RESUMEN
Rivaroxaban is a direct factor Xa inhibitor, recently implemented as a favorable alternative to warfarin in anticoagulation therapy. Rivaroxaban effectively reduces thrombin generation, which plays a major role in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) to TAFIa. Based on the antifibrinolytic role of TAFIa, we hypothesized that rivaroxaban would consequently induce more rapid clot lysis. In vitro clot lysis assays were used to explore this hypothesis and additionally determine the effects of varying TAFI levels and a stabilizing Thr325Ile polymorphism (rs1926447) in the TAFI protein on the effects of rivaroxaban. Rivaroxaban was shown to decrease thrombin generation, resulting in less TAFI activation, thus enhancing lysis. These effects were also shown to be less substantial in the presence of greater TAFI levels or the more stable Ile325 enzyme. These findings suggest a role for TAFI levels and the Thr325Ile polymorphism in the pharmacodynamics and pharmacogenomics of rivaroxaban.
Asunto(s)
Carboxipeptidasa B2 , Humanos , Carboxipeptidasa B2/genética , Carboxipeptidasa B2/farmacología , Rivaroxabán/farmacología , Fibrinólisis , Trombina/metabolismo , MutaciónRESUMEN
BACKGROUND: The mechanisms underlying trauma-induced coagulopathy remain elusive. Hyperfibrinolysis has been linked to increased plasminogen activation and antiprotease consumption; however, the mechanistic players in its counterpart, fibrinolysis shutdown, remain unclear. We hypothesize that thrombin-activatable fibrinolysis inhibitor (TAFI) plays a major role in fibrinolytic shutdown after injury. METHODS: As part of this observational cohort study, whole blood was collected from trauma activation patients at a single, level 1 trauma center. Citrated rapid thrombelastography and the following enzyme-linked immunosorbent assays were conducted: thrombin, antithrombin, thrombin-antithrombin complex, TAFI, plasminogen, antiplasmin, plasmin-antiplasmin (PAP), tissue plasminogen activator, plasminogen activator inhibitor 1, and tissue plasminogen activator-plasminogen activator inhibitor 1 complex. Univariate and cluster analysis were performed. RESULTS: Overall, 56 patients (median age, 33.5 years; 70% male) were included. The majority (57%) presented after blunt mechanism and with severe injury (median New Injury Severity Score, 27). Two clusters of patients were identified: Group 1 (normal fibrinolysis, n = 21) and Group 2 (fibrinolysis shutdown, n = 35). Group 2 had significantly lower fibrinolysis with a median LY30 of 1.1% (interquartile range [IQR], 0.1-1.9%) versus 2.1% (IQR, 0.5-2.8%) in Group 1; while the median LY30 was within physiologic range, 45% of patients in Group 2 were in shutdown (vs. 24% in Group 1, p = 0.09). Compared with Group 1, Group 2 had significantly higher PAP (median, 4.7 [IQR, 1.7-9.3] vs. 1.4 [1.0-2.1] µg/mL in Group 1; p = 0.002) and higher TAFI (median, 152.5% [IQR, 110.3-190.7%] vs. 121.9% [IQR, 93.2-155.6%]; p = 0.04). There was a strong correlation between PAP and TAFI ( R2 = 0.5, p = 0.0002). CONCLUSION: The presented data characterize fibrinolytic shutdown, indicating an initial plasmin burst followed by diminished fibrinolysis, which is distinct from hypofibrinolysis (inadequate plasmin burst and fibrinolysis). After an initial thrombin and plasmin burst (increased PAP), fibrinolysis is inhibited, mediated in part by increased TAFI.
