RESUMEN
BACKGROUND: Cholestatic liver diseases induce local and systemic hypercoagulation, with neutrophil extracellular traps (NETs) serving as major drivers. These NETs have been linked to decreased liver function in patients with obstructive jaundice. However, the impact of NETs on liver hypercoagulation in cholestatic liver disease remains unknown. METHODS: We utilized bile duct ligation to create experimental mice and analyzed NETs formation in the liver. Fibrin deposition, tissue factor expression, and inflammation in the liver were visualized through western blot and immunohistochemical techniques. LSECs were incubated with isolated NETs, and we detected endothelial procoagulant activity using coagulation protein production assays and measuring endothelial permeability. In both in vivo and in vitro settings, DNase I was applied to clarify the effect of NETs on intrahepatic hypercoagulability, hepatotoxicity, LSEC, and macrophage activation or injury. RESULTS: Bile duct ligation mice exhibited significantly increased levels of NETs in liver tissue, accompanied by neutrophil infiltration, tissue necrosis, fibrin deposition, and thrombophilia compared to sham mice. Notably, NETs resulted in phosphatidylserine and tissue factor exposure on LSEC, enhancing coagulation Factor Xa and thrombin production. The enhanced procoagulant activity could be reversed by degrading NETs with DNase I. Additionally, NETs-induced permeability changes in LSECs, characterized by increased VE-cadherin expression and F-actin retraction, which could be rescued by DNase I. Meanwhile, NET formation is associated with KC activation and the formation of inflammatory factors. CONCLUSIONS: NETs promote intrahepatic activation of coagulation and inflammation, leading to liver tissue injury. Strategies targeting NET formation may offer a potential therapeutic approach for treating cholestatic liver disease.
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Trampas Extracelulares , Hígado , Trombosis , Trampas Extracelulares/metabolismo , Animales , Ratones , Hígado/patología , Hígado/metabolismo , Trombosis/etiología , Trombosis/patología , Colestasis/patología , Colestasis/complicaciones , Modelos Animales de Enfermedad , Masculino , Tromboplastina/metabolismo , Trombofilia/etiología , Trombofilia/sangre , Fibrina/metabolismo , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Humanos , Infiltración Neutrófila , Factor Xa/metabolismo , Trombina/metabolismoRESUMEN
BACKGROUND: The high metastasis rate is one of the main reasons for the poor prognosis of patients with hepatocellular carcinoma (HCC). Coagulation factor Xa (FXa) and its receptor proteinase-activated receptor-2 (PAR-2) proven to promote tumor metastasis in other forms of cancer. Here, we explore the role and mechanism of FXa in the regulation of resistance of anoikis and immune escape of HCC. METHODS: In vitro and in vivo experiments were conducted to explore the role of FXa in HCC metastasis and its potential mechanism. The effects of FXa inhibitor rivaroxaban on HCC immunotherapy were evaluated using intrahepatic metastasis animal models and clinical trial (No. ChiCTR20000040540). We investigated the potential of FXa inhibition as a treatment for HCC. RESULTS: FXa was highly expressed in HCC and promoted metastasis by activating PAR-2. Mechanistically, FXa-activated PAR-2 endows HCC cells with the ability of anoikis resistance to survive in the circulating blood by inhibiting the extrinsic apoptosis pathway. Furthermore, suspension stimulation-induced phosphorylation of STAT2, which promotes programmed death-ligand 1 (PD-L1) transcription and inhibits the antitumor effects of immune cells by inhibiting the infiltration of CD8+T cells in tumors and the levels of secreted cytokines. In vivo inhibition of FXa with rivaroxaban reduced HCC metastasis by decreasing PD-L1 expression and exhausting tumor-infiltrating lymphocytes. Notably, the combination of rivaroxaban and anti-programmed death-1 monoclonal antibody (anti-PD-1) programmed Death-1 monoclonal antibody (anti-PD-1) induced synergistic antitumor effects in animal models. Most importantly, rivaroxaban improved the objective response rate of patients with HCC to immune checkpoint inhibitors and prolonged overall survival time. CONCLUSIONS: FXa-activated PAR-2 promotes anoikis resistance and immune escape in HCC, suggesting the potential for combining coagulation inhibitors and PD-1/PD-L1 immune checkpoint blockade to enhance the therapeutic efficacy of HCC.
