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Nigel Mackman - One of the best experts on this subject based on the ideXlab platform.
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Tissue Factor Past, Present, and Future
2015Co-Authors: Nigel Mackman, Mark TaubmanAbstract:This issue of Arteriosclerosis, Thrombosis, and Vascular Biology contains 4 reviews on Tissue Factor (TF) and 1 on Tissue Factor pathway inhibitor (TFPI). One review on TF will be published in a later issue. In this editorial, we will briefly revisit the major advances in the field, highlight some of the current controversies, and discuss some of the future challenges. TF (also known as Tissue thromboplastin or coagulation Factor III) was first identified as a constituent of Tissue that when added to plasma activated the clotting cascade — hence the name Tissue Factor. TF was first purified in 1985,1 and this subsequently led to the cloning of the TF cDNA and gene. 2-5 In 1989 Drake and colleagues6 proposed that TF around blood vessels forms a “hemostatic envelope ” that initiates clotting after vessel injury. The crystal structure of the extracellular domain of TF bound to Factor VIIa (FVIIa) was reported in 1996.7 In the same year it was discovered that inactivation of the mouse TF gene resulted in embryonic lethality.8-10 Taken together, these studies indicated that TF was essential for hemostasis. Activation of the clotting cascade leads to the generation of thrombin that cleaves fibrinogen to fibrin as well as activates platelets (Figure). In 1999, the late Yale Nemerson and colleagues11 reported that there was TF in blood of health
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Tissue Factor and Tissue Factor Pathway Inhibitor as Key Regulators of Global Hemostasis: Measurement of Their Levels
2015Co-Authors: In Coagulation Assays, Nigel Mackman, Raj S. Kasthuri, Sam L. Glover, Jeremiah Boles, Ph. DAbstract:The Tissue Factor (TF)/Factor (F)VIIa complex is the primary initiator of coagulation in vivo. Tissue Factor pathway inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Deficiencies of either TF or TFPI have not been reported in humans, and a complete absence of either of these two proteins in mice is embryonically lethal. To maintain normal hemostasis, levels of TF and TFPI need to be balanced. Increased levels of TF can overwhelm the inhibitory capacity of TFPI, resulting in thrombosis. Decreased levels of TF are associated with bleeding. Global assays of coagulation are defined as tests capable of evaluating all components of the clotting cascade that are present in plasma. In these tests the thrombogenic surface is either provided by platelets or exogenous phospholipids. Clotting assays currently used in clinical practice are not designed to measure endogenous levels of TF and TFPI. Therefore, there is a need to develop sensitive and specific assays for measuring levels of functional TF and TFPI in whole blood and plasma. These assays could be useful in patient management in many scenarios. Keywords Tissue Factor; Tissue Factor pathway inhibitor; thrombosis; thromboelastograph
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rosuvastatin lowers circulating Tissue Factor antigen bearing microparticles in metastatic breast cancer a phase ii multi dose randomized trial microstat trial
Blood, 2013Co-Authors: Jeffrey I. Zwicker, Donna Neuberg, Erica L Mayer, Federico Campigotto, Julia E Geddings, Herold Christina, Sheryl R Bowley, Gerburg M Wulf, Gary L Horowitz, Nigel MackmanAbstract:Background Elevated numbers of circulating bloodborne Tissue Factor antigen-bearing microparticles represent a significant risk Factor for the development of venous thromboembolic events in cancer patients. We recently demonstrated the efficacy of low molecular weight heparin to prevent thrombosis in cancer patients with high levels of circulating Tissue Factor bearing microparticles. Enthusiasm for prophylactic anticoagulation in cancer cohorts is tempered by the requirement for daily self-injection and potential risk for hemorrhage. Statins significantly reduce the incidence of venous thromboembolic