Asunto(s)
Antifibrinolíticos , Trastornos de la Coagulación Sanguínea , Carboxipeptidasa B2 , Humanos , Masculino , Adulto , Femenino , Activador de Tejido Plasminógeno , Fibrinolisina , Carboxipeptidasa B2/farmacología , Inhibidor 1 de Activador Plasminogénico , Trombina , Antifibrinolíticos/farmacología , Fibrinólisis , Trastornos de la Coagulación Sanguínea/etiología , PlasminógenoRESUMEN
This review contains the data concerning the mechanisms of spontaneous fibrinolysis in pulmonary vessels and possibilities of its acceleration in pulmonary embolism. The spontaneous fibrinolysis system is known to be sequential and multifactorial, with the interaction of accelerators (t-PA and u-PA) and inhibitors (alpha-2-antiplasmin, PAI-1, TAFI). The fibrinolytic processes take place in case of prevailing reactions of accelerating factors over inhibiting ones. The endothelium of pulmonary vessels possesses pronounced antithrombogenic and profibrinolytic properties, therefore, the processes of fibrinolysis in the pulmonary vascular bed normally occur more intensively than in the vessels of the systemic circulation. The membrane proteins of the endothelium annexins A2 activate plasminogen, whereas thrombomodulin inhibits the activity of PAI-1. The main approaches to increase the fibrinolysis intensity in conditions of pulmonary embolism may be aimed at elevating the activity of fibrinolytic enzymes (enhancing the synthesis of annexins A2, the use of NMDA-receptor antagonists) and suppressing its inhibitors (the use of monoclonal antibodies to alpha-2-antiplasmin, as well as plasminogen activator inhibitor-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI). Promising directions for future research can be the synthesis of a new generation of tissue-type plasminogen activators, and investigations of the possibility of clinical application of antithrombin and thrombomodulin, angiotensin converting enzyme inhibitors and cortisol antagonists. To meet these challenges, it is necessary to develop new models of venous thrombosis and acute pulmonary embolism in different animal species, with the assessment of the changes in the venous haemodynamics and pulmonary microcirculation on the background of administration of a new class of fibrinolytic agents.
Asunto(s)
Carboxipeptidasa B2 , Embolia Pulmonar , Aceleración , Animales , Carboxipeptidasa B2/farmacología , Fibrinólisis , Fibrinolíticos/farmacología , Embolia Pulmonar/tratamiento farmacológicoRESUMEN
OBJECTIVES/HYPOTHESIS: The objective of this study was to determine the role of thrombin-activatable fibrinolysis inhibitor (TAFI) as a candidate biomarker for therapeutic efficacy of sublingual immunotherapy (SLIT) and to identify the role of TAFI in the pathogenesis of allergic rhinitis (AR). STUDY DESIGN: Retrospective cohort study and laboratory study. METHODS: Serum was collected from patients with allergies to Japanese cedar pollen before, during, and after treatment with SLIT. We measured the levels of immunoreactive TAFI, C3a, and C5a in serum by enzyme-linked immunosorbent assay (ELISA) and assessed their relative impact on a combined symptom-medication score. We also examined the impact of TAFI on mast cells and fibroblasts in experiments performed in vitro. RESULTS: Serum levels of TAFI increased significantly in response to SLIT. By contrast, serum C3a levels decreased significantly over time; we observed a significant negative correlation between serum levels of TAFI versus C3a and symptom-medication score. Mast cell degranulation was inhibited in response to TAFI, as it was the expression of both CCL11 and CCL5 in cultured fibroblasts. CONCLUSIONS: High serum levels of TAFI may be induced by SLIT. TAFI may play a critical protective role in pathogenesis of AR by inactivating C3a and by inhibiting mast cell degranulation and chemokines expression in fibroblasts. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2413-2420, 2021.
Asunto(s)
Carboxipeptidasa B2/sangre , Carboxipeptidasa B2/farmacología , Rinitis Alérgica/sangre , Rinitis Alérgica/terapia , Inmunoterapia Sublingual/métodos , Adulto , Anafilatoxinas/efectos de los fármacos , Anafilatoxinas/metabolismo , Biomarcadores/metabolismo , Carboxipeptidasa B2/metabolismo , Quimiocina CCL11/metabolismo , Quimiocina CCL5/metabolismo , Complemento C3a/metabolismo , Cryptomeria/efectos adversos , Cryptomeria/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Mastocitos/patología , Persona de Mediana Edad , Estudios Retrospectivos , Rinitis Alérgica/inmunología , Resultado del TratamientoRESUMEN
INTRODUCTION: Plasma-derived FVIII/VWF complex was reported to be less sensitive to inhibitors than FVIII preparations devoid of VWF. AIM: To compare the efficacy of FVIII/VWF complex (Fanhdi) and five different VWF-free FVIII preparations in restoring thrombin generation and activation of thrombin-activatable fibrinolysis inhibitor (TAFI) in haemophilic plasma, with and without inhibitor, and in cell-based models. METHODS: Experiments were performed in haemophilic plasma supplemented with inhibitory IgG or in plasma samples obtained from haemophilia A patients without (n = 11) and with inhibitor (n = 12). Thrombin generation was evaluated by calibrated automated thrombography (CAT) under standard conditions, in the presence of activated protein C (APC) or thrombomodulin (TM), and in cell-based models including endothelial cells, either alone or in combination with platelets or tissue factor-expressing blood mononuclear cells. The kinetics of TAFI activation was determined by a two-stage functional assay in the absence and in the presence of APC. RESULTS: In haemophilic plasma without inhibitor, Fanhdi enhanced thrombin generation and TAFI activation as well as recombinant (2nd-4th generation) and plasma-derived FVIII preparations devoid of VWF. On the contrary, in plasma with inhibitor, Fanhdi displayed a greater ability to restore thrombin generation and TAFI activation under all tested conditions. Notably, in cell-based models including endothelial cells, Fanhdi proved more efficient than all other preparations in improving thrombin generation even in the absence of inhibitor. CONCLUSION: The greater pro-haemostatic activity of FVIII/VWF complex, either in haemophilic plasma with inhibitor or in the presence of endothelial cells, may offer therapeutic advantages.
