RESUMEN
OBJECTIVE: To investigate the characteristic of circulating microparticle in patients with acute myocardial infarction (AMI) and its possible mechanism of promoting coagulation. METHODS: A prospective case-control study was conducted. The patients with coronary heart disease admitted to the second department of cardiology in Harbin First Hospital from June to November 2023 were enrolled, and they were grouped according to whether the patients occurred AMI or not. On the day of admission, disseminated intravascular coagulation (DIC) score was calculated. At the same time, fasting venous blood was collected, and the levels of D-dimer, fibrin degradation product (FDP) and the activities of major coagulation factors were detected. The level of circulating microparticle was determined by microparticle trapping method. The microparticle carrying tissue factor (TF+MP) level was detected by tissue factor (TF) dependent F Xa production assay. Spearman correlation method was used to analyze the correlation among the indicators. RESULTS: A total of 52 patients with coronary heart disease were enrolled, including 26 patients in AMI group and 26 patients in non-AMI group. There was no significant difference in gender, age, body mass index (BMI), underlying diseases, smoking history, and pre-admission treatment of patients between the two groups, indicating that the baseline data of the two groups were balanced and comparable. Compared with the non-AMI group, the DIC score and D-dimer, FDP levels in the AMI group were significantly increased [DIC score: 3 (3, 4) vs. 3 (2, 3), D-dimer (mg/L): 8.80 (6.84, 15.66) vs. 2.13 (1.64, 3.86), FDP (mg/L): 30.13 (19.30, 52.54) vs. 20.00 (13.51, 28.37), all P < 0.01], indicating that the degree of coagulation activation in AMI patients was more severe. The consumption of major coagulation factors in the coagulation pathway in the AMI group was heavier than that in the non-AMI group [F II: 59.45% (49.65%, 71.25%) vs. 63.65% (49.98%, 73.22%), F V: 96.95% (73.50%, 112.78%) vs. 105.05% (73.48%, 131.48%), F VII: 42.30% (36.98%, 51.98%) vs. 53.40% (46.58%, 69.88%), F X: 60.90% (48.22%, 80.82%) vs. 73.50% (56.80%, 85.98%), F XI: 82.45% (62.90%, 99.10%) vs. 92.40% (73.90%, 114.25%), F XII: 29.90% (12.42%, 42.38%) vs. 34.65% (16.32%, 48.20%), all P < 0.05]. The circulating TF+MP level in the AMI group was significantly higher than that in the non-AMI group [nmol/L: 0.13 (0.06, 0.20) vs. 0.08 (0.04, 0.15), P < 0.05]. There was no significant difference in the level of circulating microparticle between AMI group and non-AMI group [nmol/L: 1.24 (0.71, 3.77) vs. 1.35 (0.73, 2.14), P > 0.05]. Correlation analysis showed that circulating TF+MP level in the patients with coronary heart disease was significantly positively correlated with coagulation indicator DIC score (r = 0.307, P = 0.027), D-dimer (r = 0.696, P < 0.001) and FDP (r = 0.582, P < 0.001), and there was a strong negative correlation with exogenous pathway factor F VII (r = -0.521, P < 0.001) and common pathway factor F X (r = -0.332, P = 0.016). CONCLUSIONS: The circulating TF+MP level in AMI patients was significantly higher than that in the non-AMI patients. TF+MP may play an important role in activating the extrinsic coagulation pathway, exacerbating coagulation factor consumption, and promoting clot formation during AMI occurrence.