events in healthy individuals by an uncertain mechanism. Statins reduce the generation of Tissue Factor bearing microparticles in vitro and in animal models. Whether statins affect Tissue Factor bearing microparticles in cancer patients has not been previously assessed. We performed a randomized phase II study to evaluate whether rosuvastatin reduces the number of circulating Tissue Factor bearing microparticles in women with metastatic breast cancer. Methods Eligibility requirements included women with metastatic breast cancer on stable endocrine therapy (no changes within previous 6 weeks) with preserved bone marrow function. Patients were excluded if actively receiving chemotherapy (within prior 5 weeks) or currently taking statins. Baseline laboratory testing was performed at days 0 and 28 following enrollment. Study subjects were randomized to rosuvastatin 20 mg or 40 mg to be initiated from days 28 to 56. Repeat laboratory testing was performed on days 42 and 56 to assess for a reduction of Tissue Factor bearing microparticles and D-dimer. The number of circulating Tissue Factor bearing microparticles was measured by impedance-based flow cytometry and Tissue Factor activity was measured by a chromogenic Xa assay. Two-sided Wilcoxon signed rank test was used to compare the median levels at baseline and post treatment of laboratory endpoints within group, and Fisher exact test and Wilcoxon rank sum test to compare baseline characteristics between groups. Results A total of 19 women have been enrolled. Nine women were randomized to rosuvastatin 20 mg and ten were randomized to 40 mg. There were no significant differences between the two groups in baseline characteristics (e.g. age, body mass index, performance status, blood counts, renal function or number of Tissue Factor bearing microparticles). For the group randomized to rosuvastatin 20 mg, we observed no change in the number of Tissue Factor bearing microparticles (1576/μl vs 1888/μl , P= 0.55), microparticle-associated Tissue Factor activity, D-dimer or C-reactive protein (CRP), (P>0.05). In the group of women who received 40 mg rosuvastatin, the median decrease in the number of circulating Tissue Factor bearing microparticles per patient was -28%, with a median baseline value of 1763/μl versus a post-treatment value of 1216/μl (P=0.047). The group receiving 40mg rosuvastatin also demonstrated a non-significant median change in D-dimer of -21% (P=0.44), microparticle-associated Tissue Factor activity of -2% (P=0.84), and CRP of -10% (P= 0.81). Conclusions In a randomized phase II study in women with metastatic breast cancer, we observed a statistically significant 28% decrease in circulating Tissue Factor bearing microparticles following initiation of rosuvastatin 40 mg. Furthermore, these results emphasize that assay of Tissue Factor antigen-bearing microparticles does not correlate with assay of Tissue Factor activity. Based on the association between Tissue Factor-antigen bearing microparticles and cancer-associated thrombosis, these data provide additional support for a large scale clinical trial to assess the efficacy of statins to prevent thrombosis in cancer patients. Disclosures: Off Label Use: rosuvastatin to lower Tissue Factor bearing microparticles.
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role of Tissue Factor in thrombosis in antiphospholipid antibody syndrome
Lupus, 2010Co-Authors: J Boles, Nigel MackmanAbstract:Antiphospholipid syndrome (APS) is an acquired autoimmune disorder defined by the presence of an antiphospholipid antibody (aPL) and the occurrence of at least one associated clinical condition that includes venous thrombosis, arterial thrombosis or pregnancy morbidity. The aPL detected in APS have long been thought to have a direct prothrombotic effect in vivo. However, the pathophysiology underlying their coagulopathic effect has not been defined. Emerging data suggest a role for the procoagulant protein Tissue Factor (TF). In this review we provide an overview of TF, describe mouse models used in the evaluation of the role of TF in thrombosis, as well as summarize recent work on TF and APS.