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Factor VIII/farmacología , Hemofilia A/tratamiento farmacológico , Factor de von Willebrand/farmacología , Carboxipeptidasa B2/efectos de los fármacos , Carboxipeptidasa B2/metabolismo , Carboxipeptidasa B2/farmacología , Coagulantes/farmacología , Coagulantes/uso terapéutico , Terapia Combinada , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Factor VIII/uso terapéutico , Fibrinólisis/efectos de los fármacos , Hemofilia A/sangre , Hemostasis/efectos de los fármacos , Hemostasis/fisiología , Humanos , Inmunoglobulina G/metabolismo , Cinética , Plasma/metabolismo , Proteína C/metabolismo , Trombina/efectos de los fármacos , Trombina/metabolismo , Trombomodulina/metabolismo , Tromboplastina/metabolismo , Resultado del Tratamiento , Factor de von Willebrand/uso terapéuticoRESUMEN
A direct oral anticoagulant, edoxaban, is as effective as vitamin K antagonists for the treatment of venous thromboembolism (VTE). However, the mechanism underlying the treatment effect on VTE remains to be determined. The aims of this study were to evaluate the effect of edoxaban on tissue plasminogen activator (t-PA)-induced clot lysis in human plasma and to determine the roles of plasmin and thrombin-activatable fibrinolysis inhibitor (TAFI) in the profibrinolytic effect by edoxaban. Pooled human normal plasma or TAFI-deficient plasma (containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban or an activated TAFI inhibitor, potato tuber carboxypeptidase inhibitor (PCI). Clot was induced by adding tissue factor and phospholipids. Clot lysis time and plasma plasmin-α2 antiplasmin complex (PAP) concentration were determined. Clot structure was imaged with a scanning electron microscope. In normal plasma, edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) and PCI significantly shortened clot lysis time. PCI increased PAP concentration and a correlation between PAP concentration and percent of clot lysis was observed. Edoxaban also dose-dependently elevated PAP concentration. In TAFI-deficient plasma, the effects of edoxaban and PCI on clot lysis and PAP concentration were markedly diminished as compared with normal plasma. Fibrin fibers were thinner in clots formed in the presence of edoxaban. In conclusion, edoxaban at clinically relevant concentrations accelerates t-PA-induced fibrinolysis via increasing plasmin generation in human plasma. The effects of edoxaban is mainly dependent on TAFI. The profibrinolytic effect of edoxaban might contribute to the efficacy for the treatment of VTE.
