RESUMEN
BACKGROUND: QiShen YiQi (QSYQ) dropping pill, a component-based Chinese medicine consisting of benefiting Qi (YQ) and activating blood (HX) components, has been reported to exert a beneficial effect on cerebral ischemia-induced stroke. However, its efficacy and pharmacological mechanism on acute thromboembolic stroke is not clear. PURPOSE: This study is to explore the preventative effect and pharmacological mechanism of QSYQ and its YQ/HX components on the formation of platelet-leukocyte aggregation (PLA) in acute thromboembolic stroke. STUDY DESIGN AND METHODS: In vivo thromboembolic stroke model and FeCl3-induced carotid arterial occlusion models were used. Immunohistochemistry, Western blot, RT-qPCR, and flow cytometry experiments were performed to reveal the pharmacological mechanisms of QSYQ and its YQ/HX components. RESULTS: In thromboembolic stroke rats, QSYQ significantly attenuated infarct area, improved neurological recovery, reduced PLA formation, and inhibited P-selection (CD62P)/ P-selectin glycoprotein ligand-1 (PSGL-1) expressions. The YQ component preferentially down-regulated PSGL-1 expression in leukocyte, while the HX component preferentially down-regulated CD62P expression in platelet. In carotid arterial thrombosis mice, QSYQ and its YQ/HX components inhibited thrombus formation, prolonged vessel occlusion time, reduced circulating leukocytes and P-selectin expression. PLA formation and platelet/leukocyte adhesion to endothelial cell were also inhibited by QSYQ and its YQ/HX components in vitro. CONCLUSION: QSYQ and YQ/HX components attenuated thromboembolic stroke and carotid thrombosis by decreasing PLA formation via inhibiting CD62P/PSGL-1 expressions. This study shed a new light on the prevention of thromboembolic stroke.
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Trombosis de las Arterias Carótidas , Accidente Cerebrovascular , Trombosis , Ratas , Animales , Ratones , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Trombosis de las Arterias Carótidas/metabolismo , Selectina-P/metabolismo , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismo , Leucocitos/metabolismo , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , PoliésteresRESUMEN
ABSTRACT: Desialylation, governed by sialidases or neuraminidases, is strongly implicated in a wide range of human disorders, and accumulative data show that inhibition of neuraminidases, such as neuraminidases 1 sialidase, may be useful for managing atherosclerosis. Several studies have reported promising effects of oseltamivir phosphate, a widely used anti-influenza sialidase inhibitor, on human cancer cells, inflammation, and insulin resistance. In this study, we evaluated the effects of oseltamivir phosphate on atherosclerosis and thrombosis and potential liver toxicity in LDLR-/- mice fed with high-fat diet. Our results showed that oseltamivir phosphate significantly decreased plasma levels of LDL cholesterol and elastin fragmentation in aorta. However, no effect was observed on both atherosclerotic plaque size in aortic roots and chemically induced thrombosis in carotid arteries. Importantly, oseltamivir phosphate administration had adverse effects on the liver of mice and significantly increased messenger RNA expression levels of F4/80, interleukin-1ß, transforming growth factor-ß1, matrix metalloproteinase-12, and collagen. Taken together, our findings suggest that oseltamivir phosphate has limited benefits on atherosclerosis and carotid thrombosis and may lead to adverse side effects on the liver with increased inflammation and fibrosis.
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Antivirales/toxicidad , Enfermedades de la Aorta/tratamiento farmacológico , Aterosclerosis/tratamiento farmacológico , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hígado/efectos de los fármacos , Oseltamivir/toxicidad , Receptores de LDL/deficiencia , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Trombosis de las Arterias Carótidas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Femenino , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones Noqueados , Placa Aterosclerótica , Receptores de LDL/genética , Medición de RiesgoRESUMEN
BACKGROUND: The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis. METHODS: The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays. FINDINGS: The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively. INTERPRETATION: NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions. FUNDING: This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).