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Coagulación Sanguínea , Micropartículas Derivadas de Células , Productos de Degradación de Fibrina-Fibrinógeno , Infarto del Miocardio , Tromboplastina , Humanos , Infarto del Miocardio/sangre , Infarto del Miocardio/diagnóstico , Estudios Prospectivos , Estudios de Casos y Controles , Micropartículas Derivadas de Células/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Tromboplastina/metabolismo , Factores de Coagulación Sanguínea/metabolismo , Factores de Coagulación Sanguínea/análisis , Femenino , Masculino , Coagulación Intravascular Diseminada/sangre , Persona de Mediana Edad , Enfermedad Coronaria/sangreAsunto(s)
COVID-19 , Monocitos , SARS-CoV-2 , Tromboplastina , Trombosis , Humanos , COVID-19/inmunología , COVID-19/sangre , COVID-19/complicaciones , Monocitos/inmunología , Monocitos/metabolismo , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/inmunología , Trombosis/etiología , SARS-CoV-2/inmunologíaRESUMEN
Ovarian cancer (OC) is a leading cause of death among gynaecological malignancies. The haemostatic system, which controls blood flow and prevents clotting disorders, paradoxically drives OC progression while increasing the risk of venous thromboembolism (VTE). MicroRNAs (miRNAs) have emerged as crucial in understanding VTE pathogenesis. Exploring the connection between cancer and thrombosis through these RNAs could lead to novel biomarkers of cancer-associated thrombosis (CAT) and OC, as well as potential therapeutic targets for tumour management. Thus, this study examined the impact of eight plasma miRNAs targeting the tissue factor (TF) coagulation pathway-miR-18a-5p, -19a-3p, -20a-5p, -23a-3p, -27a-3p, -103a-3p, -126-5p and -616-3p-in 55 OC patients. Briefly, VTE occurrence post-OC diagnosis was linked to shorter disease progression time (log-rank test, p = 0.024) and poorer overall survival (OS) (log-rank test, p < 0.001). High pre-chemotherapy levels of miR-20a-5p (targeting coagulation factor 3 (F3) and tissue factor pathway inhibitor 2 (TFPI2)) and miR-616-3p (targeting TFPI2) predicted VTE after OC diagnosis (χ2, p < 0.05). Regarding patients' prognosis regardless of VTE, miR-20a-5p independently predicted OC progression (adjusted hazard ratio (aHR) = 6.13, p = 0.005), while miR-616-3p significantly impacted patients' survival (aHR = 3.72, p = 0.020). Further investigation is warranted for their translation into clinical practice.
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MicroARNs , Neoplasias Ováricas , Tromboplastina , Humanos , Femenino , Neoplasias Ováricas/sangre , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/genética , MicroARNs/sangre , MicroARNs/genética , Tromboplastina/metabolismo , Tromboplastina/genética , Persona de Mediana Edad , Pronóstico , Anciano , Trombosis/sangre , Trombosis/etiología , Tromboembolia Venosa/sangre , Tromboembolia Venosa/etiología , Tromboembolia Venosa/genética , Adulto , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: The efficacy of antibody-targeted therapy of solid cancers is limited by the lack of consistent tumour-associated antigen expression. However, tumour-associated antigens shared with non-malignant cells may still be targeted using conditionally activated-antibodies, or by chimeric antigen receptor (CAR) T cells or CAR NK cells activated either by the tumour microenvironment or following 'unlocking' via multiple antigen-recognition. In this study, we have focused on tissue factor (TF; CD142), a type I membrane protein present on a range of solid tumours as a basis for future development of conditionally-activated BiTE or CAR T cells. TF is frequently upregulated on multiple solid tumours providing a selective advantage for growth, immune evasion and metastasis, as well as contributing to the pathology of thrombosis via the extrinsic coagulation pathway. METHODS: Two well-characterised anti-TF monoclonal antibodies (mAb) were cloned into expression or transposon vectors to produce single chain (scFv) BiTE for assessment as CAR and CD28-CD3-based CAR or CD3-based BiTE. The affinities of both scFv formats for TF were determined by surface plasmon resonance. Jurkat cell line-based assays were used to confirm the activity of the BiTE or CAR constructs. RESULTS: The anti-TF mAb hATR-5 and TF8-5G9 mAb were shown to maintain their nanomolar affinities following conversion into a single chain (scFv) format and could be utilised as CD28-CD3-based CAR or CD3-based BiTE format. CONCLUSION: Because of the broad expression of TF on a range of solid cancers, anti-TF antibody formats provide a useful addition for the development of conditionally activated biologics for antibody and cellular-based therapy.
