ABSTRACT
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of events initiating the blood coagulation pathway. Tfpi-/- mice die during embryonic development. The absence of protease-activated receptor (PAR) 4, the major thrombin receptor on mouse platelets, rescues Tfpi-/-mice to adulthood. Among the 3 TFPI isoforms in mice, TFPIα is the only isoform within platelets (pltTFPIα) and the only isoform that inhibits prothrombinase, the enzymatic complex that converts prothrombin to thrombin. OBJECTIVES: To determine biological functions of pltTFPIα. METHODS: Tfpi-/-/Par4-/- mice were irradiated and transplanted with bone marrow from mice lacking or containing pltTFPIα. Thus, PAR4 expression was restored in the recipient mice, which differed selectively by the presence or absence of pltTFPIα and lacked other forms of TFPI. RESULTS: Recipient mice lacking pltTFPIα had reduced survival over the 200-day posttransplant period. Necropsy revealed radiation injury associated with large intraventricular platelet-rich thrombi, whereas other organs were not affected. Thrombi were associated with fibrotic presentations, including increased collagen deposition, periostin-positive activated fibroblasts, myofibroblasts, and macrophage infiltrates. Recipient mice containing pltTFPIα showed evidence of radiation injury but lacked heart pathology. CONCLUSIONS: Tfpi-/-/Par4-/- mice develop severe cardiac fibrosis following irradiation and transplantation with bone marrow lacking pltTFPIα. This pathology is markedly reduced when the mice are transplanted with bone marrow containing pltTFPIα. Thus, in this model system pltTFPIα has an important physiological role in dampening pathological responses mediated by activated platelets within the heart tissue.
Subject(s)
Blood Platelets , Thrombosis , Mice , Animals , Blood Platelets/metabolism , Thrombosis/metabolism , Thrombin/metabolism , Protein Isoforms , FibrosisABSTRACT
BACKGROUND: The factor V east Texas bleeding disorder (FVETBD) is caused by increased plasma tissue factor pathway inhibitor-α (TFPIα) concentration. The underlying cause is a variant in F5 causing alternative splicing within exon 13 and producing FV-short, which tightly binds the C-terminus of TFPIα, prolonging its circulatory half-life. OBJECTIVES: To diagnose a family presenting with variable bleeding and laboratory phenotypes. PATIENTS/METHODS: Samples were obtained from 17 family members for F5 exon 13 sequencing. Plasma/platelet TFPI and platelet FV were measured by ELISA and/or western blot. Plasma thrombin generation potential was evaluated using calibrated automated thrombography. RESULTS: The FVET variant was identified in all family members with bleeding symptoms and associated with elevated plasma TFPIα (4.5- to 13.4-fold) and total TFPI (2- to 3-fold). However, TFPIα and FV-short were not elevated in platelets. TF-initiated thrombin generation in patient plasma was diminished but was restored by a monoclonal anti-TFPI antibody or factor VIIa. TFPIα localized within vascular extracellular matrix in an oral lesion biopsy from an affected family member. CONCLUSIONS: Factor V east Texas bleeding disorder was diagnosed in an extended family. The variant was autosomal dominant and highly penetrant. Elevated plasma TFPIα, rather than platelet TFPIα, was likely the primary cause of bleeding. Plasma FV-short did not deplete TFPIα from extracellular matrix. In vitro thrombin generation was restored with an anti-TFPI antibody or factor VIIa suggesting effective therapies may be available. Increased awareness of, and testing for, bleeding disorders associated with F5 exon 13 variants and elevated plasma TFPI are needed.
Subject(s)
Blood Coagulation Disorders , Factor V , Blood Coagulation/genetics , Blood Coagulation Disorders/genetics , Blood Coagulation Tests , Factor V/genetics , Humans , Thrombin/metabolismABSTRACT
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets. OBJECTIVE: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response. METHODS: TFPIα-mutant (TfpiΔα/Δα ) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies. RESULTS: TfpiΔα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the TfpiΔα/Δα mice, no TFPI protein or activity was present in their platelets and thrombin-antithrombin complex levels were indistinguishable from Tfpi+/+ littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the TfpiΔα/Δα mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro- or macrovasculature. CONCLUSION: Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.