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Coagulación Sanguínea , Plaquetas/metabolismo , Trombosis de las Arterias Carótidas/metabolismo , Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Accidente Cerebrovascular/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Trombosis de las Arterias Carótidas/sangre , Arteria Carótida Interna/patología , Micropartículas Derivadas de Células/metabolismo , Femenino , Fibrina/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Fosfatidilserinas/metabolismo , Activación Plaquetaria , Accidente Cerebrovascular/sangre , Sulfonamidas/farmacología , Trombina/metabolismoRESUMEN
In spite of current treatment strategies, myocardial infarction and stroke are still major causes of death worldwide. These events are triggered by damage of an atherosclerotic plaque, resulting in occlusive thrombus formation. Mouse studies have significantly contributed to our understanding of the mechanisms of atherogenesis and of thrombosis following plaque injury, but the extent to which the mouse serves as an accurate model of human disease is open to discussion. In this review, we provide a detailed overview and comparison of the described mouse models for atherothrombosis including their (dis)advantages. Herein guidance is provided on how to select a suitable atherothrombosis model for research questions primarily relevant to the field of thrombosis.
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Trombosis de las Arterias Carótidas/etiología , Modelos Animales de Enfermedad , Ratones , Placa Aterosclerótica , Animales , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Coagulación Sanguínea , Plaquetas/metabolismo , Plaquetas/fisiología , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/metabolismo , Cloruros/toxicidad , Dieta Alta en Grasa , Compuestos Férricos/toxicidad , Humanos , Ligadura , Ratones Noqueados para ApoE , Placa Aterosclerótica/inducido químicamente , Placa Aterosclerótica/patología , Ondas UltrasónicasRESUMEN
The ferric chloride models of arterial thrombosis are useful tools with which to investigate the cellular and molecular mechanisms that contribute to arterial thrombosis. Recent insights have, however, revealed the complex and multifaceted mechanism by which ferric chloride induces thrombus formation. Here, we discuss the strengths and weaknesses of the ferric chloride models of arterial thrombosis. Particular focus is given to the phenotypes of different knockout mice in the ferric chloride models and how these compare to other models with independent modes of initiation. Further, we discuss the relevance of the ferric chloride models to the human pathology of atherothrombotic disease.
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Trombosis de las Arterias Carótidas/metabolismo , Cloruros/toxicidad , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Compuestos Férricos/toxicidad , Animales , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Plaquetas/patología , Trombosis de las Arterias Carótidas/inducido químicamente , Cloruros/metabolismo , Compuestos Férricos/metabolismo , Humanos , Ratones , Ratones NoqueadosRESUMEN
Platelet activation requires fully functional mitochondria, which provide a vital energy source and control the life span of platelets. Previous reports have shown that both general autophagy and selective mitophagy are critical for platelet function. However, the underlying mechanisms remain incompletely understood. Here, we show that Nix, a previously characterized mitophagy receptor that plays a role in red blood cell maturation, also mediates mitophagy in platelets. Genetic ablation of Nix impairs mitochondrial quality, platelet activation, and FeCl3-induced carotid arterial thrombosis without affecting the expression of platelet glycoproteins (GPs) such as GPIb, GPVI, and αIIbß3 Metabolic analysis revealed decreased mitochondrial membrane potential, enhanced mitochondrial reactive oxygen species level, diminished oxygen consumption rate, and compromised adenosine triphosphate production in Nix -/- platelets. Transplantation of wild-type (WT) bone marrow cells or transfusion of WT platelets into Nix-deficient mice rescued defects in platelet function and thrombosis, suggesting a platelet-autonomous role (acting on platelets, but not other cells) of Nix in platelet activation. Interestingly, loss of Nix increases the life span of platelets in vivo, likely through preventing autophagic degradation of the mitochondrial protein Bcl-xL. Collectively, our findings reveal a novel mechanistic link between Nix-mediated mitophagy, platelet life span, and platelet physiopathology. Our work suggests that targeting platelet mitophagy Nix might provide new antithrombotic strategies.
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Plaquetas/metabolismo , Proteínas de la Membrana/metabolismo , Mitofagia , Activación Plaquetaria , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Biomarcadores , Tiempo de Sangría , Plaquetas/ultraestructura , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/metabolismo , Trombosis de las Arterias Carótidas/patología , Supervivencia Celular/genética , Humanos , Inmunofenotipificación , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Fenotipo , Activación Plaquetaria/genética , Pruebas de Función Plaquetaria , Proteínas Proto-Oncogénicas/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Dysfunction of vascular endothelial cells causes the risk of thrombosis. Aim of this study is to evaluate the antithrombotic effect of Okinawa mozuku (brown seaweed, Cladosiphon okamuranus) extract by using cultured vascular endothelial cells and rat carotid arterial thrombosis model induced by ferric chloride (FeCl3). The cell line (TKM-33) established from human umbilical vein endothelial cells were cultured with or without Okinawa mozuku extract. After incubation for 24 h, the conditioned medium was collected to evaluate urokinase-type plasminogen activator (u-PA) activity. Next, rats were fed with water or water containing 5% of Okinawa mozuku extract for 8 wk. After 8 wk of treatments, the rats were provided for the carotid arterial thrombosis model, and fibrinolytic factor and coagulation factor in blood were measured. Okinawa mozuku extract significantly augmented u-PA activity in the conditioned medium. The decrease of carotid artery blood flow induced by 40% FeCl3 injury in rats fed with Okinawa mozuku extract was less than that in control rats. Thus, oral administration of Okinawa mozuku extract prevented thrombus formation in this model. Oral administration of Okinawa mozuku extract significantly increased u-PA activity in euglobulin fraction, compared with control group. On the other hand, platelet aggregation activity, activated partial thromboplastin time, and active PAI-1 level in plasma exhibited no significant differences between control and Okinawa mozuku groups. These results indicate that oral administration of Okinawa mozuku enhances fibrinolytic activity in plasma and prevents thrombus formation which is induced by injury of vascular endothelial cells.