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Receptores Quiméricos de Antígenos , Linfocitos T , Tromboplastina , Humanos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Tromboplastina/inmunología , Tromboplastina/metabolismo , Linfocitos T/inmunología , Inmunoterapia Adoptiva/métodos , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/genética , Neoplasias/inmunología , Neoplasias/terapia , Células JurkatRESUMEN
Prothrombinase complex, composed of coagulation factors Xa (FXa) and Va (FVa) is a major enzyme of the blood coagulation network that produces thrombin via activation of its inactive precursor prothrombin (FII) on the surface of phospholipid membranes. However, pathways and mechanisms of prothrombinase formation and substrate delivery are still discussed. Here we designed a novel mathematical model that considered different potential pathways of FXa or FII binding (from the membrane or from solution) and analyzed the kinetics of thrombin formation in the presence of a wide range of reactants concentrations. We observed the inhibitory effect of large FVa concentrations and this effect was phospholipid concentration-dependent. We predicted that efficient FII activation occurred via formation of the ternary complex, in which FVa, FXa and FII were in the membrane-bound state. Prothrombin delivery was mostly membrane-dependent, but delivery from solution was predominant under conditions of phospholipid deficiency or FXa/FVa excess. Likewise, FXa delivery from solution was predominant in the case of FVa excess, but high FII did not switch the FXa delivery to the solution-dependent one. Additionally, the FXa delivery pathway did not depend on the phospholipid concentration, being the membrane-dependent one even in case of the phospholipid deficiency. These results suggest a flexible mechanism of prothrombinase functioning which utilizes different complex formation and even inhibitory mechanisms depending on conditions.
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Factor Xa , Protrombina , Cinética , Humanos , Factor Xa/metabolismo , Protrombina/metabolismo , Modelos Biológicos , Fosfolípidos/metabolismo , Coagulación Sanguínea/fisiología , Trombina/metabolismo , Factor Va/metabolismo , Tromboplastina/metabolismo , Especificidad por Sustrato , Factor VAsunto(s)
COVID-19 , Monocitos , SARS-CoV-2 , Tromboplastina , Trombosis , Humanos , COVID-19/inmunología , COVID-19/complicaciones , COVID-19/sangre , Trombosis/sangre , Trombosis/inmunología , Trombosis/etiología , Monocitos/inmunología , Monocitos/metabolismo , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Tromboplastina/metabolismoRESUMEN
Complement and extracellular vesicles (EVs) association with thrombogenic tendencies is acknowledged, but limited evidence exists for their link to COVID-19 venous thromboembolism. This study aims to examine the relationship between pulmonary embolism and the expression of complement and other proteins related to thrombogenesis in severe Covid-19 patients. We included prospectively 207 severe COVID-19 patients and retrospectively screened for pulmonary embolism (PE). This analysis comprises 20 confirmed PE cases and 20 matched patients without PE. Blood samples taken at the admission in the intensive care unit were analyzed for complement using ELISA. EVs derived from neutrophils, endothelium, or platelets, as well carrying complement or tissue factor were analyzed using flow cytometry. Complement levels were markedly elevated, with a notable increase in C3a and Terminal Complement Complex. The most prevalent EV population was identified as tissue factor (TF)-carrying EVs which peaked in patients with PE during ICU days 4-9. However, for both the complement and analyzed EV populations, no statistically significant differences were found between the patients who developed pulmonary embolism and those who did not. In conclusion, complement factors and EVs expressing tissue factor, along with EVs derived from endothelial cells and platelets, are elevated in severe COVID-19 patients, regardless of the presence of pulmonary embolism. However, the involvement of complement and procoagulant EVs in peripheral plasma in the development of pulmonary embolism is still unclear and requires further investigation.
