Tissue factor: beyond coagulation in the cardiovascular system

2009 ◽  
Vol 118 (3) ◽  
pp. 159-172 ◽  
Author(s):  
Alexander Breitenstein ◽  
Giovanni G. Camici ◽  
Felix C. Tanner
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor X ◽  
Signalling Molecules ◽  
Smooth Muscle Cell Migration ◽  
Mitogen Activated Protein ◽  
Coronary Syndromes ◽  
Phosphoinositide 3 Kinase

TF (tissue factor) is the main trigger of the coagulation cascade; by binding Factor VIIa it activates Factor IX and Factor X, thereby resulting in fibrin formation. Various stimuli, such as cytokines, growth factors and biogenic amines, induce TF expression and activity in vascular cells. Downstream targets of these mediators include diverse signalling molecules such as MAPKs (mitogen-activated protein kinases), PI3K (phosphoinositide 3-kinase) and PKC (protein kinase C). In addition, TF can be detected in the bloodstream, known as circulating or blood-borne TF. Many cardiovascular risk factors, such as hypertension, diabetes, dyslipidaemia and smoking, are associated with increased expression of TF. Furthermore, in patients presenting with acute coronary syndromes, elevated levels of circulating TF are found. Apart from its role in thrombosis, TF has pro-atherogenic properties, as it is involved in neointima formation by inducing vascular smooth muscle cell migration. As inhibition of TF action appears to be an attractive target for the treatment of cardiovascular disease, therapeutic strategies are under investigation to specifically interfere with the action of TF or, alternatively, promote the effects of TFPI (TF pathway inhibitor).

Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly

1999 ◽  
Vol 82 (08) ◽  
pp. 209-217 ◽  
Author(s):  
Patrick Celie ◽  
Joost Kolkman ◽  
Peter Lenting ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Three Dimensional ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Factor X Activation

IntroductionThe activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.

scholarly journals Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 204-212
Author(s):  
NL Sanders ◽  
SP Bajaj ◽  
A Zivelin ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Additional Material ◽  
Plasma Reaction ◽  
Progress Curve ◽  
Peptide Release ◽  
Release Data

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

scholarly journals Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 204-212 ◽  
Author(s):  
NL Sanders ◽  
SP Bajaj ◽  
A Zivelin ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Additional Material ◽  
Plasma Reaction ◽  
Progress Curve ◽  
Peptide Release ◽  
Release Data

Abstract A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

scholarly journals Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 645-651 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Mechanism Of Inhibition ◽  
Peptide Release ◽  
Release Assay

Abstract We have extended earlier studies (Blood 66:204, 1985) of a mechanism of inhibition of factor VIIa/tissue factor activity that requires a plasma component (called herein extrinsic pathway inhibitor or EPI) and factor Xa. An activated peptide release assay using 3H-factor IX as a substrate was used to evaluate inhibition. Increasing the tissue factor concentration from 20% to 40% (vol/vol) overcame the inhibitory mechanism in normal plasma but not in factor VII-deficient plasma supplemented with a low concentration of factor VII. A second wave of factor IX activation obtained by a second addition of tissue factor to plasma with a normal factor VII concentration was almost abolished by supplementing the reaction mixture with additional EPI and factor X. Factor Xa's active site was necessary for factor Xa's contribution to inhibition, but preliminary incubation of factor Xa with EPI in the absence of factor VIIa/tissue factor complex or of factor VIIa/tissue factor complex in the absence of EPI did not replace the need for the simultaneous presence of factor Xa, factor VIIa/tissue factor, calcium, and EPI in an inhibitory reaction mixture. Inhibition of factor VIIa/tissue factor was reversible; both tissue factor and factor VIIa activity could be recovered from a dissociated, inhibited factor VIIa/tissue factor complex. EPI appeared to bind to a factor VIIa/tissue factor complex formed in the presence of factor Xa but not to a factor VIIa/tissue factor complex formed in the absence of factor Xa.

Model of a Ternary Complex between Activated Factor VII, Tissue Factor and Factor IX

2002 ◽  
Vol 88 (07) ◽  
pp. 74-82 ◽  
Author(s):  
Shu-wen Chen ◽  
Jean-François Schved ◽  
Jean-Luc Pellequer ◽  
Muriel Giansily-Blaizot
Keyword(s):  
Tissue Factor ◽  
Ternary Complex ◽  
Search Algorithm ◽  
Factor Ix ◽  
Site Directed Mutagenesis ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Factor X ◽  
Three Dimensional Model ◽  
Inhibitory Peptide

SummaryUpon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

scholarly journals A Soluble Tissue Factor Mutant Is a Selective Anticoagulant and Antithrombotic Agent