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Anoicis , Antígeno B7-H1 , Carcinoma Hepatocelular , Inmunoterapia , Neoplasias Hepáticas , Receptor PAR-2 , Escape del Tumor , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Humanos , Receptor PAR-2/metabolismo , Animales , Ratones , Inmunoterapia/métodos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Factor Xa/metabolismo , Factor Xa/farmacología , Factor Xa/uso terapéutico , Masculino , Femenino , Línea Celular Tumoral , Inhibidores del Factor Xa/farmacología , Inhibidores del Factor Xa/uso terapéuticoRESUMEN
The hemostatic response to vascular injury entails a sequence of proteolytic events where several inactive zymogens of the trypsin family are converted to active proteases. The cascade starts with exposure of tissue factor from the damaged endothelium and culminates with conversion of prothrombin to thrombin in a reaction catalyzed by the prothrombinase complex composed of the enzyme factor Xa, cofactor Va, Ca2+, and phospholipids. This cofactor-dependent activation is paradigmatic of analogous reactions of the blood coagulation and complement cascades, which makes elucidation of its molecular mechanism of broad significance to the large class of trypsin-like zymogens to which prothrombin belongs. Because of its relevance as the most important reaction in the physiological response to vascular injury, as well as the main trigger of pathological thrombotic complications, the mechanism of prothrombin activation has been studied extensively. However, a molecular interpretation of this mechanism has become available only recently from important developments in structural biology. Here we review current knowledge on the prothrombin-prothrombinase interaction and outline future directions for the study of this key reaction of the coagulation cascade.
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Coagulación Sanguínea , Protrombina , Tromboplastina , Humanos , Protrombina/metabolismo , Protrombina/química , Tromboplastina/metabolismo , Tromboplastina/química , Coagulación Sanguínea/fisiología , Animales , Unión Proteica , Factor Xa/metabolismo , Factor VRESUMEN
The advantageous optical properties of quantum dots (QDs) motivate their use in a wide variety of applications related to imaging and bioanalysis, including the detection of proteases and their activity. Recent studies have shown that surface chemistry on QDs is able to modulate protease activity, but only nonspecifically. Here, we present a strategy to selectively accelerate the activity of a particular target protease by as much as two orders of magnitude. Exosite-binding "bait" peptides were derived from proteins that span a range of biological rolesâsubstrate, receptor, and inhibitorâand were used to increase the affinity of the QD-peptide conjugates for either thrombin or factor Xa, resulting in increased rates of proteolysis for coconjugated substrates. Unlike effects from QD surface chemistry, the acceleration was specific to the target protease with negligible acceleration of other proteases. Benefits of this "bait and cleave" sensing approach included detection limits that improved by more than an order of magnitude, reenabled detection of target protease against an overwhelming background of nontarget proteolysis, and mitigation of the action of inhibitors. The cumulative results point to a generalizable strategy, where the mechanism of acceleration, considerations for the design of bait peptides and conjugates, and routes to expanding the scope of this approach are discussed. Overall, this research represents a major step forward in the rational design of nanoparticle-based enzyme sensors that enhance sensitivity and selectivity.