Asunto(s)
Carboxipeptidasa B2/farmacología , Fibrinolisina/efectos de los fármacos , Fibrinólisis/efectos de los fármacos , Piridinas/farmacología , Tiazoles/farmacología , Anticoagulantes/farmacología , Coagulación Sanguínea , Carboxipeptidasa B2/deficiencia , Relación Dosis-Respuesta a Droga , Tiempo de Lisis del Coágulo de Fibrina , Fibrinolisina/análisis , Fibrinolisina/biosíntesis , Fibrinolisina/farmacología , Humanos , Activador de Tejido Plasminógeno , Tromboembolia Venosa/tratamiento farmacológico , alfa 2-Antiplasmina/análisis , alfa 2-Antiplasmina/farmacologíaRESUMEN
: No rodent models are currently available for evaluating inhibitors of the activated form of thrombin-activatable fibrinolysis inhibitor (TAFIa) without exogenous supplementation of tissue-type plasminogen activator (tPA). Characterization of tPA transgenic rats as a tool for the nonclinical evaluation of TAFIa inhibitors is the objective of the current study. tPA transgenic rats were subjected to rat models of tissue-factor-induced thromboembolism, FeCl3-induced deep vein thrombosis (DVT) and arterial thrombosis, and tail bleeding. Potato tuber carboxypeptidase inhibitor (PCI), a selective TAFIa inhibitor, was used as an experimental compound at doses of 0.1, 1, or 10âmg/kg, and its antithrombotic effects and bleeding prolongation effect were compared with nontransgenic rats. Intravenous PCI showed significant and dose-related increase in plasma D-dimer levels in the tissue-factor-induced thromboembolism model. Intravenous PCI also significantly and dose-dependently reduced thrombus weights in the two thrombosis models only in the tPA transgenic rats. These results suggest that sensitive in-vivo evaluation of TAFIa inhibitors can be achieved using tPA transgenic rats without exogenous supplementation of recombinant tPA. On the other hand, no statistically significant prolongation of bleeding times by PCI was observed in either strain, whereas increased bleeding times were observed with 10âmg/kg of intravenous recombinant tPA, suggesting that the low bleeding risk of TAFIa inhibitors is further confirmed in the tPA transgenic rats whose basal tPA levels are elevated. tPA transgenic rats may be beneficial for the pharmacological and toxicological evaluation of TAFIa inhibitors and further confirm that TAFIa is a promising target for various thrombotic disorders.
Asunto(s)
Carboxipeptidasa B2/farmacología , Trombosis/tratamiento farmacológico , Activador de Tejido Plasminógeno/farmacología , Animales , Tiempo de Sangría , Carboxipeptidasa B2/uso terapéutico , Productos de Degradación de Fibrina-Fibrinógeno/efectos de los fármacos , Ratas , Ratas Transgénicas , Trombosis/inducido químicamente , Activador de Tejido Plasminógeno/genéticaRESUMEN
Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Carboxipeptidasa B2/farmacología , Fibrinólisis/efectos de los fármacos , Microscopía Intravital/métodos , Microscopía Confocal/métodos , Activación Plaquetaria/efectos de los fármacos , Plasma Rico en Plaquetas/diagnóstico por imagen , Plaquetas/enzimología , Carboxipeptidasa B2/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Fibrina/metabolismo , Humanos , Fosfatidilserinas/metabolismo , Plasma Rico en Plaquetas/citología , Plasma Rico en Plaquetas/enzimología , Inhibidores de Proteasas/farmacología , Trombomodulina/metabolismo , Factores de TiempoRESUMEN
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen present in blood plasma. Proteolytic activation of TAFI by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin, or plasmin results in an enzyme (TAFIa) that removes carboxyl-terminal lysine residues from protein and peptide substrates, including cell-surface plasminogen receptors. TAFIa is therefore capable of inhibiting plasminogen activation in the pericellular milieu. Since plasminogen activation has been linked to angiogenesis, TAFIa could therefore have anti-angiogenic properties, and indeed TAFIa has been shown to inhibit endothelial tube formation in a fibrin matrix. In this study, the TAFI pathway was manipulated by providing exogenous TAFI or TAFIa or by adding a potent and specific inhibitor of TAFIa. We found that TAFIa elicited a series of anti-angiogenic responses by endothelial cells, including decreased endothelial cell proliferation, cell invasion, cell migration, tube formation, and collagen degradation. Moreover, TAFIa decreased tube formation and proteolysis in endothelial cell culture grown alone and in co-culture with breast cancer cell lines. In accordance with these findings, inhibition of TAFIa increased secretion of matrix metalloprotease proenzymes by endothelial and breast cancer cells. Finally, treatment of endothelial cells with TAFIa significantly inhibited plasminogen activation. Taken together our results suggest a novel role for TAFI in inhibiting tumour angiogenic behaviors in breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Carboxipeptidasa B2/fisiología , Células Endoteliales/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Carboxipeptidasa B2/antagonistas & inhibidores , Carboxipeptidasa B2/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Colágeno Tipo IV/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/farmacología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Plasminógeno/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis. METHODS: To assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM. RESULTS: Inhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa. CONCLUSIONS: Taken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells.