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COVID-19 , Proteínas del Sistema Complemento , Vesículas Extracelulares , Embolia Pulmonar , SARS-CoV-2 , Humanos , Embolia Pulmonar/sangre , COVID-19/complicaciones , COVID-19/sangre , Vesículas Extracelulares/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Anciano , Proteínas del Sistema Complemento/metabolismo , Estudios Retrospectivos , Plaquetas/metabolismo , Plaquetas/patología , Tromboplastina/metabolismo , Adulto , Estudios ProspectivosRESUMEN
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
INTRODUCTION: Tafamidis is a molecular chaperone that stabilizes the transthyretin (TTR) homo-tetramer, preventing its dissociation and consequent deposition as amyloid fibrils in organ tissues. Tafamidis reduces mortality and the incidence of hospitalization for cardiovascular causes in patients with TTR amyloid (ATTR) cardiomyopathy. As ATTR cardiomyopathy is associated with a high risk of thromboembolic complications, we hypothesized that tafamidis may have a direct ancillary anti-thrombotic effect. METHODS: Primary human aortic endothelial cells (HAECs) were treated with tafamidis at clinically relevant concentrations and with plasma of patients, before and after the initiation of treatment with tafamidis. The expression of TF was induced by incubation with Tumor Necrosis Factor α (TNFα). Intracellular expression of tissue factor (TF) was measured by western blot. TF activity was measured by a colorimetric assay. Gene expressions of TF were measured by quantitative polymerase chain reaction. RESULTS: Treatment with tafamidis dose-dependently reduced the expression and activity of TNFα-induced TF. This effect was confirmed in cells treated with patients' plasma. Signal Transducer and Activator of Transcription 3 (STAT3) phosphorylation was significantly inhibited by tafamidis. Incubation of HAECs with tafamidis and the STAT3 activator colivelin partially rescued the expression of TF. CONCLUSIONS: Treatment with tafamidis lowers the thrombotic potential in human primary endothelial cells by reducing TF expression and activity. This previously unknown off-target effect may provide a novel mechanistic explanation for the lower number of thromboembolic complications in ATTR cardiomyopathy patients treated with tafamidis.
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Neuropatías Amiloides Familiares , Benzoxazoles , Cardiomiopatías , Células Endoteliales , Factor de Transcripción STAT3 , Tromboplastina , Factor de Necrosis Tumoral alfa , Humanos , Cardiomiopatías/metabolismo , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/prevención & control , Cardiomiopatías/patología , Cardiomiopatías/genética , Benzoxazoles/farmacología , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Células Cultivadas , Neuropatías Amiloides Familiares/tratamiento farmacológico , Neuropatías Amiloides Familiares/metabolismo , Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/patología , Tromboplastina/metabolismo , Tromboplastina/genética , Factor de Transcripción STAT3/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Fibrinolíticos/farmacología , Fosforilación , Relación Dosis-Respuesta a Droga , Prealbúmina/metabolismo , Prealbúmina/genética , Masculino , Transducción de Señal/efectos de los fármacos , Femenino , Aorta/metabolismo , Aorta/efectos de los fármacos , Aorta/patología , Anciano , Persona de Mediana EdadRESUMEN
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
Background: The intimal hyperplasia (IH) and vascular remodelling that follows endovascular injury, for instance after post-angioplasty re-stenosis, results in downstream ischaemia and progressive end organ damage. Interferon gamma (IFNγ) is known to play a critical role in this process. In mouse models we have previously shown that fibrocytes expressing tissue factor (TF) are recruited early to the site of injury. Through thrombin generation and protease activated receptor-1 (PAR-1) activation, fibrocytes secrete angiopoietin-2, stimulate neointimal cell proliferation, inhibit apoptosis and induce CXCL-12 production, all of which contribute to the progressive IH that then develops. In this study we investigated the relationship between TF, angiopoietin-2 and IFNγ. Methods and results: IH developing in carotid arteries of wild-type mice 4 weeks after endoluminal injury contained a significant proportion of IFNγ+ fibrocytes and macrophages, which we show, using a previously defined adoptive transfer model, were derived from circulating CD34+ cells. IH did not develop after injury in IFNγ-deficient mice, except after transplantation of WT bone marrow or adoptive transfer of WT CD34+ cells. In vitro, CD34+ cells isolated from post-injury mice did not express IFNγ, but this was induced when provided with FVIIa and FX, and enhanced when prothrombin was also provided: In both cases IFNγ secretion was TF-dependent and mediated mainly through protease activated PAR-1. IFNγ was predominantly expressed by fibrocytes. In vivo, all IFNγ+ neointimal cells in WT mice co-expressed angiopoietin-2, as did the small numbers of neointimal cells recruited in IFNγ-/- mice. Adoptively transferred WT CD34+ cells treated with either an anti-TIE-2 antibody, or with siRNA against angiopoetin-2 inhibited the expression of IFNγ and the development of IH. Conclusion: TF-dependent angiopoietin-2 production by newly recruited fibrocytes, and to a lesser extent macrophages, switches on IFNγ expression, and this is necessary for the IH to develop. These novel findings enhance our understanding of the pathophysiology of IH and expose potential targets for therapeutic intervention.