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early growth response gene 1 regulates hypoxia induced expression of Tissue Factor in glioblastoma multiforme through hypoxia inducible Factor 1 independent mechanisms
Cancer Research, 2006Co-Authors: Yuan Rong, Nigel Mackman, Ruo Pan Huang, Jonathan M Horowitz, Randy L Jensen, Donald L Durden, Erwin G Van Meir, Daniel J BratAbstract:Hypoxia strongly up-regulates Tissue Factor and promotes plasma clotting by glioblastoma multiforme, but transcriptional mechanisms remain undefined. Here, we investigated the potential roles of early growth response gene-1 (Egr-1), Sp1, nuclear Factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and hypoxia-inducible Factor-1 (HIF-1) in the hypoxic regulation of Tissue Factor by glioblastoma multiforme cells in vitro. Hypoxia (1% O2) strongly induced Egr-1 mRNA within 1 hour and led to nuclear localization of Egr-1 protein. Using luciferase reporter plasmids in glioma cells, we found that hypoxia dramatically increased luciferase activity in cells with constructs containing Egr-1-binding sites but not in cells with constructs containing AP-1- or NF-kappaB-binding sites. Electrophoretic mobility shift assays revealed hypoxia-induced Egr-1, but not Sp1, binding to oligonucleotides containing the Egr-1/Sp1 motif of Tissue Factor gene promoter. Using an expression vector containing the minimal Tissue Factor promoter (-111 to +14 bp) and small interfering RNA (siRNA) directed at Egr-1 and Sp1 mRNAs, we found that Egr-1 was required for maximal hypoxic induction of promoter activity. Forced overexpression of Egr-1 but not Sp1 by cDNA transfection caused up-regulation of Tissue Factor in glioma cells under normoxia (21% O2), whereas siRNA directed at Egr-1 strongly attenuated hypoxia-induced Tissue Factor expression. To examine the effects of HIF-1alpha on Tissue Factor expression, we used glioma cells stably transfected with a HIF-1alpha siRNA expression vector and found that HIF-1alpha mRNA silencing did not affect Tissue Factor expression under hypoxia. We conclude that hypoxic up-regulation of Tissue Factor in glioblastoma multiforme cells depends largely on Egr-1 and is independent of HIF-1.
Harold R Roberts - One of the best experts on this subject based on the ideXlab platform.
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platelet activity of high dose Factor viia is independent of Tissue Factor
British Journal of Haematology, 1997Co-Authors: Dougald M Monroe, Maureane Hoffman, Julie A Oliver, Harold R RobertsAbstract:High-dose recombinant Factor VIIa has been successfully used as therapy for haemophiliacs with inhibitors. The mechanism by which high-dose Factor VIIa supports haemostasis is the subject of some controversy. Postulating a mechanism in which activity is dependent on Tissue Factor at the site of injury explains the localization of activity but not the requirement for high doses. Postulating a mechanism in which Factor VIIa acts on available lipid independently of Tissue Factor explains the requirement for high doses but not the lack of systemic procoagulant activity. We report that Factor VIIa bound weakly to activated platelets (Kd ∼ 90 nm). This Factor VIIa was functionally active and could initiate thrombin generation in the presence of plasma concentrations of prothrombin, Factor X, Factor V, antithrombin III and Tissue Factor pathway inhibitor. The activity was not dependent on Tissue Factor. The concentration of Factor VIIa required for detectable thrombin generation agreed well with the lowest concentration of Factor VIIa required for efficacy in patients. High-dose Factor VIIa may function on the activated platelets that form the initial haemostatic plug in haemophilic patients. These observations are in agreement with clinical trials which have shown that high-dose Factor VIIa was haemostatically effective without causing systemic activation of coagulation.
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platelet activity of high dose Factor viia is independent of Tissue Factor
British Journal of Haematology, 1997Co-Authors: Dougald M Monroe, Maureane Hoffman, Julie A Oliver, Harold R RobertsAbstract:High-dose recombinant Factor VIIa has been successfully used as therapy for haemophiliacs with inhibitors. The mechanism by which high-dose Factor VIIa supports haemostasis is the subject of some controversy. Postulating a mechanism in which activity is dependent on Tissue Factor at the site of injury explains the localization of activity but not the requirement for high doses. Postulating a mechanism in which Factor VIIa acts on available lipid independently of Tissue Factor explains the requirement for high doses but not the lack of systemic procoagulant activity. We report that Factor VIIa bound weakly to activated platelets (Kd approximately 90 nM). This Factor VIIa was functionally active and could initiate thrombin generation in the presence of plasma concentrations of prothrombin, Factor X, Factor V, antithrombin III and Tissue Factor pathway inhibitor. The activity was not dependent on Tissue Factor. The concentration of Factor VIIa required for detectable thrombin generation agreed well with the lowest concentration of Factor VIIa required for efficacy in patients. High-dose Factor VIIa may function on the activated platelets that form the initial haemostatic plug in haemophilic patients. These observations are in agreement with clinical trials which have shown that high-dose Factor VIIa was haemostatically effective without causing systemic activation of coagulation.