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Neoplasias de la Mama/tratamiento farmacológico , Carboxipeptidasa B2/farmacología , Movimiento Celular , Plasminógeno/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/secundario , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Proteolisis , Trombomodulina/metabolismo , Células Tumorales CultivadasRESUMEN
INTRODUCTION: Bleeding symptoms in severe thrombocytopenia range from mild to severe. The aim of this in vitro study was to improve blood clotting and protect against fibrinolysis in reconstituted severe thrombocytopenia blood. METHODS: Thrombocytopenia [(16 ± 4) × 10(6) /mL] was created by high-speed centrifugation of normal blood with subsequent mixing plasma with packed cells. The blood samples were subjected to clotting by CaCl2 and tissue factor and to fibrinolysis by the addition of tissue plasminogen activator. Blood was spiked with fibrinogen, activated prothrombin complex concentrate (FEIBA), thrombin activatable fibrinolysis inhibitor (TAFI), or their combinations. To mimic the situation that may occur in patients subjected to massive transfusion of plasma substitutes, blood was diluted by 40% of TRIS/saline buffer. Clotting time (CT), α-Angle, maximum clot firmness (MCF), and lysis onset time (LOT) were evaluated using rotation thromboelastometry. RESULTS: Spiking thrombocytopenia blood with FEIBA led to reduction of CT. Fibrinogen and FEIBA enhanced α-Angle and MCF both in the absence and in the presence of tPA. LOT values were prolonged by TAFI and to less extent by FEIBA. Dilution of thrombocytopenia blood was followed by reduction of α-Angle and MCF compared to nondiluted blood which partly reversed by either fibrinogen or FEIBA being higher using fibrinogen and FEIBA together. Clot strength was enhanced, and fibrinolysis was inhibited by TAFI. CONCLUSION: The results of this study suggest that combined spiking of blood with fibrinogen and FEIBA may be enough to correct the clot formation disorder in severe thrombocytopenia, whereas in thrombocytopenia and blood dilution, additive inhibition of fibrinolysis may be needed.
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Factores de Coagulación Sanguínea/farmacología , Pruebas de Coagulación Sanguínea/métodos , Coagulación Sanguínea/efectos de los fármacos , Carboxipeptidasa B2/farmacología , Fibrinógeno/farmacología , Pruebas de Coagulación Sanguínea/instrumentación , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Cloruro de Calcio/farmacología , Humanos , Modelos Biológicos , Cultivo Primario de Células , Rotación , Índice de Severidad de la Enfermedad , Tromboelastografía/instrumentación , Trombocitopenia/sangre , Trombocitopenia/patología , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Thrombin activatable fibrinolysis inhibitor (TAFI) was discovered two decades ago consequent to the identification of an unstable carboxypeptidase (CPU) formed upon thrombin activation of its proenzyme. The antifibrinolytic effects of the activated form (TAFIa, CPU) are linked with its capacity to remove C-terminal lysines from the surface of the fibrin clot. A distinctive characteristic of TAFIa is its temperature-dependent conformational instability: TAFIa activity spontaneously decays with an apparent half-life of 8 to 15 minutes at 37°C. A variety of studies has demonstrated a role for TAFI/TAFIa in venous and arterial diseases. In addition, a role for TAFI/TAFIa in inflammation and cell migration has also been shown. Because TAFI/TAFIa is a potential risk factor for thrombotic disorders, many inhibitors, both at the level of activation or at the level of activity, have been developed and were proven to exhibit a profibrinolytic effect in animal models. Pharmacologically active inhibitors of the TAFI/TAFIa system may open new ways for the prevention of thrombotic diseases or for the establishment of adjunctive treatments during thrombolytic therapy.