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Angiopoyetina 2 , Hiperplasia , Interferón gamma , Macrófagos , Ratones Noqueados , Neointima , Tromboplastina , Animales , Ratones , Interferón gamma/metabolismo , Angiopoyetina 2/metabolismo , Neointima/patología , Neointima/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Tromboplastina/metabolismo , Tromboplastina/genética , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Masculino , Fibroblastos/metabolismo , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/metabolismoRESUMEN
We studied the effectiveness of Xe/O2 mixture inhalation (30% Xe and 70% O2, 20 min for 5 days) in a model of experimental thromboplastin pneumonitis. Inhalation of the studied mixture decreased the intensity of the inflammatory process in the lung tissue assessed by the temperature response of animals, changed lung weight and lung weight coefficient. At acute stage of pneumonitis, an increase in xenon consumption was recorded due to its retention in the gas exchange zone and a natural decrease in oxygen consumption due to partial alveolar/capillary block. The formation of pneumonitis was accompanied by a pronounced procoagulant shift in the regulation system of the aggregate state of blood. The Xe/O2 inhalations ensured physiologically optimal levels of prothrombin and activated partial thromboplastin time against the background of a moderate decrease in fibrinogen level throughout the experiment. At the same time, the activity of the natural anticoagulant antithrombin III increased from day 5 to day 14.
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Oxígeno , Neumonía , Xenón , Animales , Neumonía/sangre , Neumonía/patología , Masculino , Oxígeno/metabolismo , Xenón/administración & dosificación , Xenón/farmacología , Hemostasis/efectos de los fármacos , Administración por Inhalación , Fibrinógeno/metabolismo , Tiempo de Tromboplastina Parcial , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Antitrombina III/metabolismo , Ratas , Tromboplastina/metabolismo , Protrombina/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Coagulación Sanguínea/efectos de los fármacosRESUMEN
Coagulopathy, microvascular alterations and concomitant organ dysfunctions are hallmarks of sepsis. Attempts to attenuate coagulation activation with an inhibitor of tissue factor (TF), i.e. tissue factor pathway inhibitor (TFPI), revealed no survival benefit in a heterogenous group of sepsis patients, but a potential survival benefit in patients with an international normalized ratio (INR) < 1.2. Since an increased TF/TFPI ratio determines the procoagulant activity specifically on microvascular endothelial cells in vitro, we investigated whether TF/TFPI ratio in blood is associated with INR alterations, organ dysfunctions, disseminated intravascular coagulation (DIC) and outcome in septic shock. Twenty-nine healthy controls (HC) and 89 patients with septic shock admitted to a tertiary ICU were analyzed. TF and TFPI in blood was analyzed and related to organ dysfunctions, DIC and mortality. Patients with septic shock had 1.6-fold higher levels of TF and 2.9-fold higher levels of TFPI than HC. TF/TFPI ratio was lower in septic shock compared to HC (0.003 (0.002-0.005) vs. 0.006 (0.005-0.008), p < 0.001). Non-survivors had higher TFPI levels compared to survivors (43038 (29354-54023) vs. 28041 (21675-46582) pg/ml, p = 0.011). High TFPI levels were associated with acute kidney injury, liver dysfunction, DIC and disease severity. There was a positive association between TF/TFPI ratio and troponin T (b = 0.531 (0.309-0.754), p < 0.001). A high TF/TFPI ratio is exclusively associated with myocardial injury but not with other organ dysfunctions. Systemic TFPI levels seem to reflect disease severity. These findings point towards a pathophysiologic role of TF/TFPI in sepsis-induced myocardial injury.