Valentin Fuster - One of the best experts on this subject based on the ideXlab platform.
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role of risk Factors in the modulation of Tissue Factor activity and blood thrombogenicity
Circulation, 2003Co-Authors: Antonia Sambola, Yale Nemerson, Valentin Fuster, James J Hathcock, Julio I Osende, Michael Degen, Jill P Crandall, Juan J BadimonAbstract:Background— Several studies suggest a role for an increased circulating pool of Tissue Factor (TF) in atherothrombotic diseases. Furthermore, certain cardiovascular risk Factors, such as diabetes, hyperlipemia, and smoking, are associated with a higher incidence of thrombotic complications. We hypothesized that the observed increased blood thrombogenicity (BT) observed in patients with type 2 diabetes mellitus may be mediated via an increased circulating Tissue Factor activity. We have extended our study to smokers and hyperlipidemic subjects. Methods and Results— Poorly controlled patients with type 2 diabetes mellitus (n=36), smokers (n=10), and untreated hyperlipidemic subjects (n=10) were studied. Circulating TF was immunocaptured from plasma, relipidated, and quantified by Factor Xa (FXa) generation in the presence of Factor VIIa. BT was assessed as thrombus formation on the Badimon perfusion chamber. Patients with improvement in glycemic control showed a reduction in circulating TF (362±135 versus 2...
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local inhibition of Tissue Factor reduces the thrombogenicity of disrupted human atherosclerotic plaques effects of Tissue Factor pathway inhibitor on plaque thrombogenicity under flow conditions
Circulation, 1999Co-Authors: Juan J Badimon, Vincenzo Toschi, Maddalena Lettino, Maria Berrozpe, James H. Chesebro, Valentin Fuster, Lina BadimonAbstract:Background—Plaque disruption and subsequent thrombus formation lead to acute coronary syndromes and progression of atherosclerotic disease. Tissue Factor (TF) appears to mediate plaque thrombogenicity. Tissue Factor pathway inhibitor (TFPI) is the major physiological inhibitor of TF. This study analyzes the role of TF on thrombogenicity of disrupted human atherosclerotic plaques and the therapeutic possibilities of its specific inhibition. Methods and Results—Human atherosclerotic and normal arterial segments were exposed to heparinized blood at flow conditions modeling medium-grade coronary stenosis in the Badimon perfusion chamber. The antithrombotic effects of the specific inhibition of plaque TF was assessed by reduction in the deposition of radiolabeled platelets and fibrin(ogen) and immunohistochemical analysis of perfused arteries. TF activity was inhibited by both recombinant TFPI and a polyclonal antibody against human TF. Human lipid-rich plaques were more thrombogenic than less advanced atheros...
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Tissue Factor modulates the thrombogenicity of human atherosclerotic plaques
Circulation, 1997Co-Authors: Vincenzo Toschi, Richard Gallo, Maddalena Lettino, S D Gertz, Antonio Fernandezortiz, Yale Nemerson, James H. Chesebro, Lina Badimon, John T Fallon, Valentin FusterAbstract:BACKGROUND: The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic Factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of Tissue Factor in the thrombogenicity of different types of atherosclerotic plaques and their components. METHODS AND RESULTS: Fifty human arterial segments (5 foam cell-rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by 111In-labeled platelets. After perfusion, specimens were stained for Tissue Factor by use of an in situ binding assay for Factor VIIa. Tissue Factor in specimens was semiquantitatively assessed on a scale of 0 to 3. Platelet deposition on the lipid-rich atheromatous core was significantly higher than on all other substrates (P = .0002). The lipid-rich core also exhibited the most intense Tissue Factor staining (3 +/- 0.1 arbitrary units) compared with other arterial components. Comparison of all specimens showed a positive correlation between quantitative platelet deposition and Tissue Factor staining score (r = .35, P < .01). CONCLUSIONS: Our results show that Tissue Factor is present in lipid-rich human atherosclerotic plaques and suggest that it is an important determinant of the thrombogenicity of human atherosclerotic lesions after spontaneous or mechanical plaque disruption.