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Carboxipeptidasa B2/fisiología , Animales , Carboxipeptidasa B2/química , Carboxipeptidasa B2/farmacología , Fibrinólisis , HumanosRESUMEN
Induction chemotherapy is associated with increased thrombosis risk in children with acute lymphoblastic leukemia (ALL). In this prospective study, we explored the effects of ALL and induction chemotherapy on the procoagulant, anticoagulant, and fibrinolytic systems in 20 children with ALL. The levels of d-dimer, factor VIII, von Willebrand factor, protein C, antithrombin III, and thrombin-activated fibrinolysis inhibitor (TAFI) were elevated at diagnosis. These changes were not related with peripheral blast count. The levels of fibrinogen, d-dimer, coagulation inhibitors, and plasminogen decreased, whereas the levels of tissue factor pathway inhibitor and plasminogen activator inhibitor 1 increased progressively following prednisolone monotherapy, administration of vincristine plus daunorubicin, and l-asparaginase. The levels of factor VIII, d-dimer, and TAFI remained elevated during the study period. In conclusion, coagulation activation and impaired fibrinolysis already exist at diagnosis, whereas induction chemotherapy leads to reactivation of coagulation and progressive impairment in fibrinolytic and anticoagulant capacities in childhood ALL.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Coagulación Sanguínea/efectos de los fármacos , Carboxipeptidasa B2/uso terapéutico , Fibrinólisis/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Adolescente , Anticoagulantes/uso terapéutico , Proteína C-Reactiva/metabolismo , Carboxipeptidasa B2/farmacología , Niño , Preescolar , Femenino , Humanos , Quimioterapia de Inducción , Masculino , Factores de Riesgo , Trombosis/sangre , Trombosis/patologíaRESUMEN
Available therapies for thromboembolic disorders include thrombolytics, anticoagulants, and antiplatelets, but these are associated with complications such as bleeding. To develop an alternative drug which is clinically safe, we focused on activated thrombin-activatable fibrinolysis inhibitor (TAFIa) as the target molecule. TAFIa is a zinc-containing carboxypeptidase that significantly inhibits fibrinolysis. Here we designed and synthesized selenium-containing compounds 5-13 to discover novel TAFIa inhibitors having a superior zinc-coordinating group. Compounds 5-13 significantly inhibited TAFIa activity (IC(50) 2.2 × 10(-12) M - 2.6 × 10(-6) M). We found that selenol is a better functional group than thiol for coordinating to zinc at the active site of TAFIa. Furthermore, compound 12, which has an amino-chloro-pyridine ring, was found to be a potent and selective TAFIa inhibitor that lacks carboxypeptidase N inhibitory activity. Therefore, compound 12 is a promising candidate for the treatment of thromboembolic disorders. This is the first report of a selenium-containing inhibitor for TAFIa.
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Carboxipeptidasa B2/farmacología , Diseño de Fármacos , Selenio/análisis , Carboxipeptidasa B2/química , Espectroscopía de Resonancia Magnética , Espectrometría de MasasRESUMEN
Patients suffering major traumatic or surgical bleeding are often exposed to hemodilution resulting in dilutional coagulopathy. The aim of this study was to evaluate in vitro the effects of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and resistance to fibrinolysis in hemodilution conditions. Citrated whole blood from 36 healthy volunteers was diluted to 30 and 60% with lactated Ringer's solution. Blood samples were subsequently supplemented with fibrinogen, FXIII, TAFI or their combinations. Rotation thromboelastometry (ROTEM) in whole blood and thrombin generation in plasma were performed in the presence of CaCl2 and tissue factor/EXTEM reagent, and fibrinolysis was induced by tissue plasminogen activator (tPA). Hemodilution was expressed by decrease of peak height in thrombin generation and α-angle and maximum clot firmness (MCF) in ROTEM. Fibrinogen, FXIII or TAFI did not correct the decrease in thrombin generation peak height. In ROTEM, spiking of diluted blood with fibrinogen stimulated clot propagation. In tPA-treated blood fibrinogen, FXIII and TAFI increased clot firmness and inhibited fibrinolysis. Stronger protection against fibrinolysis was achieved combining FXIII with TAFI. Hemodilution was associated with inhibition of thrombin generation; however, this effect was not sensitive to blood spiking with fibrinogen, FXIII and TAFI. In ROTEM, these hemostasis agents improved clot strength and decreased clot susceptibility to tPA in nondiluted and to more extent in diluted blood. The maximal protection against fibrinolysis was caused by TAFI. Combining FXIII with TAFI exerted synergistic inhibitory effect on fibrinolysis.