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Lipoproteínas , Choque Séptico , Tromboplastina , Humanos , Choque Séptico/sangre , Choque Séptico/metabolismo , Tromboplastina/metabolismo , Masculino , Femenino , Lipoproteínas/sangre , Lipoproteínas/metabolismo , Persona de Mediana Edad , Anciano , Insuficiencia Multiorgánica/sangre , Insuficiencia Multiorgánica/etiología , Coagulación Intravascular Diseminada/sangre , Estudios de Casos y Controles , Adulto , Biomarcadores/sangreRESUMEN
We recently reported the potential application of recombinant prothrombin activator ecarin (RAPClot™) in blood diagnostics. In a new study, we describe RAPClot™ as an additive to develop a novel blood collection prototype tube that produces the highest quality serum for accurate biochemical analyte determination. The drying process of the RAPClot™ tube generated minimal effect on the enzymatic activity of the prothrombin activator. According to the bioassays of thrombin activity and plasma clotting, γ-radiation (>25 kGy) resulted in a 30-40% loss of the enzymatic activity of the RAPClot™ tubes. However, a visual blood clotting assay revealed that the γ-radiation-sterilized RAPClot™ tubes showed a high capacity for clotting high-dose heparinized blood (8 U/mL) within 5 min. This was confirmed using Thrombelastography (TEG), indicating full clotting efficiency under anticoagulant conditions. The storage of the RAPClot™ tubes at room temperature (RT) for greater than 12 months resulted in the retention of efficient and effective clotting activity for heparinized blood in 342 s. Furthermore, the enzymatic activity of the RAPClot™ tubes sterilized with an electron-beam (EB) was significantly greater than that with γ-radiation. The EB-sterilized RAPClot™ tubes stored at RT for 251 days retained over 70% enzyme activity and clotted the heparinized blood in 340 s after 682 days. Preliminary clinical studies revealed in the two trials that 5 common analytes (K, Glu, lactate dehydrogenase (LD), Fe, and Phos) or 33 analytes determined in the second study in the γ-sterilized RAPClot™ tubes were similar to those in commercial tubes. In conclusion, the findings indicate that the novel RAPClot™ blood collection prototype tube has a significant advantage over current serum or lithium heparin plasma tubes for routine use in measuring biochemical analytes, confirming a promising application of RAPClot™ in clinical medicine.
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Proteínas Recombinantes , Humanos , Coagulación Sanguínea/efectos de los fármacos , Suero/química , Suero/metabolismo , Tromboplastina/metabolismo , Recolección de Muestras de Sangre/métodos , Tromboelastografía/métodos , Rayos gamma , Anticoagulantes/farmacología , Anticoagulantes/químicaRESUMEN
Blood coagulation is a network of biochemical reactions wherein dozens of proteins act collectively to initiate a rapid clotting response. Coagulation reactions are lipid-surface dependent, and this dependence is thought to help localize coagulation to the site of injury and enhance the association between reactants. Current mathematical models of coagulation either do not consider lipid as a variable or do not agree with experiments where lipid concentrations were varied. Since there is no analytic rate law that depends on lipid, only apparent rate constants can be derived from enzyme kinetic experiments. We developed a new mathematical framework for modeling enzymes reactions in the presence of lipid vesicles. Here the concentrations are such that only a fraction of the vesicles harbor bound enzymes and the rest remain empty. We call the lipid vesicles with and without enzyme TF:VIIa+ and TF:VIIa- lipid, respectively. Since substrate binds to both TF:VIIa+ and TF:VIIa- lipid, our model shows that excess empty lipid acts as a strong sink for substrate. We used our framework to derive an analytic rate equation and performed constrained optimization to estimate a single, global set of intrinsic rates for the enzyme-substrate pair. Results agree with experiments and reveal a critical lipid concentration where the conversion rate of the substrate is maximized, a phenomenon known as the template effect. Next, we included product inhibition of the enzyme and derived the corresponding rate equations, which enables kinetic studies of more complex reactions. Our combined experimental and mathematical study provides a general framework for uncovering the mechanisms by which lipid mediated reactions impact coagulation processes.