Subject(s)
Hemostatics , Animals , Blood Coagulation , Hemostasis , Hemostatics/pharmacology , Mice , Platelet Activation , Thrombin/pharmacologyABSTRACT
[Figure: see text].
Subject(s)
Endothelial Cells/metabolism , Extracellular Matrix/metabolism , Kidney/blood supply , Lipoproteins/metabolism , Proteoglycans/metabolism , Binding Sites , Binding, Competitive , Cell Adhesion Molecules/metabolism , Cell Line , Endothelial Cells/drug effects , Extracellular Matrix/drug effects , Glypicans/metabolism , Heparin/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Protein Binding , Protein Isoforms , Syndecan-4/metabolism , Thrombospondins/metabolism , KalininABSTRACT
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein required for murine embryonic development. Intrauterine lethality of Tfpi-/- mice occurs at mid- and late gestation, the latter of which is associated with severe cerebrovascular defects. Megakaryocytes produce only the TFPIα isoform, which is stored within platelets and released upon activation. OBJECTIVES: To examine biological activities of platelet TFPIα (pTFPIα) by characterizing effects of pTFPIα overexpression in Tfpi-/- mice. METHODS: Transgenic mice overexpressing pTFPIα were generated and crossed onto the Tfpi-/- background. Genetic and histological analyses of embryos were performed to investigate the function of pTFPIα during embryogenesis. RESULTS: The transgene (Tg) increased pTFPIα 4- to 5-fold without altering plasma TFPI in adult Tfpi+/+ and Tfpi+/- mice but did not rescue Tfpi-/- mice to wean. Analyses of the impact of pTFPIα overexpression on Tfpi-/- survival, however, were complicated by linkage between the Tg integration site and the endogenous Tfpi locus on chromosome 2. Strain-specific genetic interactions also modulated Tfpi-/- embryonic survival. After accounting for these underlying genetic factors, pTFPIα overexpression completely suppressed mid-gestational lethality of Tfpi-/- embryos but had no effect on development of cerebrovascular defects during late gestation resulting in their lack of survival to wean. CONCLUSIONS: pTFPIα overexpression rescued Tfpi-/- embryos from mid-gestational but not late gestational lethality. The prevalence of underlying genetic factors complicating analyses within our study illustrates the importance of meticulously characterizing transgenic mouse models to avoid spurious interpretation of results.
Subject(s)
Blood Platelets , Lipoproteins , Animals , Embryonic Development , Female , Lipoproteins/genetics , Mice , Pregnancy , Protein IsoformsABSTRACT
Tissue factor pathway inhibitor (TFPI) inhibits proteases in the blood coagulation cascade that lead to the production of thrombin, including prothrombinase (factor Xa [FXa]/FVa), the catalytic complex that directly generates thrombin. Thus, TFPI and FV are directly linked in regulating the procoagulant response. Studies using knockout mice indicate that TFPI and FV are necessary for embryogenesis, but their contributions to vascular development are unclear. We performed extensive histological analyses of Tfpi-/- and Tfpi-/-F5-/- mouse embryos to investigate the importance of the interplay between TFPI and FV in regulating hemostasis and vascular development during embryogenesis. We observed normal tissue development throughout Tfpi-/- embryos, except in the central nervous system (CNS). The CNS displayed stunted brain growth, delayed development of the meninges, and severe vascular pathology characterized by the formation of glomeruloid bodies surrounding areas of cellular death, fibrin deposition, and hemorrhage. Removing FV from Tfpi-/- embryos completely ameliorated their brain pathology, suggesting that TFPI dampens FV-dependent procoagulant activity in a manner that modulates cerebrovascular development. Thus, we have identified a previously unrecognized role for TFPI activity within the CNS. This TFPI activity likely diminishes an effect of excess thrombin activity on signaling pathways that control cerebral vascular development.