Asunto(s)
Carboxipeptidasa B2/farmacología , Factor XIII/farmacología , Fibrinógeno/farmacología , Activador de Tejido Plasminógeno/farmacología , Adulto , Fibrinólisis/efectos de los fármacos , Hemodilución , Humanos , Soluciones Isotónicas/química , Lactato de Ringer , Tromboelastografía , Trombina/metabolismo , Tiempo de Coagulación de la Sangre TotalRESUMEN
Carbon monoxide, derived from carbon monoxide-releasing molecules, has been recently demonstrated to enhance the velocity of formation and strength of plasma thrombi. We tested the hypothesis that carbon monoxide-releasing molecule-2 would modulate fibrinolysis of plasma thrombi. Normal plasma was exposed to 0, 25, 50, 100 or 200 micromol/l carbon monoxide-releasing molecule-2, with coagulation activated with tissue factor and fibrinolysis initiated with tissue-type plasminogen activator. Additional experiments utilized factor XIII, plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor or alpha2-antiplasmin-deficient plasmas. Thrombus growth/disintegration kinetics was monitored with thrombelastography. Carbon monoxide-releasing molecule-2, in a concentration-dependent fashion, increased the velocity of thrombus formation and strength, and markedly attenuated fibrinolysis in normal plasma. In factor XIII-deficient plasma, carbon monoxide-releasing molecule-2 mediated effects on thrombus growth/disintegration kinetics were similar to that seen with normal plasma; however, carbon monoxide-releasing molecule-2 had a less marked effect on thrombus growth/disintegration in both plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor-deficient plasma, with even less carbon monoxide-releasing molecule-2-mediated effects noted in alpha2-antiplasmin-deficient plasma. Carbon monoxide-releasing molecule-2 attenuated fibrinolysis by enhancing the velocity of clot growth and strength while augmenting the effects of plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor and alpha2-antiplasmin. These findings serve as the rationale for further investigations to determine if carbon monoxide-releasing molecules could be utilized as hemostatic agents.
Asunto(s)
Monóxido de Carbono/farmacología , Fibrinólisis/efectos de los fármacos , Hemostáticos/farmacología , Compuestos Organometálicos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Carboxipeptidasa B2/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Factor XIII/farmacología , Fibrinógeno/análisis , Hemostáticos/administración & dosificación , Humanos , Compuestos Organometálicos/administración & dosificación , Tiempo de Tromboplastina Parcial , Inhibidor 1 de Activador Plasminogénico/farmacología , Tiempo de Protrombina , Tromboelastografía , Tromboplastina/farmacología , Activador de Tejido Plasminógeno/farmacología , alfa 2-Antiplasmina/farmacologíaAsunto(s)
Permeabilidad Capilar/efectos de los fármacos , Carboxipeptidasa B2/farmacología , Sepsis/tratamiento farmacológico , Adenosina Monofosfato/metabolismo , Animales , Permeabilidad Capilar/fisiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Humanos , Lipopolisacáridos/farmacología , Sensibilidad y Especificidad , Sepsis/enzimología , Sepsis/fisiopatología , Quinasas Asociadas a rho/metabolismoRESUMEN
OBJECTIVE: Although thrombin-activatable fibrinolysis inhibitor (TAFI) has been implicated as a negative regulator of fibrinolysis, its pathophysiological significance remains to be unveiled. We performed the pharmacologic study to assess the effect of EF6265, a specific inhibitor of activated form of TAFI (TAFIa) on sepsis-induced organ dysfunction models. DESIGN: A controlled, in vivo laboratory study. SETTING: Company research laboratory. SUBJECTS: Wistar and Sprague-Dawley rats. INTERVENTIONS: Endotoxemia and sepsis models were induced by intravenous injection of lipopolysaccharide and Pseudomonas aeruginosa, respectively. MEASUREMENTS AND MAIN RESULTS: In the endotoxemia model, posttreatment (1 hour) with EF6265 reduced fibrin deposits in the kidney and liver accompanied by no significant changes in platelet count and fibrinogen concentration in plasma. This compound also significantly decreased levels of plasma lactate dehydrogenase and aspartate aminotransferase, markers of organ dysfunction. In the sepsis model, EF6265, simultaneously administered with ceftazidime (CAZ) 2 hours after Pseudomonas aeruginosa injection, showed no influence on the antibiotic activity of CAZ. Meanwhile, it dramatically potentiated the interleukin-6-reducing effect of CAZ in plasma, suggesting that inhibition of TAFIa leads to the reduction in systemic inflammatory response associated with bacterial infection. This combined treatment also lowered plasma lactate dehydrogenase and blood urea nitrogen more potently than single treatment with CAZ. CONCLUSIONS: These results clearly suggest that TAFI plays an important role in the deterioration of organ dysfunction in sepsis and the inhibitor of TAFIa protects against sepsis-induced tissue damage through regulation of fibrinolysis and inflammation.