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Trombosis de las Arterias Carótidas/metabolismo , Fibrinolíticos/farmacología , Phaeophyceae , Extractos Vegetales/farmacología , Administración Oral , Animales , Modelos Animales de Enfermedad , Fibrinolíticos/administración & dosificación , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Extractos Vegetales/administración & dosificación , Ratas , Ratas Sprague-Dawley , Activador de Plasminógeno de Tipo Uroquinasa/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Lumbrokinase (LK) has strong fibrinolytic and thrombolytic activities, but it has a short half-life, can be easily inactivated, and may cause hemorrhage as a side effect. This study develops a potential thrombolytic therapy by fabricating N,N,N-Trimethyl Chitosan (TMC) nanoparticles modified with the cyclic Arg-Gly-Asp-Phe-Lys peptide (c-RGD) and loaded with LK (i.e. c-RGD-LK-NPs). The binding of c-RGD to platelet membrane GPIIb/IIIa receptors is expected to enable targeted delivery of the c-RGD-conjugated TMC to the thrombus. The synthesized c-RGD-LK-NPs had a mean particle size of 232.0 nm, zeta potential of 19.8 mV, entrapment efficiency of 52.7% ± 2.5%, and loading efficiency of 17.4% ± 0.65%. Transmission electron microscopy showed that they were generally spherical. The c-RGD-LK-NPs gave a cumulative in vitro LK release of 80.6% over 8 h, and the activity of LK was close to 80%, indicating that the nanoparticles protected the activity of LK. In vitro blood clot lysis assays were carried out and in vivo thrombolysis effect was tested in Sprague-Dawley rats carotid artery thrombus model. In all cases, the c-RGD-LK-NPs showed superior performance compared with the free LK and the unmodified TMC nanoparticles loaded with LK. The c-RGD-LK-NPs reagent is expected to be potentially useful in treating thromboembolic diseases.
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Quitosano/administración & dosificación , Portadores de Fármacos/administración & dosificación , Endopeptidasas/administración & dosificación , Nanopartículas/administración & dosificación , Péptidos Cíclicos/administración & dosificación , Animales , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Trombosis de las Arterias Carótidas/metabolismo , Quitosano/síntesis química , Quitosano/metabolismo , Portadores de Fármacos/síntesis química , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Endopeptidasas/síntesis química , Endopeptidasas/metabolismo , Fibrinolíticos/administración & dosificación , Fibrinolíticos/síntesis química , Fibrinolíticos/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Salvia deserta Schang (SDS) belongs to the same family as Salvia miltiorrhiza bunge, one of the antithrombotic Chinese herbal medicines. In our study, EtOAc root extracts were analyzed for their effects on adenosine diphosphate (ADP)-induced platelet aggregation in rabbits and FeCl3-induced rat common carotid artery thrombosis as well as on rat blood plasma concentrations of thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1α), antithrombin-III (AT-III), protein C (PC), plasminogen (PLG), plasminogen activator inhibitor (PAI-1), von Willebrand factor (vWF) and tissue-type plasminogen activator (t-PA). EtOAc extracts from SDS roots had significant inhibitory effects on ADP-induced maximum platelet aggregation rate (10.2 ± 2.6 vs control 35.7 ± 5.2; P < 0.05), reduced the FeCl3-induced rat common carotid artery thrombus weight and thrombus area ratio (P < 0.05), significantly decreased plasma TXB2, vWF and PAI-1 levels and increased 6-keto-PGF1α and t-PA levels in a dose dependent manner (all P < 0.05). Thus, the ratio of TXB2/6-keto-PGF1α was significantly decreased (P < 0.05), while the ratio of t-PA/PAI-1 was significantly increased (P < 0.05). In addition, enhanced AT-III and PC activities indicated coagulation inactivation effects of EtOAc SDS root extracts. EtOAc extraction from SDS showed antithrombotic effects, which are likely due to platelet adhesion and aggregation inhibition as well as anticoagulant activities.
