Tissue Factor Deficiency Research Articles

TLR3-Dependent Induction of TF Is Required to Maintain Lung Hemostasis during Poly IC-Induced ARDS-like Lung Dysfunction in Mice

Background: Respiratory virus infection causes damage to the epithelial-endothelial barrier of the lung. Mice challenged with influenza A virus (IAV) exhibit bleeding in the airspace. IAV increases tissue factor (TF) expression in epithelial cells (EpC) of the lung and deletion of lung EpC TF is associated with increased bleeding. aThe ssRNA viruses IAV undergo a dsRNA genomic phase during viral replication. Poly IC is a chemical mimetic of dsRNA. dsRNA acts like PAMPs and can activate the innate immune receptor, toll-like receptor 3 (TLR3). TLR3 stimulation of endothelial cells increased TF expression. However, the role of TLR3 in virus-induced TF expression in the lung TF has not been studied. Aim: To investigate the role of TLR3 in induction of TF during poly IC induced acute respiratory distress syndrome (ARDS)-like lung dysfunction in mice Methods: The murine lung EpC line, E10, was treated with the dsRNA mimetic (5 µg/mL), poly IC, for 0-6h. TF expression was measured by western blotting and a one-stage plasma clotting assay. Wild-type (WT) mice, mice with lung EpC TF deletion (TF fl/fl;SPCCre +, TFΔEpC) and their controls (TFfl/fl) as wells as TLR3 +/+ and TLR3 -/- mice were used in the study. To induce ARDS, poly IC (100 µg in 50 µL PBS) was administered intranasally daily for 3 days to adult mice (both sexes). Lung function was recorded noninvasively using the Buxco whole-body plethysmography system and plasma and bronchoalveolar lavage fluid (BALF) were collected before and 24 hours after the last administration of poly IC. Cellular composition of the BALF and blood was analyzed by an automated cell counter. Lung TF activity was quantified by one-stage plasma clotting and TF activity of extracellular vesicles (EV-TF) was analyzed by two-stage FXa generation assay. Results: Poly IC stimulation induced NFκB-dependent TF expression in E10 cells in dose and time-dependent manner. In addition, poly IC administration induced ARDS-like lung dysfunction, local lung inflammation and coagulation activation in WT mice compared to PBS-treated WT mice. Lung TF activity was increased 2-fold after poly IC administration compared to PBS-treated mice. Mice with lung EpC TF deficiency (TFΔEpC) did not exhibit an increase in lung TF or EV-TF in BALF after poly IC administration compared to controls. Importantly, TFΔEpC mice exhibited reduced activation of coagulation, impaired pulmonary hemostasis and increased hemorrhage into the airspace after poly IC compared to their littermate controls. In addition, TFΔEpC mice showed increased signs of poly IC-induced ARDS and had reduced lung function, increased lung injury and local inflammation compared to their control mice after poly IC. TLR3 -/- mice had significantly less EV-TF activity and less thrombin-antithrombin complexes (TAT) in BALF compared to TLR3 +/+ mice after poly IC. Conclusion: We show that the dsRNA mimetic poly IC induces lung EpC TF expression in vitro and vivo in part via TLR3-dependent NFκB pathway activation. TLR3 stimulation by intranasal poly IC results in sterile inflammation, and ARDS-like phenotype in mice similarly to that observed after IAV infection. Importantly, a deficiency in lung EpC TF leads to impaired lung hemostasis that is associated with an increased poly IC-induced ARDS phenotype as seen previously in our studies using IAV infection of mice. dsRNA induces induction of lung TF via TLR3. We proposed that induction of TF expression in the lung during respiratory viral infections protects against alveolar hemorrhage but can also contribute to thrombosis.

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.

Inflammasome activation promotes venous thrombosis through pyroptosis

Crosstalk between coagulation and innate immunity contributes to the progression of many diseases, including infection and cardiovascular disease. Venous thromboembolism (VTE), including pulmonary embolism and deep vein thrombosis (DVT), is among the most common causes of cardiovascular death. Here, we show that inflammasome activation and subsequent pyroptosis play an important role in the development of venous thrombosis. Using a flow restriction-induced mouse venous thrombosis model in the inferior vena cava (IVC), we show that deficiency of caspase-1, but not caspase-11, protected against flow restriction-induced thrombosis. Interleukin-1β expression increased in the IVC following ligation, indicating that inflammasome is activated during injury. Deficiency of gasdermin D (GSDMD), an essential mediator of pyroptosis, protected against restriction-induced venous thrombosis. After induction of venous thrombosis, fibrin was deposited in the veins of wild-type mice, as detected using immunoblotting with a monoclonal antibody that specifically recognizes mouse fibrin, but not in the caspase-1-deficient or GSDMD-deficient mice. Depletion of macrophages by gadolinium chloride or deficiency of tissue factor also protected against venous thrombosis. Our data reveal that tissue factor released from pyroptotic monocytes and macrophages following inflammasome activation triggers thrombosis.