Asunto(s)
Aminoácidos/farmacología , Carboxipeptidasa B2/farmacología , Insuficiencia Multiorgánica/prevención & control , Ácidos Fosfínicos/farmacología , Infecciones por Pseudomonas/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Análisis de Varianza , Animales , Análisis Químico de la Sangre , Ceftazidima/farmacología , Modelos Animales de Enfermedad , Fibrinógeno/efectos de los fármacos , Fibrinógeno/metabolismo , Heparina/farmacología , Interleucina-6/sangre , Recuento de Leucocitos , Lipopolisacáridos/farmacología , Masculino , Insuficiencia Multiorgánica/tratamiento farmacológico , Recuento de Plaquetas , Probabilidad , Infecciones por Pseudomonas/sangre , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Sensibilidad y Especificidad , Sepsis/sangre , Células MadreRESUMEN
Inflammation and disturbances of the hemostatic system may play a role in pathogenesis and complications of ischemic heart disease. More and more reports indicate that apart from their cholesterol-lowering effect statins also exert other beneficial effects in cardiovascular diseases. Taking this into consideration, the aim of the study was to assess the influence of simvastatin (20 mg per day) on a marker of inflammation - CRP and some parameters of coagulation and fibrinolysis in 22 patients with ischaemic heart disease. Serum lipids, levels of hsCRP, thrombomodulin (TM), vWF, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), thrombin activatable fibrinolysis inhibitor (TAFI), t-PA, plasmin-antiplasmin complex (PAP) and TAFI activity were assessed before and after one, three and six months simvastatin treatment. After one month therapy of simvastatin, there have been significant reduction of levels of total cholesterol, LDL-cholesterol and triglycerides and these values have remained until the end of the study. No influence on the level of HDL-cholesterol has been observed. After 6 months of treatment significant decrease in the level of hsCRP and increase of the levels TM and vWF with reference to baselines results have been observed. After a 1-and 6-month therapy, the level of TAFI have been significantly increased. Other hemostatic parameters, i.e. levels of F1+2, TAT, t-PA, PAP and TAFI activity have not changed significantly. This prospective study has confirmed high efficacy of lipid-lowering effect and anti-inflammatory properties of simvastatin. Simvastatin influenced some hemo-static parameters, however, these effects were not, in majority, significant.
Asunto(s)
Proteína C-Reactiva/efectos de los fármacos , Hemostasis/efectos de los fármacos , Isquemia Miocárdica/prevención & control , Simvastatina/farmacología , Anciano , Carboxipeptidasa B2/metabolismo , Carboxipeptidasa B2/farmacología , Colesterol/sangre , LDL-Colesterol/sangre , LDL-Colesterol/efectos de los fármacos , Femenino , Humanos , Masculino , Isquemia Miocárdica/etiología , Miocarditis/complicaciones , Miocarditis/metabolismo , Miocarditis/prevención & control , Trombomodulina/efectos de los fármacos , Triglicéridos/sangreRESUMEN
To clarify the role of TAFI in hypertensive disorders in pregnancy, 22 subjects, including 10 with pre-eclampsia (PE) and 12 with gestational hypertension were examined for the levels of TAFI and thrombin-antithrombin (TAT) complex. Thirty normal pregnant women served as controls. ELISA was employed for the detection. The results showed that the TAFI antigen levels in normal pregnancy group, gestational hypertension group and PE group were (85.35+/-24.69)%, (99.65+/-18.27)%, (110.12+/-23.36)%; (97.06+/-21.40)%, (114.08+/-27.76)%, (125.49+/-24.70)%; (106.6+/-19.21)%, (129.2+/-25.07)%, (139.1+/-30.12)%, in the 1st, 2nd and 3rd trimester respectively. No significant differences were found between the normal pregnancy group and gestational hypertension group but significant difference existed between normal pregnancy group and PE group in each trimester (P<0.05). TAT complexes were significantly higher in patients with PE than that in controls (P<0.05), but no correlation was found between TAT and TAFI. It is concluded that TAFI may contributed to the impairment of fibrinolysis in the patients with PE and may serves as a sensitive indicator for PE, but it may not help in the diagnosis of the gestational hypertension.