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Péptidos , Puntos Cuánticos , Trombina , Puntos Cuánticos/química , Péptidos/química , Péptidos/metabolismo , Trombina/metabolismo , Trombina/análisis , Trombina/química , Factor Xa/metabolismo , Factor Xa/química , Proteolisis , Humanos , Propiedades de Superficie , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/químicaRESUMEN
Cell-cell fusion mediated by most paramyxovirus requires fusion protein (F) and attachment protein (H, HN, or G). The F protein is proteolytic cleaved to be fusogenically active. J paramyxovirus (JPV) has a unique feature in the family Paramyxoviridae: It encodes an integral membrane protein, syncytial protein (SP, formerly known as transmembrane protein, TM), which is essential in JPV-promoted cell-cell fusion (i.e., syncytial). In this study, we report that cleavage of SP is essential for its syncytial-promoting activity. We have identified the cleavage site of SP at amino acid residues 172 to 175, LKTG, and deletion of the "LKTG" residues abolished SP protein cleavage and its ability to promote cell-cell fusion. Replacing the cleavage site LKTG with a factor Xa protease cleavage site allows cleavage of the SP with factor Xa protease and restores its ability to promote cell-cell fusion. Furthermore, results from a hemifusion assay indicate that cleavage of SP plays an important role in the progression from the intermediate hemifusion state to a complete fusion. This work indicates that SP has many characteristics of a fusion protein. We propose that SP is likely a cell-cell fusion-promoting protein.
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Fusión Celular , Proteínas Virales de Fusión , Animales , Proteínas Virales de Fusión/metabolismo , Chlorocebus aethiops , Proteolisis , Células Vero , Internalización del Virus , Factor Xa/metabolismo , Humanos , Línea CelularRESUMEN
Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.
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Aptámeros de Nucleótidos , Dominio Catalítico , Hirudinas , Trombina , Humanos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Trombina/antagonistas & inhibidores , Trombina/metabolismo , Trombina/química , Hirudinas/química , Hirudinas/farmacología , Anticoagulantes/farmacología , Anticoagulantes/química , Factor Xa/metabolismo , Factor Xa/química , Inhibidores del Factor Xa/química , Inhibidores del Factor Xa/farmacología , Animales , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacosRESUMEN
BACKGROUND: The use of extracorporeal membrane oxygenation (ECMO) is associated with complex hemostatic changes. Systemic anticoagulation is initiated to prevent clotting in the ECMO system, but this comes with an increased risk of bleeding. Evidence on the use of anti-Xa-guided monitoring to prevent bleeding during ECMO support is limited. Therefore, we aimed to analyze the association between anti-factor Xa-guided anticoagulation and hemorrhage during ECMO. METHODS: A systematic review and meta-analysis was performed (up to August 2023). PROSPERO: CRD42023448888. RESULTS: Twenty-six studies comprising 2293 patients were included in the analysis, with six works being part of the meta-analysis. The mean anti-Xa values did not show a significant difference between patients with and without hemorrhage (standardized mean difference -0.05; 95% confidence interval [CI]: -0.19; 0.28, p = .69). We found a positive correlation between anti-Xa levels and unfractionated heparin dose (UFH; pooled estimate of correlation coefficients 0.44; 95% CI: 0.33; 0.55, p < .001). The most frequent complications were any type of hemorrhage (pooled 36%) and thrombosis (33%). Nearly half of the critically ill patients did not survive to hospital discharge (47%). CONCLUSIONS: The most appropriate tool for anticoagulation monitoring in ECMO patients is uncertain. Our analysis did not reveal a significant difference in anti-Xa levels in patients with and without hemorrhagic events. However, we found a moderate correlation between anti-Xa and the UFH dose, supporting its utilization in monitoring UFH anticoagulation. Given the limitations of time-guided monitoring methods, the role of anti-Xa is promising and further research is warranted.