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Coagulación Sanguínea , Factor VIIa , Coagulación Sanguínea/fisiología , Factor VIIa/metabolismo , Modelos Biológicos , Humanos , Cinética , Lípidos , Tromboplastina/metabolismoRESUMEN
Blood coagulation mediated by pig tissue factor (TF), which is expressed in pig tissues, causes an instant blood-mediated inflammatory reaction during pig-to-human xenotransplantation. Previously, we generated a soluble pig tissue factor pathway inhibitor α fusion immunoglobulin (TFPI-Ig) which inhibits pig TF activity more efficiently than human TFPI-Ig in human plasma. In this study, we generated several pig TFPI-Ig mutants and tested the efficacy of these mutants in preventing pig-to-human xenogeneic blood coagulation. Structurally important amino acid residues of pig TFPI-Ig were changed into different residues by site-directed mutagenesis. Subsequently, a retroviral vector encoding each cDNA of several pig TFPI-Ig mutants was cloned and transduced into CHO-K1 cells. After establishing stable cell lines expressing each of the pig TFPI-Ig mutants, soluble proteins were produced and purified for evaluating their inhibitory effects on pig TF-mediated blood coagulation in human plasma. The replacement of K36 and K257 with R36 and H257, respectively, in pig TFPI-Ig more efficiently blocked pig TF activity in human plasma when compared with the wild-type pig TFPI-Ig. These results may provide additional information to understand the structure of pig TFPIα, and an improved pig TFPI-Ig variant that more efficiently blocks pig TF-mediated blood coagulation during pig-to-human xenotransplantation.
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Coagulación Sanguínea , Lipoproteínas , Trasplante Heterólogo , Animales , Humanos , Porcinos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Coagulación Sanguínea/genética , Células CHO , Cricetulus , Tromboplastina/genética , Tromboplastina/metabolismo , Mutagénesis Sitio-Dirigida , Análisis Mutacional de ADNRESUMEN
AIMS: To detect the expression of autophagy components, p38 MAPK (p38) and phosphorylated forkhead box transcription factor O-1 (pFoxO1) in pulmonary vascular endothelial cells of chronic thromboembolic pulmonary hypertension (CTEPH) rats and to investigate the possible mechanism through which tissue factor (TF) regulates autophagy. METHODS: Pulmonary artery endothelial cells (PAECs) were isolated from CTEPH (CTEPH group) and healthy rats (control group (ctrl group)) which were cocultured with TF at different time points including 12 h, 24 h, 48 h and doses including 0 nM,10 nM, 100 nM, 1µM, 10µM, 100µM and cocultured with TFPI at 48 h including 0 nM, 2.5 nM, 5 nM. The expression of forkhead box transcription factor O-1 (FoxO1), pFoxO1, p38, Beclin-1 and LC3B in PAECs was measured. Coimmunoprecipitation (co-IP) assays were used to detect the interaction between FoxO1 and LC3. RESULTS: The protein expression of p-FoxO1/FoxO1 was significantly lower in the CTEPH groups (cocultured with TF from 0 nM to 100 µM) than in the ctrl group at 12 h, 24 h, and 48 h (P < 0.05) and was significantly lower in the CTEPH groups (cocultured with TFPI from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of p38 in the CTEPH groups treated with 0 nM, 10 nM, 100 nM or 1 µM TF for 48 h significantly increased than ctrl groups (P < 0.05) and was significantly increased in the CTEPH groups (cocultured with TFPI concentration from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of Beclin1 at the same concentration (cocultured with TF from 0 nM to 100 µM) was significantly lower in the CTEPH groups than ctrl groups after 24 h and 48 h (P < 0.05) and was significantly decreased in the CTEPH groups (cocultured with TFPI concentration from 2.5 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). The protein expression of LC3-II/LC3-I at the same concentration (cocultured with TF 0 nM, 1 µM, 10 µM, and 100 µM) was significantly lower in the CTEPH than in the ctrl groups after 12 h (P < 0.05) and was significantly lower in the CTEPH groups (cocultured with TFPI concentration from 0 nM to 5 nM) than in the ctrl group at 48 h (P < 0.05). There were close interactions between FoxO1 and LC3 in the control and CTEPH groups at different doses and time points. CONCLUSION: The autophagic activity of PAECs from CTEPH rats was disrupted. TF, FoxO1 and p38 MAPK play key roles in the autophagic activity of PAECs. TF may regulate autophagic activity through the p38 MAPK-FoxO1 pathway.