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Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Fibrinolíticos/farmacología , Raíces de Plantas/química , Salvia miltiorrhiza/química , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Canfanos , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Trombosis de las Arterias Carótidas/metabolismo , Femenino , Masculino , Panax notoginseng , Agregación Plaquetaria/efectos de los fármacos , Conejos , Ratas , Ratas Sprague-Dawley , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Tromboxano B2/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
ADAMTS18 is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) that are known for their crucial role in development, angiogenesis, inflammation and coagulation. It was previously reported that ADAMTS18 cleaved by thrombin induced platelet fragmentation, through which thrombus were dissolved. However, it remains unclear whether this represents a dominant physiologic mechanism controlling thrombus growth in vivo. Here, we used an established Adamts18 knockout (KO) mouse model to determine its function in thrombus formation. ADAMTS18 deficiency accelerated FeCl3-induced carotid artery thrombosis and aggravated postischemic cerebral infarction in mice. However, this accelerated thrombus phenotype in Adamts18 KO mice was not due to the lack of ADAMTS18-mediated-platelet fragmentation. Moreover, Adamts18 deficiency exerted little effects on mouse platelet functions. The underlying molecular mechanisms could be attributed in part to the abnormal vascular remodeling, including deficiency of carotid body (glomus) and aberrant carotid basal lamina. These results indicate a novel function of ADAMTS18 in vascular remodeling and associated thrombus formation.
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Proteínas ADAMTS/metabolismo , Trombosis de las Arterias Carótidas/metabolismo , Infarto Cerebral/metabolismo , Trombosis/metabolismo , Trombosis/patología , Proteínas ADAMTS/genética , Animales , Trombosis de las Arterias Carótidas/patología , Infarto Cerebral/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
INTRODUCTION: Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid ß-protein precursor (AßPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AßPP are abundant in brain. Regions of AßPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AßPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. METHODS: To determine the extent of KPI function of AßPP in regulating cerebral thrombosis we generated a recombinant reactive center KPIR13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AßPP knock out and AßPP/KPIR13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. RESULTS: Recombinant mutant KPIR13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AßPP/KPIR13I mutant mice were similarly deficient as AßPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. CONCLUSIONS: We demonstrate that the anti-thrombotic function of AßPP primarily resides in the KPI activity of the protein.
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Precursor de Proteína beta-Amiloide/metabolismo , Coagulación Sanguínea , Trombosis Intracraneal/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animales , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/genética , Trombosis de las Arterias Carótidas/metabolismo , Hemorragia Cerebral/sangre , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Técnicas de Sustitución del Gen , Humanos , Trombosis Intracraneal/sangre , Trombosis Intracraneal/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A thrombus (blood clot) is formed in injured vessels to maintain the integrity of vasculature. However, obstruction of blood vessels by thrombosis slows blood flow, leading to death of tissues fed by the artery and is the main culprit of various life-threatening cardiovascular diseases. Herein, we report a rationally designed nanomedicine that could specifically image obstructed vessels and inhibit thrombus formation. On the basis of the physicochemical and biological characteristics of thrombi such as an abundance of fibrin and an elevated level of hydrogen peroxide (H2O2), we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN, as a theranostic system for obstructive thrombosis. FTIAN inhibited the generation of H2O2 and suppressed the expression of tumor necrosis factor-alpha (TNF-α) and soluble CD40 ligand (sCD40L) in activated platelets, demonstrating its intrinsic antioxidant, anti-inflammatory, and antiplatelet activity. In a mouse model of ferric chloride (FeCl3)-induced carotid thrombosis, FTIAN specifically targeted the obstructive thrombus and significantly enhanced the fluorescence/photoacoustic signal. When loaded with the antiplatelet drug tirofiban, FTIAN remarkably suppressed thrombus formation. Given its thrombus-specific imaging along with excellent therapeutic activities, FTIAN offers tremendous translational potential as a nanotheranostic agent for obstructive thrombosis.