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.

Genetic Duplication of Tissue Factor Leads to Partial Specialization of Function in Zebrafish

Tissue factor (TF) is a critical factor for hemostasis in response to tissue injury. Among mouse knockouts of procoagulant factors, those lacking TF have the most severe phenotype, with complete lethality by midgestation. Furthermore, complete loss of TF has never been described in humans. Together, these suggest additional roles in embryonic development beyond coagulation. Zebrafish are a small freshwater teleost fish with a well described hemostatic system, including conservation of the coagulation cascade. Zebrafish are prolific breeders that reproduce through external fertilization, with subsequent rapid and transparent development, allowing studies not possible in mammals. Due to an ancient genomic event, 30-40% of the teleost genome is duplicated, resulting in two TF paralogs (TFa and TFb) with unknown functions. Here we use CRISPR/Cas9 to produce null alleles of TFa and TFb and uncover partial subspecialization of these duplicates. It has been shown previously that both TFs are expressed before the initiation of blood circulation, between 24 and 48 hours post fertilization, yet complete loss of TFa and TFb yielded no gross abnormalities. Embryos and larvae were able to develop normally through juvenile stages but succumbed to hemorrhage by early adulthood at 9 weeks of age. Surprisingly, a single allele of either TFa or TFb was able to rescue survival in the context of complete loss of the other gene. To evaluate for hemostatic effects of TF deficiency, laser-mediated endothelial injury was used in the venous and arterial systems at 3 and 5 days post fertilization (dpf), respectively. Loss of TFb alone at 3 dpf resulted in no observable hemostatic defects. Conversely, loss of TFa led to a 50% increase in the time to venous occlusion (TTO), which was exacerbated by concomitant loss of one allele of TFb. Total TF deficiency led to a complete inability to form occlusive venous thrombi, indicating that both TFs can trigger coagulation but TFa is able to completely compensate for the loss of TFb. Concordant with these data, loss of TFb resulted in transcriptional upregulation of TFa but not vice versa. Interestingly, the roles are reversed in the arterial vasculature. Loss of TFa had no effect, loss of TFb lead to a 60% reduction in the number of occlusive thrombi, and complete deficiency resulted in no arterial occlusion. Combined with the venous results, these data point to differentiated roles of TFa and TFb in the venous and arterial systems. In order to test whether these differences were functional, recombinant TF (rTF) molecules were expressed in E. coli, purified, and incorporated into 80% phosphatidylcholine/20% phosphatidylserine liposomes. Ex vivo tube-tilt clotting assays were performed by using each rTF to activate citrated plasma from lake trout. rTFa triggered stable clot formation within 1-2 minutes of recalcification. rTFb usually failed to induce clot formation, with occasional delayed fibrin thrombi that appeared to be grossly disorganized and were easily disrupted following agitation. Taken with the in vivo data, this hints at an altered kinetic profile, with TFa being a more potent cofactor for factor VIIa in low flow (venous) settings. The laboratory is an artificially safe environment, so a synthetic chemical stress test was performed on 3 dpf larvae. Prolonged treatment with cortisol and epinephrine led to the development of cardiac tamponade in larvae with complete TF deficiency (61%), but similar results were only found at low levels in wild type siblings (2-5%). The same assay in prothrombin mutants also revealed a high rate of tamponade (75%), but lower levels in fibrinogen-deficient larvae (20%). These data suggest an extrahemostatic risk factor for tamponade that is modified by prothrombin and tissue factor levels and is independent of fibrin formation. Our results intimate that TFa and TFb have overlapping procoagulant functions but differential kinetic profiles in venous vs arterial systems. We also find that the duplication provides a layer of quantitative regulation and creates a titratable level for regulation of hemostatic and extrahemostatic roles of TF. Overall, this novel model provides new structural and physiologic information about TF function in vivo, including potential previously unknown roles in perivascular development, cardiovascular stability, remodeling and/or regeneration. Disclosures Morrissey: PrevThro Pharmaceuticals: Equity Ownership; Cayuga Pharmaceuticals: Equity Ownership; Kerafast, Inc: Research Funding; Issued and pending patent applications relating to medical uses of polyphosphate and polyphosphate inhibitors: Patents & Royalties. Shavit:Bayer: Consultancy; Sanofi: Consultancy; Shire/Takeda: Consultancy; Spark Therapeutics: Consultancy; CSL-Behring: Consultancy; Novo Nordisk: Consultancy.