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3219-3227 ◽  
Author(s):  
Robert F. Kelley ◽  
Canio J. Refino ◽  
Mark P. O'Connell ◽  
Nishit Modi ◽  
Pat Sehl ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Arterial Thrombosis ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Cascade ◽  
Factor X ◽  
Rabbit Plasma ◽  
Extrinsic Pathway ◽  
Antithrombotic Agent ◽  
Thrombotic Disease

Abstract One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF ) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule “hTFAA.” hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA⋅FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF⋅FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant (“rTFAA”) was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF⋅FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.

scholarly journals A candidate activation pathway for coagulation factor VII

2019 ◽  
Vol 476 (19) ◽  
pp. 2909-2926
Author(s):  
Tina M. Misenheimer ◽  
Kraig T. Kumfer ◽  
Barbara E. Bates ◽  
Emily R. Nettesheim ◽  
Bradford S. Schwartz
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Contact Activation ◽  
Coagulation Factor Vii ◽  
Factor Viiia

Abstract The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

Extrinsic Activation of Human Blood Coagulation Factors IX and X

1990 ◽  
Vol 63 (02) ◽  
pp. 224-230 ◽  
Author(s):  
V J J Bom ◽  
J H Reinalda-Poot ◽  
R Cupers ◽  
R M Bertina
Keyword(s):  
Tissue Factor ◽  
Kinetic Data ◽  
Factor Viia ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Lag Phase ◽  
Factor X ◽  
Extrinsic Factor ◽  
Factor Ixa ◽  
Factor X Activation

SummaryWe studied activation of human coagulation factors IX and X by factor VIIa in the presence of calcium ions, phospholipid (phosphatidylserine/phosphatidylcholine, 50/50, mol/mol) and purified tissue factor apoprotein. Activation of factor IX and factor X was found to occur without a measurable lag-phase and hence initial rates of factor IXa and factor Xa formation could be determined. Like previously observed for the activation of factor X, the activation of factor IX was saturable with respect to factor VIIa, tissue factor apoprotein and phospholipid. The results suggested that in the presence of a Ca2+ ions the same ternary complex of factor VIIa-tissue factor apoprolein-phospholipid is responsible for the activation of factor IX and factor X. Roth the apparent Km of 22 nM-factor IX and the apparent Kcat of 28 min−1 were about 3-fold lower than the coiicsponding parameters of factor X activation by this complex. Hence, the catalytic efficiency (Kcat/Km) of factor IX and factor X activation was about equal. However, the two substrates inhibited the activation of each other by competition for the same catalytic sites. The apparent Kinh of factor IX for inhibition of extrinsic factor X activation is 30 nM. The apparent Kinh of factor X for inhibition of extrinsic factor IX activation is 116 nM. From these kinetic data it was calculated that at plasma concentration of factors IX and X, the rate of extrinsic factor IX activation would be half the rate of factor X activation. These relative rates of extrinsic factor IX and factor X activation in combination with previously reported kinetic data on the activation of factor X by factor IXa in the presence of factor VIIIa provide support for the concept that at low levels of tissue factor, factor IXa formation might play an important role in the extiinsic pathway of coagulation in vivo.

scholarly journals Human plasma extrinsic pathway inhibitor activity: II. Plasma levels in disseminated intravascular coagulation and hepatocellular disease

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 994-998 ◽  
Author(s):  
TA Warr ◽  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Liver Disease ◽  
Tissue Factor ◽  
Disseminated Intravascular Coagulation ◽  
Factor Viia ◽  
Test Sample ◽  
Factor Ix ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Intravascular Coagulation ◽  
Hepatocellular Disease

Abstract Plasma or serum extrinsic pathway inhibitor (EPI) activity was measured in 24 patients with disseminated intravascular coagulation (DIC) and in 23 patients with severe hepatocellular disease. EPI was measured as activity in a test sample that inhibited factor VIIa/tissue factor (TF)- catalyzed activation of 3H-factor IX (activation peptide release) in the presence of factor X. Of the 24 patients with DIC, 13 had sepsis and five had metastatic carcinoma, disorders in which tissue factor is believed to initiate DIC. EPI activity ranged from 68% to 300% (mean 134% +/- 50%). Serial measurements in nine patients failed to show depletion of EPI activity coincident with worsening DIC. DIC induced by tissue factor or other activating materials may progress despite normal EPI levels. In the patients with liver disease, of whom 15 had decompensated chronic hepatocellular disease (two fatal cases) and eight had acute fulminant liver failure (seven fatal cases), plasma or serum EPI activity varied from less than 20% to 194%. Values were distributed in a bimodal fashion. EPI activity could not be correlated with either the etiology of the liver disease or the degree of prolongation of the prothrombin time. Patients with chronic hepatocellular disease who survived had normal or elevated EPI activity. Patients with fatal hepatic dysfunction had low, normal, or high values for EPI activity. This must mean that secretion of EPI from cells other than hepatocytes can maintain normal plasma EPI levels.

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