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Tissue Factor modulates the thrombogenicity of human atherosclerotic plaques
Circulation, 1997Co-Authors: Vincenzo Toschi, Maddalena Lettino, S D Gertz, Antonio Fernandezortiz, Yale Nemerson, James H. Chesebro, Lina Badimon, John T Fallon, Richard L Gallo, Valentin FusterAbstract:Background The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic Factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of Tissue Factor in the thrombogenicity of different types of atherosclerotic plaques and their components. Methods and Results Fifty human arterial segments (5 foam cell–rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by 111In-labeled platelets. After perfusion, specimens were stained for Tissue Factor by use of an in situ binding assay for Factor VIIa. Tissue Factor...
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macrophages smooth muscle cells and Tissue Factor in unstable angina implications for cell mediated thrombogenicity in acute coronary syndromes
Circulation, 1996Co-Authors: Pedro R Moreno, Yale Nemerson, Victor Bernardi, Julio Lopezcuellar, Alvaro M Murcia, Igor F Palacios, Herman K Gold, Roxana Mehran, Samin K Sharma, Valentin FusterAbstract:Background Macrophage expression of Tissue Factor may be responsible for coronary thrombogenicity in patients with plaque rupture. In patients without plaque rupture, smooth muscle cells may be the thrombogenic substrate. This study was designed to identify the cellular correlations of Tissue Factor in patients with unstable angina. Methods and Results Tissue from 50 coronary specimens (1560 pieces) from patients with unstable angina and 15 specimens from patients with stable angina were analyzed. Total and segmental areas (in square millimeters) were identified with trichrome staining. Macrophages, smooth muscle cells, and Tissue Factor were identified by immunostaining. Tissue Factor content was larger in unstable angina (42±3%) than in stable angina (18±4%) (P=.0001). Macrophage content was also larger in unstable angina (16±2%) than in stable angina (5±2%) (P=.002). The percentage of Tissue Factor located in cellular areas was larger in coronary samples from patients with unstable angina (67±8%) than ...
Yale Nemerson - One of the best experts on this subject based on the ideXlab platform.
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alternatively spliced human Tissue Factor a circulating soluble thrombogenic protein
Nature Medicine, 2003Co-Authors: Vladimir Y Bogdanov, Viji Balasubramanian, James J Hathcock, Oana Vele, Mark Lieb, Yale NemersonAbstract: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.
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role of risk Factors in the modulation of Tissue Factor activity and blood thrombogenicity
Circulation, 2003Co-Authors: Antonia Sambola, Yale Nemerson, Valentin Fuster, James J Hathcock, Julio I Osende, Michael Degen, Jill P Crandall, Juan J BadimonAbstract:Background— Several studies suggest a role for an increased circulating pool of Tissue Factor (TF) in atherothrombotic diseases. Furthermore, certain cardiovascular risk Factors, such as diabetes, hyperlipemia, and smoking, are associated with a higher incidence of thrombotic complications. We hypothesized that the observed increased blood thrombogenicity (BT) observed in patients with type 2 diabetes mellitus may be mediated via an increased circulating Tissue Factor activity. We have extended our study to smokers and hyperlipidemic subjects. Methods and Results— Poorly controlled patients with type 2 diabetes mellitus (n=36), smokers (n=10), and untreated hyperlipidemic subjects (n=10) were studied. Circulating TF was immunocaptured from plasma, relipidated, and quantified by Factor Xa (FXa) generation in the presence of Factor VIIa. BT was assessed as thrombus formation on the Badimon perfusion chamber. Patients with improvement in glycemic control showed a reduction in circulating TF (362±135 versus 2...
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transfer of Tissue Factor from leukocytes to platelets is mediated by cd15 and Tissue Factor
Blood, 2000Co-Authors: Ursula Rauch, Juan J Badimon, Diana Bonderman, Bernd Bohrmann, Jacques Himber, Markus A Riederer, Yale NemersonAbstract:We describe thrombogenic Tissue Factor (TF) on leukocyte-derived microparticles and their incorporation into spontaneous human thrombi. Polymorphonuclear leukocytes and monocytes transfer TF+particles to platelets, thereby making them capable of triggering and propagating thrombosis. This phenomenon calls into question the original dogma that vessel wall injury and exposure of TF within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF+ leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Both the inhibition of TF transfer to platelets by antagonizing the interaction CD15 with P-selectin and the direct interaction of TF itself suggest a novel therapeutic approach to prevent thrombosis.