Intrahepatic fibrin(ogen) deposition drives liver regeneration after partial hepatectomy in mice and humans

AbstractPlatelets play a pivotal role in stimulating liver regeneration after partial hepatectomy in rodents and humans. Liver regeneration in rodents is delayed when platelets are inhibited. However, the exact mechanisms whereby platelets accumulate and promote liver regeneration remain uncertain. Thrombin-dependent intrahepatic fibrin(ogen) deposition was recently reported after partial hepatectomy (PHx) in mice, but the role of fibrin(ogen) deposits in liver regeneration has not been investigated. We tested the hypothesis that fibrin(ogen) contributes to liver regeneration by promoting intrahepatic platelet accumulation and identified the trigger of rapid intrahepatic coagulation after PHx. PHx in wild-type mice triggered rapid intrahepatic coagulation, evidenced by intrahepatic fibrin(ogen) deposition. Intrahepatic fibrin(ogen) deposition was abolished in mice with liver-specific tissue factor deficiency, pinpointing the trigger of coagulation after PHx. Direct thrombin activation of platelets through protease-activated receptor-4 did not contribute to hepatocyte proliferation after PHx, indicating that thrombin contributes to liver regeneration primarily by driving intrahepatic fibrin(ogen) deposition. Fibrinogen depletion with ancrod reduced both intrahepatic platelet accumulation and hepatocyte proliferation after PHx, indicating that fibrin(ogen) contributes to liver regeneration after PHx by promoting intrahepatic platelet accumulation. Consistent with the protective function of fibrin(ogen) in mice, low postoperative plasma fibrinogen levels were associated with liver dysfunction and mortality in patients undergoing liver resection. Moreover, increased intrahepatic fibrin(ogen) deposition was evident in livers of patients after liver resection but was remarkably absent in patients displaying hepatic dysfunction postresection. The results suggest a novel mechanism whereby coagulation-dependent intrahepatic fibrin(ogen) deposition drives platelet accumulation and liver regeneration after PHx.

Myeloid but not epithelial tissue factor exerts protective anti‐inflammatory effects in acid aspiration‐induced acute lung injury

Essentials Tissue factor (TF) represents a central link between hemostasis and inflammation.We studied the roles of myeloid and airway epithelial TF in acid‐caused acute lung injury (ALI).TF on myeloid cells displays a non‐coagulatory role regulating the inflammatory response in ALI.Airway epithelial TF contributes to hemostatic functions, but is dispensable in ALI pathogenesis. SummaryIntroductionAcute lung injury (ALI) is a life‐threatening condition characterized by damaged alveolar–capillary structures and activation of inflammatory and hemostatic processes. Tissue factor (TF) represents a crucial link between inflammation and coagulation, as inflammatory mediators induce myeloid TF expression, and TF initiates extrinsic coagulation.ObjectiveAs pulmonary inflammation stimulates TF expression and TF modulates immune responses, we aimed to elucidate its impact on ALI. In particular, we wanted to distinguish the contributions of TF expressed on airway epithelial cells and TF expressed on myeloid cells.MethodsMice with different cell type‐specific TF deficiency and wild‐type littermates were intratracheally treated with hydrochloric acid, and leukocyte recruitment, cytokine levels, thrombin–antithrombin (TAT) complexes and pulmonary protein‐rich infiltrates were analyzed.ResultsOur data demonstrate that a lack of epithelial TF did not influence acute responses, as bronchoalveolar neutrophil accumulation 8 h after ALI induction was unaltered. However, it led to mild, prolonged inflammation, as pulmonary leukocyte and erythrocyte numbers were still increased after 24 h, whereas those in wild‐type mice had returned to basal levels. In contrast, myeloid TF was primarily involved in regulating the acute phase of ALI without affecting local coagulation, as indicated by increased bronchoalveolar neutrophil infiltration, pulmonary interleukin‐6 levels, and edema formation, but equal TAT complex formation, 8 h after ALI induction. This augmented inflammatory response associated with myeloid TF deficiency was confirmed in vitro, as lipopolysaccharide‐stimulated TF‐deficient alveolar macrophages released increased levels of chemokine (C‐X‐C motif) ligand 1 and tumor necrosis factor‐α as compared with wild‐type macrophages.ConclusionWe conclude that myeloid TF dampens inflammation in acid‐induced ALI.