Subject(s)
Blood Vessels/embryology , Brain/blood supply , Brain/embryology , Embryonic Development/physiology , Lipoproteins/metabolism , Animals , Factor V/metabolism , Mice , Mice, KnockoutABSTRACT
Comprehensive and spatially accurate poultry population demographic data do not currently exist in the United States; however, these data are critically needed to adequately prepare for, and efficiently respond to and manage disease outbreaks. In response to absence of these data, this study developed a national-level poultry population dataset by using a novel combination of remote sensing and probabilistic modelling methodologies. The Farm Location and Agricultural Production Simulator (FLAPS) (Burdett et al., 2015) was used to provide baseline national-scale data depicting the simulated locations and populations of individual poultry operations. Remote sensing methods (identification using aerial imagery) were used to identify actual locations of buildings having the characteristic size and shape of commercial poultry barns. This approach was applied to 594 U.S. counties with > 100,000 birds in 34 states based on the 2012 U.S. Department of Agriculture (USDA), National Agricultural Statistics Service (NASS), Census of Agriculture (CoA). The two methods were integrated in a hybrid approach to develop an automated machine learning process to locate commercial poultry operations and predict the number and type of poultry for each operation across the coterminous United States. Validation illustrated that the hybrid model had higher locational accuracy and more realistic distribution and density patterns when compared to purely simulated data. The resulting national poultry population dataset has significant potential for application in animal disease spread modelling, surveillance, emergency planning and response, economics, and other fields, providing a versatile asset for further agricultural research.
Subject(s)
Poultry , Remote Sensing Technology , Animals , Datasets as Topic , Disease Outbreaks , Influenza in Birds/epidemiology , Poultry Diseases/epidemiology , United StatesABSTRACT
INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is a naturally occurring anticoagulant found in plasma, where it circulates bound to lipoproteins, factor V (FV) or Protein S (PS), and in platelets. Therapeutic agents targeting TFPI are under development for the treatment of haemophilia A and haemophilia B. AIM: To begin to understand how TFPI, FV and PS interact to modulate haemophilia bleeding. METHODS: Plasma and platelet antigen concentrations of these factors were determined in 73 people with haemophilia A and 18 with haemophilia B. Using multiple regression models, these were compared to the same analytes measured in 224 male blood donors. RESULTS: There were no differences in plasma or platelet TFPI, FV or PS concentrations between haemophilia types or severities. However, compared to blood donors, people with haemophilia had approximately one-third lower plasma PS, 9% lower plasma TFPIα, 50% higher platelet FV and 26% lower platelet Protein S. CONCLUSION: Together, the presented data suggest that individuals with haemophilia may have a compensatory procoagulant response of both plasma and platelet proteins to the decreased concentrations of FVIII or FIX.
Subject(s)
Blood Platelets/metabolism , Factor V/metabolism , Hemophilia A/blood , Hemophilia B/blood , Lipoproteins/blood , Plasma/metabolism , Protein S/metabolism , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
The mammalian blood coagulation system was designed to restrict blood loss due to injury as well as keep the blood fluid within the blood vessels of the organism. Blood coagulation activity in inbred mouse strains varies widely among strains, suggesting that many genomic variants affect hemostasis. Some of these molecules have been discovered and characterized; however, many are still unknown. Genetically modified mouse technologies are providing a plethora of new mouse models for investigating the regulation of blood coagulation. Here we provide a protocol for the tail bleeding time as a primary assessment of in vivo blood coagulation, as well as in vitro methods such as the prothrombin time, activated partial thromboplastin time, and thrombin generation assay. We also provide protocols for the assessment of the activities of specific known factors involved in blood coagulation. © 2019 by John Wiley & Sons, Inc.