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Tissue Factor modulates the thrombogenicity of human atherosclerotic plaques
Circulation, 1997Co-Authors: Vincenzo Toschi, Richard Gallo, Maddalena Lettino, S D Gertz, Antonio Fernandezortiz, Yale Nemerson, James H. Chesebro, Lina Badimon, John T Fallon, Valentin FusterAbstract:BACKGROUND: The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic Factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of Tissue Factor in the thrombogenicity of different types of atherosclerotic plaques and their components. METHODS AND RESULTS: Fifty human arterial segments (5 foam cell-rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by 111In-labeled platelets. After perfusion, specimens were stained for Tissue Factor by use of an in situ binding assay for Factor VIIa. Tissue Factor in specimens was semiquantitatively assessed on a scale of 0 to 3. Platelet deposition on the lipid-rich atheromatous core was significantly higher than on all other substrates (P = .0002). The lipid-rich core also exhibited the most intense Tissue Factor staining (3 +/- 0.1 arbitrary units) compared with other arterial components. Comparison of all specimens showed a positive correlation between quantitative platelet deposition and Tissue Factor staining score (r = .35, P < .01). CONCLUSIONS: Our results show that Tissue Factor is present in lipid-rich human atherosclerotic plaques and suggest that it is an important determinant of the thrombogenicity of human atherosclerotic lesions after spontaneous or mechanical plaque disruption.
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Tissue Factor modulates the thrombogenicity of human atherosclerotic plaques
Circulation, 1997Co-Authors: Vincenzo Toschi, Maddalena Lettino, S D Gertz, Antonio Fernandezortiz, Yale Nemerson, James H. Chesebro, Lina Badimon, John T Fallon, Richard L Gallo, Valentin FusterAbstract:Background The thrombogenicity of a disrupted atherosclerotic lesion is dependent on the nature and extent of the plaque components exposed to flowing blood together with local rheology and a variety of systemic Factors. We previously reported on the different thrombogenicity of the various types of human atherosclerotic lesions when exposed to flowing blood in a well-characterized perfusion system. This study examines the role of Tissue Factor in the thrombogenicity of different types of atherosclerotic plaques and their components. Methods and Results Fifty human arterial segments (5 foam cell–rich, 9 collagen-rich, and 10 lipid-rich atherosclerotic lesions and 26 normal, nonatherosclerotic segments) were exposed to heparinized blood at high shear rate conditions in the Badimon perfusion chamber. The thrombogenicity of the arterial specimens was assessed by 111In-labeled platelets. After perfusion, specimens were stained for Tissue Factor by use of an in situ binding assay for Factor VIIa. Tissue Factor...
Walter Kisiel - One of the best experts on this subject based on the ideXlab platform.
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Tissue Factor TRANSCRIPTION DRIVEN BY EGR-1 IS A CRITICAL MECHANISM OF MURINE PULMONARY FIBRIN DEPOSITION IN HYPOXIA
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Shi Fang Yan, Nigel Mackman, Walter Kisiel, David J. Pinsky, Yu Shan Zou, Yun Gao, Chao Zhai, Stephen L. Lee, Jeffrey Milbrandt, David M. SternAbstract:Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of Tissue Factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription Factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of Tissue Factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO2 ≈13 torr) had increased levels of Tissue Factor transcripts (≈18-fold) and an increased rate of transcription (≈15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; −111/+14 bp) of the Tissue Factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the Tissue Factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated Tissue Factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed Tissue Factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither Tissue Factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of Tissue Factor, has an unexpected and central role.
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molecular cloning expression and partial characterization of a second human Tissue Factor pathway inhibitor
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Cindy A Sprecher, Walter Kisiel, Shannon Mathewes, Donald C FosterAbstract:Abstract Previous studies have shown that Tissue-Factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit Factor Xa and Factor VIIa-Tissue Factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the Factor VIIa-Tissue Factor complex. In addition, the inhibition of Factor VIIa-Tissue Factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human Factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.