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Anticoagulantes , Oxigenación por Membrana Extracorpórea , Inhibidores del Factor Xa , Hemorragia , Oxigenación por Membrana Extracorpórea/efectos adversos , Oxigenación por Membrana Extracorpórea/métodos , Humanos , Hemorragia/inducido químicamente , Inhibidores del Factor Xa/uso terapéutico , Inhibidores del Factor Xa/efectos adversos , Anticoagulantes/efectos adversos , Coagulación Sanguínea/efectos de los fármacos , Factor Xa/metabolismo , Factores de RiesgoRESUMEN
BACKGROUND: Direct oral factor (F)Xa inhibitors are widely used as alternatives to conventional vitamin K antagonists in managing venous thromboembolism and nonvalvular atrial fibrillation. Unfortunately, bleeding-related adverse events remain a major concern in clinical practice. In case of bleeding or emergency surgery, rapid-onset reversal agents may be required to counteract the anticoagulant activity. OBJECTIVES: The ability of FXa variants to bypass the direct oral FXa inhibitors was assessed. METHODS: Human FXa variants were generated through substitution of phenylalanine 174 (F174) for either alanine, isoleucine, or serine. FXa variants were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. RESULTS: F174-substituted human FX variants demonstrated efficacy in restoring thrombin generation in plasma containing direct FXa inhibitors (apixaban, rivaroxaban, edoxaban). Their ability to bypass the anticoagulant effects stems from a significantly reduced sensitivity for the direct FXa inhibitors due to a decrease in binding affinity determined using molecular dynamics simulations and free energy computation. Furthermore, F174 modification resulted in a partial loss of inhibition by tissue factor pathway inhibitor, enhancing the procoagulant effect of F174-substituted FX. Consequently, the F174A- and F174S-substituted FX variants effectively counteracted the effects of 2 widely used anticoagulants, apixaban and rivaroxaban, in plasma of atrial fibrillation and venous thromboembolism patients. CONCLUSION: These human FX variants have the potential to serve as a rescue reversal strategy to overcome the effect of direct FXa inhibitors in case of life-threatening bleeding events or emergency surgical interventions.
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Coagulación Sanguínea , Factor X , Inhibidores del Factor Xa , Pirazoles , Piridonas , Rivaroxabán , Humanos , Inhibidores del Factor Xa/farmacología , Coagulación Sanguínea/efectos de los fármacos , Pirazoles/farmacología , Células HEK293 , Factor X/metabolismo , Piridonas/farmacología , Factor Xa/metabolismo , Piridinas/uso terapéutico , Piridinas/farmacología , Simulación de Dinámica Molecular , Tiazoles/farmacología , Trombina/metabolismo , Trombina/química , Hemorragia , Unión ProteicaRESUMEN
Direct oral anticoagulants (DOACs) targeting activated factor Xa (FXa) are used to prevent or treat thromboembolic disorders. DOACs reversibly bind to FXa and inhibit its enzymatic activity. However, DOAC treatment carries the risk of anticoagulant-associated bleeding. Currently, only one specific agent, andexanet alfa, is approved to reverse the anticoagulant effects of FXa-targeting DOACs (FXaDOACs) and control life-threatening bleeding. However, because of its mechanism of action, andexanet alfa requires a cumbersome dosing schedule, and its use is associated with the risk of thrombosis. Here, we present the computational design, engineering, and evaluation of FXa-variants that exhibit anticoagulation reversal activity in the presence of FXaDOACs. Our designs demonstrate low DOAC binding affinity, retain FXa-enzymatic activity and reduce the DOAC-associated bleeding by restoring hemostasis in mice treated with apixaban. Importantly, the FXaDOACs reversal agents we designed, unlike andexanet alfa, do not inhibit TFPI, and consequently, may have a safer thrombogenic profile.
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Inhibidores del Factor Xa , Hemorragia , Hemostasis , Pirazoles , Piridonas , Animales , Humanos , Masculino , Ratones , Anticoagulantes/farmacología , Anticoagulantes/efectos adversos , Factor Xa/metabolismo , Inhibidores del Factor Xa/farmacología , Hemorragia/tratamiento farmacológico , Hemorragia/inducido químicamente , Hemostasis/efectos de los fármacos , Pirazoles/farmacología , Piridonas/farmacología , Proteínas RecombinantesAsunto(s)
Anticoagulantes , Fibrilación Atrial , Inhibidores del Factor Xa , Accidente Cerebrovascular Isquémico , Humanos , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/complicaciones , Accidente Cerebrovascular Isquémico/diagnóstico , Accidente Cerebrovascular Isquémico/sangre , Anciano , Inhibidores del Factor Xa/uso terapéutico , Masculino , Femenino , Anticoagulantes/uso terapéutico , Anciano de 80 o más Años , Administración Oral , Factor Xa/metabolismo , Factor Xa/análisis , Arteriopatías Oclusivas/diagnóstico , Accidente Cerebrovascular , Persona de Mediana EdadRESUMEN
BACKGROUND: Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. METHODS: Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 µM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin-antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. RESULTS: In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml-1) and supplementing antithrombin (1.5-4.5 µM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml-1) and antithrombin (3 µM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin-antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. CONCLUSIONS: Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor.