Subject(s)
Blood Coagulation Factors/metabolism , Blood Coagulation Tests/methods , Blood Coagulation , Mice/blood , Animals , Blood Coagulation Tests/instrumentation , Mice, Inbred Strains , Tail , Time FactorsABSTRACT
BACKGROUND: Neonates undergoing cardiopulmonary bypass (CPB) surgery to correct congenital heart defects often experience excessive bleeding. Exposure of blood to artificial materials during CPB may activate coagulation, complement and inflammatory pathways. In addition, the surgical stress placed on the haemostatic system may result in cross-activation of other plasma proteolytic cascades, which could further complicate physiological responses to the surgical procedure and post-operative recovery. Plasma protease inhibitors undergo distinct conformational changes upon interaction with proteases, and, thereby, can serve as endogenous biosensors to identify activation of the different proteolytic cascades. We tested the hypothesis that changes in the concentration and conformation of protease inhibitors regulating plasma proteolytic cascades during neonatal CPB are associated with post-operative bleeding. PATIENTS AND METHODS: Plasma samples from 44 neonates were obtained at four time points across the surgical procedure. Anti-thrombin, antitrypsin, anti-chymotrypsin, anti-plasmin, C1-inhibitor and tissue factor pathway inhibitor (TFPI) concentrations and conformations were evaluated by enzyme-linked immunosorbent assay, transverse urea gradient gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS/CONCLUSION: The most striking changes were observed following heparin administration and were associated with the appearance of inactive forms of anti-thrombin and an increase in the plasma concentration of TFPI. Changes in anti-thrombin and TFPI remained evident throughout surgery and into the post-operative period but were not different between patients with or without post-operative bleeding. The concentration of antitrypsin decreased across surgery, but there was no significant accumulation of inactive conformations of any inhibitor besides anti-thrombin, indicating that widespread cross-activation of other plasma proteolytic cascades by coagulation proteases did not occur.
Subject(s)
Anticoagulants/blood , Blood Coagulation/drug effects , Cardiopulmonary Bypass , Heparin/therapeutic use , Lipoproteins/blood , Female , Hemorrhage/etiology , Humans , Infant, Newborn , Male , Peptide Hydrolases/blood , Plasma/metabolism , Postoperative Complications , Protein Binding , Thrombin/metabolismABSTRACT
Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength-defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 0 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-α antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-α is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.
Subject(s)
Blood Platelets/drug effects , Coagulants/chemistry , Factor VIIa/chemistry , Factor Xa/chemistry , Fibrin/chemistry , Lipoproteins/chemistry , Animals , Anticoagulants/chemistry , Blood Coagulation , Blood Platelets/cytology , Collagen/chemistry , Crosses, Genetic , Female , Healthy Volunteers , Hemophilia A/blood , Hemophilia B/blood , Heterozygote , Humans , Male , Mice , Mice, Inbred C57BL , Perfusion , Thromboplastin/chemistry , ThrombosisABSTRACT
BACKGROUND: Plasma Tissue Factor Pathway Inhibitor (TFPI) circulates bound to factor V (fV) and Protein S (PS). Estrogen therapy decreases plasma TFPI and PS. TFPI, fV, and PS circulate within platelets, and are released upon activation to modulate thrombus formation. OBJECTIVE: Identify factors affecting the concentrations of plasma and platelet TFPI, fV, and PS. METHODS: Blood samples were obtained from 435 healthy individuals. Plasma total TFPI, TFPIÉ, fV, and PS, and platelet TFPI, fV, and PS were quantified. Correlations between these protein concentrations and age, gender, race, and estrogen use were established. RESULTS: In males, only plasma fV increased with age, while in females, all plasma analytes increased with age. Males had higher plasma total TFPI, TFPIα, and PS than females. The platelet proteins in either sex remained relatively stable with increasing age. Platelet TFPI and PS were comparable in both sexes, while platelet fV was higher in females. Estrogen use was associated with decreased plasma total TFPI and TFPIα, and platelet PS, but not with platelet TFPI concentration. Racial differences in plasma and platelet proteins were observed, some of which were larger than inter-individual differences observed within racial groups. TFPI, fV and PS concentrations correlated in plasma, while only fV and PS correlated in platelets. CONCLUSIONS: Plasma and platelet TFPI, fV and PS differ in their: (i) in vivo association; (ii) demographic correlates; and (iii) alteration by estrogen therapies. Therefore, the plasma and platelet pools of these proteins may modulate hemostasis and thrombosis via different biochemical pathways.