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.

Abstract 534: Deficiency of Protease-activated Receptor 2 Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm Independent of Vascular Smooth Muscle Cell Tissue Factor

Objective: Tissue factor (TF) is constitutively expressed in subendothelial cells, including vascular smooth muscle cells (VSMCs), and maintains hemostasis. The TF/factor VIIa complex as well as factor Xa have been shown to activate protease-activated receptor 2 (PAR-2). We and others have shown that TF/FVIIa-PAR-2 signaling induces migration of VSMCs in vitro and this is reduced in TF and PAR-2 deficient cells. We previously demonstrated C57BL/6 mice with 1% of normal TF levels (Low TF mice) had increased suprarenal abdominal aortic diameter and incidence of abdominal aortic aneurysm (AAA) compared to littermate controls. However, the source of cellular TF and whether signaling occurred through PAR-2 has not been examined. Our objective was to determine the importance of VSMC-specific deletion of TF and PAR-2 deficiency on the etiology of AAA. Methods and Results: VSMC-specific TF deficiency was evaluated on a low-density lipoprotein receptor deficient (Ldlr -/- ) background. Male mice were fed a fat-enriched diet (21% milk fat) and infused with angiotensin II (AngII; 1,000 ng/kg/min) for 28 days. Ldlr -/- /TF flox/flox SM22αCre + mice had increased abdominal aortic luminal diameters (Cre + : 1.92 ± 0.16 (n = 12); Cre - : 1.22 ± 0.03 mm (n = 10); P = 0.001) and incidence of AAA (100% vs. 50%; P = 0.01) compared to Cre - littermates. Next, we determined the effects of PAR-2 deficiency in AAA. Ldlr -/- /PAR-2 +/+ (n = 12) and Ldlr -/- /PAR-2 -/- (n = 15) male mice were fed a fat-enriched diet and infused with AngII for 28 days. PAR-2 deficiency attenuated abdominal aortic luminal diameters (PAR-2 +/+ : 1.78 ± 0.06 mm; PAR2 -/- : 1.17 ± 0.03 mm; P = 0.001) and incidence of AAA (84% versus 40%; P = 0.048) when compared to littermate controls. Total plasma cholesterol concentrations, lipoprotein cholesterol distributions, or increased systolic blood pressure was not different between groups. Conclusion: These results suggest that TF limits AAA growth and incidence via VSMCs in a PAR-2-independent manner. To verify this hypothesis, studies are currently underway to determine the effect of Factor Xa (FXa) inhibitors in PAR-2 deficient mice.

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

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

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

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

Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection.

Tissue factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF(Δ) ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2-like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth.

Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury

Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury. We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation (thrombin-antithrombin complexes (TATc)) in the plasma of mice subjected to bile duct ligation for 12days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TF(flox/flox),LysMCre mice) or in hepatocytes (TF(flox/flox),AlbCre). Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc. Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients.

Role of Tissue Factor in Mycobacterium Tuberculosis-induced Inflammation and Disease Pathogenesis