Asunto(s)
Autofagia , Células Endoteliales , Hipertensión Pulmonar , Arteria Pulmonar , Ratas Sprague-Dawley , Tromboplastina , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Autofagia/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Ratas , Masculino , Células Endoteliales/metabolismo , Células Cultivadas , Tromboplastina/metabolismo , Tromboplastina/biosíntesis , Hipertensión Pulmonar/metabolismo , Embolia Pulmonar/metabolismo , Embolia Pulmonar/patología , Enfermedad Crónica , Transducción de Señal/fisiología , Proteína Forkhead Box O1RESUMEN
ABSTRACT: Interplay between platelets, coagulation factors, endothelial cells (ECs), and fibrinolytic factors is necessary for effective hemostatic plug formation. This study describes a 4-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components in mice with high spatiotemporal resolution. Fibrin accumulation after laser-induced vascular injury was observed at the platelet plug-EC interface, controlled by the antagonistic balance between fibrin generation and breakdown. We observed less fibrin accumulation in mice expressing low levels of tissue factor or F12-/-mice compared with controls, whereas increased fibrin accumulation, including on the vasculature adjacent to the platelet plug, was observed in plasminogen-deficient mice or wild-type mice treated with tranexamic acid. Phosphatidylserine (PS), a membrane lipid critical for the assembly of coagulation factors, was first detected at the platelet plug-EC interface, followed by exposure across the endothelium. Impaired PS exposure resulted in a significant reduction in fibrin accumulation in cyclophilin D-/-mice. Adoptive transfer studies demonstrated a key role for PS exposure on platelets, and to a lesser degree on ECs, in fibrin accumulation during hemostatic plug formation. Together, these studies suggest that (1) platelets are the functionally dominant procoagulant cellular surface, and (2) plasmin is critical for limiting fibrin accumulation at the site of a forming hemostatic plug.
Asunto(s)
Plaquetas , Fibrina , Hemostasis , Animales , Plaquetas/metabolismo , Ratones , Fibrina/metabolismo , Microscopía Intravital/métodos , Fosfatidilserinas/metabolismo , Células Endoteliales/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Ratones Noqueados , Coagulación Sanguínea , Tromboplastina/metabolismo , Tromboplastina/genéticaRESUMEN
Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6-40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31+) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7-40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients.
Asunto(s)
Quemaduras , Citrulinación , Histonas , Neutrófilos , Trombosis , Humanos , Quemaduras/inmunología , Quemaduras/metabolismo , Quemaduras/complicaciones , Histonas/metabolismo , Histonas/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Masculino , Femenino , Adulto , Persona de Mediana Edad , Trombosis/metabolismo , Trombosis/inmunología , Trombosis/patología , Tromboplastina/metabolismo , Anciano , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factor XII/metabolismo , Microvasos/patología , Microvasos/inmunología , Microvasos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Piel/patología , Piel/inmunología , Piel/metabolismo , Piel/irrigación sanguínea , Linfocitos/inmunología , Linfocitos/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Adulto JovenRESUMEN
Atherosclerosis, a chronic inflammatory disease, is the most relevant cause of carotid artery stenosis. Vascular endothelial cells (ECs) play a significant role in the development of atherosclerosis. In this chronic inflammatory environment, we aimed to investigate whether PCSK9 could mitigate atherosclerosis progression by reducing tissue factor expression in ECs via in vivo and in vitro assays. In vivo, we investigated the effect of PCSK9 inhibition on preventing atherosclerotic lesion formation in ApoE-/- mice fed a western diet. The results showed that inhibiting PCSK9 could significantly downregulate the protein expression of tissue factor (TF) in ECs to reduce the area of atherosclerotic plaques. In vitro, we incubated human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS). We found that LPS-induced TF elevation was suppressed by a PCSK9 inhibitor at both the mRNA and protein levels and that the TLR4/NF-κB pathway was also suppressed by a PCSK9 inhibitor. With respect to plasma samples from patients with carotid artery stenosis, we also demonstrated that the expression of TF was positively correlated with that of PCSK9. Thus, in addition to regulating lipid metabolism, the regulation of endothelial cell TF expression through the TLR4/NF-κB pathway may be a potential mechanism of PCSK9 in promoting atherosclerotic carotid stenosis.