ABSTRACT
Activated factor V (FVa) and factor X (FXa) form prothrombinase, which converts prothrombin to thrombin. The α isoform of tissue factor (TF) pathway inhibitor (TFPI) dampens early procoagulant events, partly by interacting with FV. FV Leiden (FVL) is the most common genetic thrombophilia in Caucasians. Thrombosis risk is particularly elevated in women with FVL taking oral contraceptives, which produce acquired TFPIα deficiency. In mice, FVL combined with 50% reduction in TFPI causes severe thrombosis and perinatal lethality. However, a possible interaction between FVL and TFPIα has not been defined in humans. Here, we examined this interaction using samples from patients with FVL in thrombin generation and fibrin formation assays. In dilute TF- or FXa-initiated reactions, these studies exposed a TFPI-dependent activation threshold for coagulation initiation that was greatly reduced by FVL. The reduced threshold was progressively overcome with higher concentrations of TF or FXa. Plasma assays using anti-TFPI antibodies or a TFPI peptide that binds and inhibits FVa demonstrated that the decreased activation threshold resulted from reduced TFPIα inhibition of prothrombinase. In assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL than with FV. Thus, FVL reduces the threshold for initiating coagulation, and this threshold is further reduced in situations of low TFPIα concentration. Individuals with FVL are likely prone to thrombosis in response to weak procoagulant stimuli that would not initiate blood clot formation in individuals with FV.
ABSTRACT
Hemophilia is a severe bleeding disorder treated by infusion of the missing blood coagulation protein, factor VIII or factor IX. The discovery and characterization of the anticoagulant protein tissue factor pathway inhibitor (TFPI) led to the realization that inhibition of TFPI activity could restore functional hemostasis through the extrinsic blood coagulation pathway in a manner that does not require the activity of factors VIII or IX. There are currently several therapeutic agents that inhibit TFPI in development for treatment of hemophilia. A comprehensive understanding of TFPI structure, biochemistry, and cellular expression is necessary to understand how it modulates bleeding in hemophilia and the physiological impact of therapeutic agents targeting TFPI.
Subject(s)
Blood Coagulation/drug effects , Hemophilia A/drug therapy , Hemophilia B/drug therapy , Lipoproteins/antagonists & inhibitors , Lipoproteins/metabolism , Molecular Targeted Therapy , Animals , Drug Discovery/methods , Factor IX/metabolism , Factor VIII/metabolism , Hemophilia A/blood , Hemophilia A/metabolism , Hemophilia B/blood , Hemophilia B/metabolism , Hemorrhage/blood , Hemorrhage/drug therapy , Hemorrhage/metabolism , Humans , Lipoproteins/analysis , Molecular Targeted Therapy/methodsABSTRACT
Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi(-/-)) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi(+/-) mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4(-/-)), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi(-/-) embryos, but >40% of expected Tfpi(-/-):Par4(-/-) offspring survived to adulthood. Adult Tfpi(-/-):Par4(-/-) mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi(-/-):Par4(-/-) mice have platelet and fibrin accumulation similar to Par4(-/-) mice following venous electrolytic injury but were more susceptible than Par4(-/-) mice to TF-induced pulmonary embolism. In addition, â¼30% of the Tfpi(-/-):Par4(-/-) mice were born with short tails. Tfpi(-/-):Par4(-/-) mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation.
Subject(s)
Embryonic Development/physiology , Lipoproteins/metabolism , Mice/embryology , Platelet Activation/physiology , Thrombin/metabolism , Animals , Mice, KnockoutABSTRACT
Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIß, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIß have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5' untranslated region allows for translational regulation of TFPIß expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIß has the K1 and K2 domains attached to a glycosylphosphatidyl inositol-anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIß on the cell surface. TFPIα and TFPIß inhibit both TF-factor VIIa-dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIß may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.