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Antitrombinas , Resistencia a Medicamentos , Inhibidores del Factor Xa , Factor Xa , Heparina , Humanos , Anticoagulantes/farmacología , Antitrombinas/farmacología , Puente Cardiopulmonar , Resistencia a Medicamentos/efectos de los fármacos , Factor Xa/metabolismo , Inhibidores del Factor Xa/farmacología , Heparina/farmacologíaRESUMEN
Abnormal blood coagulation is a major health problem and natural anticoagulants from blood-feeding organisms have been investigated as novel therapeutics. NAPc2, a potent nematode-derived inhibitor of coagulation, has an unusual mode of action that requires coagulation factor Xa but does not inhibit it. Molecular dynamics simulations of NAPc2 and factor Xa were generated to better understand NAPc2. The simulations suggest that parts of NAPc2 become more rigid upon binding factor Xa and reveal that two highly conserved residues form an internal salt bridge that stabilises the bound conformation. Clotting time assays with mutants confirmed the utility of the salt bridge and suggested that it is a conserved mechanism for stabilising the bound conformation of secondary structure-poor protease inhibitors.
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Anticoagulantes , Factor Xa , Simulación de Dinámica Molecular , Unión Proteica , Animales , Anticoagulantes/química , Anticoagulantes/farmacología , Factor Xa/metabolismo , Factor Xa/química , Nematodos/metabolismo , Nematodos/efectos de los fármacos , Humanos , Coagulación Sanguínea/efectos de los fármacos , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Proteínas del Helminto/genética , Sitios de UniónRESUMEN
Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10⯵g/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.
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Heparina , Inhibidor de Proteína C , Serina Proteasas , Inhibidor de Proteína C/química , Inhibidor de Proteína C/metabolismo , Heparina/química , Heparina/farmacología , Humanos , Serina Proteasas/metabolismo , Serina Proteasas/química , Trombina/metabolismo , Proteína C/metabolismo , Proteína C/química , Factor Xa/metabolismo , Factor Xa/química , Secuencia de Aminoácidos , Activación Enzimática/efectos de los fármacosRESUMEN
BACKGROUND: Until recently, the treatment of hemophilia A relied on factor (F)VIII replacement. However, up to one-third of patients with severe hemophilia A develop neutralizing alloantibodies that render replacement therapies ineffective. The development of emicizumab, a bispecific antibody that partially mimics FVIIIa, has revolutionized the treatment of these patients. However, the use of an activated prothrombin complex concentrate [FEIBA (Takeda)] to treat breakthrough bleeding in patients on emicizumab has been associated with thrombotic complications including a unique microangiopathy. OBJECTIVES: We hypothesized that the thrombotic complications observed with the combination of emicizumab and FEIBA might be due to excessive expression of procoagulant activity on the surface of endothelial cells. METHODS: We examined the ability of emicizumab to promote FX activation on endothelial cells using 2 cell culture models. RESULTS: We found that endothelial cells readily support emicizumab-mediated activation of FX by FIXa. The level of FXa generation depends on the concentration of available FIXa. The addition of FEIBA to emicizumab increased FXa generation in a dose-dependent manner on endothelial cells in both models. The rate of FXa generation was further enhanced by endothelial cell activation. However, unlike emicizumab, we found limited FXa generation in the presence of FVIII(a), which followed a significant lag time and was not dependent on FIXa concentration under these conditions. CONCLUSION: Emicizumab promotes FXa generation on the surface of endothelial cells, which is markedly enhanced in the presence of FEIBA. These findings demonstrate a potential mechanism for the thrombotic complications seen with the combined use of emicizumab and FEIBA.