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inhibitory properties of full length and truncated recombinant Tissue Factor pathway inhibitor tfpi evidence that the third kunitz type domain of tfpi is not essential for the inhibition of Factor viia Tissue Factor complexes on cell surfaces
Journal of Biological Chemistry, 1993Co-Authors: Takayoshi Hamamoto, O Nordfang, Donald C Foster, M Yamamoto, J G L Petersen, Walter KisielAbstract:Human Tissue Factor pathway inhibitor (TFPI) is a plasma protease inhibitor that consists of three tandem Kunitz-type inhibitor domains flanked by a negatively charged NH2 terminus and a positively charged COOH-terminal tail. Previous studies have shown that the first and second Kunitz-type domains in TFPI are involved in the inhibition of Factor VIIa and Factor Xa activity, respectively. In the present study, we have compared the inhibitory properties of full-length recombinant TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and COOH-terminal tail (TFPI1-161) with respect to inhibition of Factor VIIa-Tissue Factor complexes on the surface of a human bladder carcinoma cell line J82. Full-length TFPI and TFPI1-161 were kinetically indistinguishable with respect to neutralization of the proteolytic activity of preformed complexes of Factor VIIa-Tissue Factor on the J82 cell surface in the absence of Factor Xa. Equimolar amounts of Factor Xa augmented the anticoagulant activity of both preparations of TFPI to the same extent, and both preparations of TFPI were equally effective in inhibiting Factor VIIa-Tissue Factor amidolytic activity in solution phase. In addition, plasma concentrations of both forms of TFPI, in stoichiometric complex with Factor Xa, inhibited cell surface Factor VIIa-Tissue Factor proteolytic activity markedly faster than plasma levels of antithrombin III, even in the presence of 1 unit/ml heparin. The results of displacement studies suggested slight differences in the affinity of the two TFPI molecules for the cell surface in that approximately 5% of a VIIa.TF.Xa.TFPI1-161 quaternary complex on J82 cells was displaceable from the cell surface by high concentrations of Factor VIIa (10-100 nM), whereas only 1-2% of a VIIa.TF.Xa.TFPI complex was displaceable under comparable conditions. Pretreatment of the cells with TFPI/Xa alone or together with R152E Factor VII, followed by Factor VIIa treatment, revealed significant differences in the two TFPI forms with respect to the degree with which offered Factor VIIa could restore Factor X activation on the cell surface. These differences notwithstanding, our collective findings indicate that the third Kunitz-type domain and/or COOH-terminal tail of TFPI is not essential for the inhibition of cell surface Factor VIIa-Tissue Factor complexes and suggests that TFPI1-161 may be a useful therapeutic agent in the treatment of thromboembolic episodes.
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The role of phospholipids and the Factor VII Gla-domain in the interaction of Factor VII with Tissue Factor.
Thrombosis and haemostasis, 1992Co-Authors: Peter Wildgoose, Tony Jørgensen, Yutaka Komiyama, Tomohiro Nakagaki, Anders Hjelholt Pedersen, Walter KisielAbstract:Whether or not the Factor VII Gla-domain is involved in the high-affinity interaction of Factor VII and Tissue Factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the Factor VII Gla-peptide (FVII-GP) from digested recombinant human Factor VIIa and assessed its effect on Factor VII:Tissue Factor interactions. FVII-GP inhibited the activation of Factor X by Factor VIIa in the presence of either soluble or cell surface Tissue Factor half-maximally at 0.5 microM and 2.7 microM, respectively. However, FVII-GP failed to inhibit the specific binding of Factor VIIa to cell-surface Tissue Factor, and did not inhibit the ability of Tissue Factor to stimulate the amidolytic activity of Factor VIIa. Unrelipidated Tissue Factor apoprotein stimulated the amidolytic activity of Factor VIIa to the same extent as relipidated Tissue Factor apoprotein. These findings suggest that the Factor VII Gla-domain does not directly interact with Tissue Factor, but rather is important for calcium binding and concomitant expression of other Factor VII epitopes necessary for Tissue Factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative Factor VII epitope that participates in the interaction of Factor VII with cell-surface Tissue Factor (peptide 195-206) and assessed its ability to bind to Factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact Factor VIIa was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206.(ABSTRACT TRUNCATED AT 250 WORDS)