Subject(s)
Alternative Splicing , Blood Platelets/metabolism , Endothelial Cells/metabolism , Lipoproteins/genetics , Animals , Binding Sites/genetics , Factor Xa/metabolism , Humans , Lipoproteins/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
OBJECTIVE: Tissue factor pathway inhibitor (TFPI) blocks the initiation of coagulation by inhibiting TF-activated factor VII, activated factor X, and early prothrombinase. Humans produce two 3' splice variants, TFPIα and TFPIß, which are differentially expressed in endothelial cells and platelets and possess distinct structural features affecting their inhibitory function. TFPI also undergoes alternative splicing of exon 2 within its 5' untranslated region. The role of exon 2 splicing in translational regulation of human TFPI isoform expression is investigated. APPROACH AND RESULTS: Exon 2 splicing occurs in TFPIα and TFPIß transcripts. Human tissue mRNA analysis uncovered a wide variability of exon 2 expression. Polysome analysis revealed a repressive effect of exon 2 on TFPIß translation but not on TFPIα. Luciferase reporter assays further exposed strong translational repression of TFPIß (90%) but not TFPIα. Use of a Morpholino to remove exon 2 from TFPI mRNA increased cell surface expression of endogenous TFPIß. Exon 2 also repressed luciferase production (80% to 90%) when paired with the ß-actin 3' untranslated region, suggesting that it is a general translational negative element whose effects are overcome by the TFPIα 3' untranslated region. CONCLUSIONS: Exon 2 is a molecular switch that prevents translation of TFPIß. This is the first demonstration of a 5' untranslated region alternative splicing event that alters translation of isoforms produced via independent 3' splicing events within the same gene. Therefore, it represents a previously unrecognized mechanism for translational control of protein expression. Differential expression of exon 2 denotes a mechanism to provide temporal and tissue-specific regulation of TFPIß-mediated anticoagulant activity.
Subject(s)
5' Untranslated Regions , Alternative Splicing , Lipoproteins/biosynthesis , Lipoproteins/genetics , RNA, Messenger/biosynthesis , 3' Untranslated Regions , Animals , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Down-Regulation , Exons , Gene Expression Regulation , Genes, Reporter , Humans , Polyribosomes/metabolism , Protein Biosynthesis , TransfectionABSTRACT
OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is produced in 2 isoforms: TFPIα, a soluble protein in plasma, platelets, and endothelial cells, and TFPIß, a glycosylphosphatidylinositol-anchored protein on endothelium. Protein S (PS) functions as a cofactor for TFPIα, enhancing the inhibition of factor Xa. However, PS does not alter the inhibition of prothrombinase by TFPIα, and PS interactions with TFPIß are undescribed. Thus, the physiological role and scope of the PS-TFPI system remain unclear. APPROACH AND RESULTS: Here, the cofactor activity of PS toward platelet and endothelial TFPIα and endothelial TFPIß was quantified. PS enhanced the inhibition of factor Xa by TFPIα from platelets and endothelial cells and stabilized the TFPIα/factor Xa inhibitory complex, delaying thrombin generation by prothrombinase. By contrast, PS did not enhance the inhibitory activity of TFPIß or a membrane-anchored form of TFPI containing the PS-binding third Kunitz domain (K1K2K3) although PS did function as a cofactor for K1K2K3 enzymatically released from the cell surface. CONCLUSIONS: The PS-TFPI anticoagulant system is limited to plasma TFPIα and TFPIα released from platelets and endothelial cells. PS likely functions to localize solution-phase TFPIα to the cell surface, where factor Xa is bound. PS does not alter the activity of membrane-associated TFPI. Because activated platelets release TFPIα and PS, the PS-TFPIα anticoagulant system may act physiologically to dampen thrombin generation at the platelet surface.