coagulation cascade and is thought to play a key role in the generation of arterial thrombosis. Recent studies have suggested that TF mediates inflammatory processes in the arterial wall and may be an important regulator of intimal hyperplasia. We have employed genetically engineered mice (mTF(-/-) /hTF(+)) with markedly diminished TF activity ( approximately 1% normal levels) o examine the role of TF in mediating the response to arterial injury. mTF(-/-)/hTF(+) displayed a marked reduction in intimal hyperplasia (46% decrease in intimal area, 60% decrease in intimal/medial ratio) in response to femoral artery injury when compared to wild type controls. The decreased intimal hyperplasia seen in low TF mice was noted in a model of vascular injury not associated with significant thrombosis, suggesting that it may be mediated by non-procoagulant properties of TF. Smooth muscle cells from mTF(-/-)/hTF(+) mice grew normally in response to serum, but exhibited a marked defect in cell migration in a modified Boyden chamber assay. In contrast, there was no difference in platelet derived growth factor- induced migration, suggesting that the effect of TF on smooth muscle cell migration is agonist dependent. These data suggest that TF may mediate intimal hyperplasia by regulating smooth muscle cell migration.