Tuberculosis, a chronic lung infection caused by Mycobacterium tuberculosis (M.tb), affects nearly one third of the world’s population. Clinical manifestations of TB include hypercoagulable states and thrombotic complications particularly disseminated intravascular coagulation and deep vein thrombosis. Tissue factor (TF) plays an important role in the initiation of inflammation-induced coagulation. Various bacterial infections induce aberrant expression of TF on vascular cells, which contributes to intravascular coagulation and exacerbation of inflammation. Studies have shown that either a genetic deficiency of TF or blockade of TF functional activity reduces coagulopathy, proinflammatory cytokine release and infection-associated mortality. In contrast, TF-dependent coagulation activation and fibrin deposition may be protective in host-defense against certain bacterial infections via reducing pathogen burden and limiting their dissemination. In vitro M.tb infection markedly upregulates TF expression and increases procoagulant activity of macrophages. However, it is not yet known whether TF expression has any functional significance in TB pathogenesis. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth and containment of infection. Wild-type C57BL6 (WT) and transgenic mice that express either very low levels of human TF (low TF, ~1% of WT) or high levels of human TF (HTF, ~100% of WT) in place of murine TF were infected with aerosol exposure of M.tb H37Rv. Mice were sacrificed 2 and 8 weeks post-infection. An evaluation of in vivo TF expression, coagulation activation, proinflammatory cytokines and tissue bacterial burden was performed. M.tb infection did not significantly alter overall TF expression and procoagulant activity in lungs of WT and HTF mice. Although not statistically significant, M.tb infection increased TF activity substantially in the lung homogenates in low TF mice. Nonetheless, TF expression levels in lungs of low TF mice, both uninfected and M.tb.-infected, was negligible as compared to WT and HTF mice. M.tb infection markedly increased TF expression in localized areas within the granulomas of WT and HTF mice. Interestingly, these intensely stained TF positive patches were also present in the granulomas of low TF mice after M.tb infection. The increased localized expression of TF in low TF mice may be responsible for the increased TF activity in the lung homogenates in low TF mice. M.tb infection was not accompanied by systemic and pulmonary activation of coagulation in WT and HTF mice. There was no change in the plasma thrombin-anti-thrombin complexes (TATc) upon M.tb infection in all three genotypes. Although, the bronchoalveolar lavage (BAL) TATc significantly increased (10-fold) after M.tb infection in the low TF mice, still the level was 15-50 folds lower than those in HTF and WT mice. The levels of TNF-α, IFN-γ, IL-6 and IL1-β increased upon M.tb infection but no significant differences in the cytokine profiles of BAL and total lung homogenates were observed among the genotypes. Higher expression of TF in the granuloma of WT and HTF correlated with the presence of small discrete regions of fibrin islands especially extending toward outer margin of the granuloma whereas little fibrin staining was seen in the granuloma of low TF mice. Despite, marked differences in fibrin generation in the granuloma, there were no significant differences in either lung bacterial burden or dissemination to liver and spleen. In summary, our data suggest that TF-mediated coagulation and/or signaling does not appear to contribute to host defense during experimental tuberculosis. However, it is difficult to completely eliminate a role for TF in M.tb. pathogenesis since M.tb. induced significant amount of TF expression in localized areas in the granuloma even in low TF mice. It is possible that this small amount of TF expressed within the granuloma may be sufficient to mediate local coagulant and signaling functions to facilitate M.tb. growth and dissemination. DisclosuresNo relevant conflicts of interest to declare.

Fas-induced apoptosis increases hepatocyte tissue factor procoagulant activity in vitro and in vivo.

Hepatocyte (HPC) apoptosis occurs in association with hepatotoxic responses and chronic liver disease, and is coupled to activation of the blood coagulation cascade. HPCs have been shown to express tissue factor (TF), the primary activator of blood coagulation, in a form that lacks procoagulant activity. In this study, we determined the effect of inducing HPC apoptosis on the procoagulant activity of TF. Treatment of primary mouse HPCs with the Fas death receptor agonist (anti-CD95 antibody, Jo2) triggered apoptosis as shown by cleavage of caspase-3, increased caspase-3 proteolytic activity, and cell surface exposure of phosphatidylserine (PS). Jo2-induced apoptosis significantly increased TF-dependent factor Xa generation by HPCs. Moreover, Jo2 treatment was associated with increased levels of microparticle-associated TF procoagulant activity in the culture medium. Pretreatment with a caspase-3 inhibitor significantly reduced Jo2-induced HPC TF activity and prevented the increase in microparticle-associated TF procoagulant activity. Application of the high-affinity PS-binding protein lactadherin inhibited TF-dependent factor Xa generation by Jo2-treated HPCs and dramatically reduced microparticle-associated TF procoagulant activity. Treatment of wild-type mice with a sublethal dose of Jo2 was associated with a robust increase in the activation of coagulation as measured by plasma thrombin-antithrombin (TAT) levels; whereas mice with liver-specific TF deficiency had significantly lower TAT levels. Overall, the results indicate that Fas-initiated, caspase-3-dependent HPC apoptosis increases TF procoagulant activity through a mechanism involving PS externalization. This suggests that activation of liver TF likely contributes to the procoagulant state associated with HPC apoptosis in liver toxicity and disease.