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Anticuerpos Biespecíficos , Anticuerpos Monoclonales Humanizados , Factores de Coagulación Sanguínea , Células Endoteliales , Factor Xa , Humanos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Factor Xa/metabolismo , Factores de Coagulación Sanguínea/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Hemofilia A/tratamiento farmacológico , Hemofilia A/sangre , Factor IX/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Cultivadas , Coagulantes/farmacologíaRESUMEN
Atrial fibrillation (AF), characterised by irregular high-frequency contractions of the atria of the heart, is of increasing clinical importance. The reasons are the increasing prevalence and thromboembolic complications caused by AF. So-called atrial remodelling is characterised, among other things, by atrial dilatation and fibrotic remodelling. As a result, AF is self-sustaining and forms a procoagulant state. But hypercoagulation not only appears to be the consequence of AF. Coagulation factors can exert influence on cells via protease-activated receptors (PAR) and thereby the procoagulation state could contribute to the development and maintenance of AF. In this work, the influence of FXa on Heart Like-1 (HL-1) cells, which are murine adult atrial cardiomyocytes (immortalized), was investigated. PAR1, PAR2, and PAR4 expression was detected. After incubations with FXa (5-50 nM; 4-24 h) or PAR1- and PAR2-agonists (20 µM; 4-24 h), no changes occurred in PAR expression or in the inflammatory signalling cascade. There were no time- or concentration-dependent changes in the phosphorylation of the MAP kinases ERK1/2 or the p65 subunit of NF-κB. In addition, there was no change in the mRNA expression of the cell adhesion molecules (ICAM-1, VCAM-1, fibronectin). Thus, FXa has no direct PAR-dependent effects on HL-1 cells. Future studies should investigate the influence of FXa on human cardiomyocytes or on other cardiac cell types like fibroblasts.
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Fibrilación Atrial , Factor Xa , Animales , Ratones , Factor Xa/metabolismo , FN-kappa B/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transducción de SeñalRESUMEN
Background: Atrial fibrillation (AF) generates a hypercoagulable state with an increased thrombin generation and raised levels of thrombin-antithrombin complexes, which results in a high risk of stroke and thromboembolism. Aim: To evaluate the anticoagulant effect of rivaroxaban by anti-Xa factor activity and its correlation with thrombin-antithrombin complexes, thrombin generation and prothrombin time in patients newly diagnosed with non-valvular AF. Patients and Methods: Prospective study in patients with indication of anticoagulation. Demographic variables, cardiovascular risk factors, CHA2DS2-VASc and HAS-BLED scores were recorded. Blood samples were taken at baseline, at 3 and 24 hours after the administration of the drug and at 30 days. Rivaroxaban levels, anti-Xa activity, prothrombin time, thrombin generation and plasma levels of thrombin-antithrombin complexes were determined. Results: We studied 20 patients aged 76.3 ± 8.0 years (60% female) with a CHA2DS2-VASc score > 2 points. The anti-Xa factor activity correlated with rivaroxaban plasma levels at 3 hours (r = 0.61, p < 0.01), at 24 hours (r = 0.85, p < 0.01) and at 30 days (r = 0.99, p < 0.01), with prothrombin time at 3 hours (r = -0.86, p = 0.019) and at 30 days (r = -0.63, p = 0.02) and with a sustained decrease in thrombin generation at 30 days of follow-up (r = -0.74, p < 0.01). There was no correlation with thrombin-antithrombin complexes (r = -0.02, p = 0.83). Conclusions: Rivaroxaban consistently inhibited the mild pro-coagulant state found in newly diagnosed non-valvular AF patients through the first 24 hours and this effect was maintained at 30 days. Plasma levels of the drug correlated with anti-Xa factor activity, thrombin generation and prothrombin time
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Humanos , Masculino , Femenino , Anciano , Anciano de 80 o más Años , Péptido Hidrolasas/efectos de los fármacos , Fibrilación Atrial/sangre , Trombina/efectos de los fármacos , Factor Xa/efectos de los fármacos , Antitrombina III/efectos de los fármacos , Inhibidores del Factor Xa/farmacología , Rivaroxabán/farmacología , Tiempo de Protrombina , Factores de Tiempo , Trombina/metabolismo , Factor Xa/metabolismo , Administración Oral , Estudios ProspectivosRESUMEN
We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.