PAR-4 Deficiency Rescues The Embryonic Lethal Phenotype Of TFPI Null Mice

BackgroundTissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor that dampens tissue factor (TF) initiated blood coagulation. It is produced by endothelial cells and megakaryocytes. Mice lacking TFPI activity die in utero from yolk sac hemorrhage or a presumptive consumptive coagulopathy. The embryonic lethal phenotype of TFPI null mice can be rescued by TF or factor VII deficiency. Our laboratory has shown that platelet TFPI limits platelet accumulation, but not fibrin formation, in an in vivo vascular injury model. Interestingly, mice lacking PAR4, the major thrombin receptor in mouse platelets, have decreased platelet accumulation but not fibrin formation following vascular injury. Therefore, we hypothesized that PAR-4 deficiency would counterbalance the absence of TFPI in platelets and rescue the embryonic lethal phenotype of TFPI null mice. Methods/ResultsTFPI heterozygous mice (TFPI+/-; C57/Bl6) were bred with PAR4 null mice (PAR4-/-; C57/Bl6) to generate TFPI+/-/PAR4-/- mice. These mice were bred to assess rescue of TFPI lethality. Of 76 pups closely monitored following birth, 12 (15.8%) were TFPI-/-/PAR4-/-, with 8 (10.5%) surviving to wean. TFPI-/-/PAR4-/- male mice were bred with TFPI+/-/PAR4-/- females for subsequent studies. Of 447 pups generated, 101 TFPI-/-/PAR4-/- mice (22.6%) were produced. Thus, TFPI-/-/PAR4-/- pups survive to wean at approximately one-half the expected frequency. TFPI-/-/PAR4-/- mice surviving to wean appeared healthy with no overt signs of thrombosis, and have apparently normal survival to over 12 months of age. Compared to TFPI+/-/PAR4-/- littermates, TFPI-/-/PAR4-/- have reduced body weight at 8 weeks. Female TFPI-/-/PAR4-/- mice can reproduce successfully. Approximately one-third of the TFPI-/-/PAR4-/- mice have short or totally absent tails. The short tail phenotype develops in utero and changes in tail:body proportion do not occur following birth. There was no difference in blood loss following tail clip between TFPI+/+/PAR4-/-, TFPI+/-/PAR4-/- or TFPI-/-/PAR4-/- mice. There also was no difference in fibrin or platelet accumulation between TFPI+/+/PAR4-/- or TFPI-/-/PAR4-/-mice following venous electrolytic injury. ConclusionsThis is the first reported rescue of the TFPI null embryonic lethal phenotype by a platelet activation defect. Almost one-half of the TFPI-/-/PAR4-/- mice survive through adulthood despite having normal tissue factor function not regulated by TFPI. This, and our inability, thus far, to identify a procoagulant state in the adult mice, suggests that an essential TFPI anticoagulant function is to modulate PAR-4 mediated platelet function; likely by limiting thrombin generation. TFPI may do this through inhibition of TF-fVIIa, or via inhibition of early forms of prothrombinase on the platelet surface, a very recently recognized inhibitory function of TFPI. The long term survival of these mice also demonstrates that unfettered TF-fVlla-fXa mediated signaling events through PAR-2 are not solely responsible for the embryonic lethality of TFPI null mice. However, the short or absent tails that arise during development of TFPI deficient mice suggests that TF and/or TFPI may have roles in notochord development that is separate from the coagulation system. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

Low levels of tissue factor lead to alveolar haemorrhage, potentiating murine acute lung injury and oxidative stress

BackgroundSystemic blockade of tissue factor (TF) attenuates acute lung injury (ALI) in animal models of sepsis but the effects of global TF deficiency are unknown. We used mice with complete...

Abstract 42: Monocyte Tissue Factor-Dependent Activation of Coagulation in Hypercholesterolemic Mice and Monkeys Is Inhibited by Simvastatin

Hyperlipidemia is associated with a prothrombotic state that includes activation of platelets. However, the molecular pathway that leads to the activation of the coagulation cascade has not been defined. Hyperlipidemia leads to the presence of low levels of oxidized lipoproteins, such as oxidized LDL (oxLDL), in the circulation and the accumulation of oxLDL in atherosclerotic lesions.. Monocytes and macrophages are activated by oxLDL binding to a TLR4/TLR6/CD36 receptor complex. We hypothesized that oxLDL induction of monocyte tissue factor (TF) expression and the release of TF-positive microparticles (MPs) produces a prothrombotic state. In support of this hypothesis, we found that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma MP TF activity. Furthermore, a western diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Inhibition of TF or a genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity consistent with a role of the TLR4/TLR6/CD36 complex in oxLDL induction of monocyte TF expression. Importantly, simvastatin treatment of hyperlipidemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hyperlipidemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6/CD36 receptor complex. Furthermore, simvastatin appears to act at multiple levels to reduce the prothrombotic state.

Monocyte tissue factor–dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.