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Trombosis de las Arterias Carótidas/diagnóstico por imagen , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Fibrina/metabolismo , Fibrinolíticos/uso terapéutico , Colorantes Fluorescentes/química , Peróxido de Hidrógeno/metabolismo , Nanopartículas/química , Animales , Ácidos Borónicos/química , Ligando de CD40/metabolismo , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/metabolismo , Supervivencia Celular/efectos de los fármacos , Cloruros , Portadores de Fármacos , Liberación de Fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Compuestos Férricos , Fibrinolíticos/química , Humanos , Lipopéptidos/química , Ratones , Imagen Óptica , Polímeros , Células RAW 264.7 , Nanomedicina Teranóstica , Trombosis/diagnóstico por imagen , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Tirofibán/química , Tirofibán/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Arterial thrombosis and its related diseases are major healthcare problems worldwide. Blebbistatin is an inhibitor of myosin II, which plays an important role in thrombosis. The aim of our study is to explore the effect and potential mechanism of blebbistatin on arterial thrombosis. A ferric chloride (FeCl3) solution at a concentration of 5% was used to induce carotid artery thrombosis in mice. Immunohistochemistry and immunofluorescence staining were used to detect the expression or activation of non-muscle myosin heavy chain IIA (NMMHC IIA), tissue factor (TF), GSK3ß and NF-κB. Blebbistatin (1 mg/kg, i.p.) significantly reduced carotid artery thrombosis induced by FeCl3 solution in mice, inhibited NMMHC IIA expression and impeded TF expression via the GSK3ß-NF-κB signalling pathway in mouse arterial vascular tissues. The present study demonstrates that blebbistatin may impede TF expression partly via the Akt/GSK3ß-NF-κB signalling pathways in the endothelium in a FeCl3 model, shedding new insights into the pathogenesis of arterial thrombosis and providing new clues for the development of antithrombotic drugs.
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Trombosis de las Arterias Carótidas/metabolismo , Cloruros/farmacología , Compuestos Férricos/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , FN-kappa B/metabolismo , Animales , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Masculino , Ratones , FN-kappa B/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tromboplastina/metabolismoRESUMEN
La enfermedad arterial periférica es una manifestación común de la aterosclerosis sistémica que se asocia a un mayor riesgo cardiovascular. Dado que los pacientes con enfermedad arterial periférica pueden presentar asimetría o ausencia de pulsos, aún estando asintomáticos, ante un evento coronario agudo en estos pacientes debemos realizar el diagnóstico diferencial con la disección de aorta
Peripheral arterial disease is a common manifestation of systemic atherosclerosis that is associated with increased cardiovascular risk. When presented in the context of an acute coronary syndrome a differential diagnosis with aorta dissection should be made, because peripheral arterial disease may be asymptomatic despite the absence or asymmetry of femoral pulses
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Humanos , Masculino , Enfermedad Arterial Periférica/patología , Síndrome Coronario Agudo/genética , Enfermedades Vasculares Periféricas/patología , Aorta/anomalías , Trombosis de las Arterias Carótidas/sangre , Aterosclerosis/metabolismo , Enfermedad Arterial Periférica/metabolismo , Síndrome Coronario Agudo/fisiopatología , Enfermedades Vasculares Periféricas/complicaciones , Aorta/lesiones , Trombosis de las Arterias Carótidas/metabolismo , Aterosclerosis/patologíaRESUMEN
No disponible
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Femenino , Humanos , Cefaleas Primarias/metabolismo , Cefaleas Primarias/patología , Neuroimagen/instrumentación , Neuroimagen/métodos , Trombosis de las Arterias Carótidas/complicaciones , Trombosis de las Arterias Carótidas/metabolismo , Vasculitis/inducido químicamente , Vasculitis/metabolismo , Cefaleas Primarias/complicaciones , Cefaleas Primarias/diagnóstico , Neuroimagen/clasificación , Neuroimagen/enfermería , Trombosis de las Arterias Carótidas/enfermería , Trombosis de las Arterias Carótidas/patología , Vasculitis/complicaciones , Vasculitis/diagnósticoRESUMEN
AIMS: Thymic stromal lymphopoietin (TSLP) plays an important role in inflammatory diseases and is over-expressed in human atherosclerotic artery specimens. The present study investigated the role of TSLP in platelet activation and thrombosis models in vitro and in vivo, as well as the underlying mechanism and signaling pathway. METHODS AND RESULTS: Western blotting and flow cytometry demonstrated that the TSLP receptor was expressed on murine platelets. According to flow cytometry, platelet stimulation with TSLP induced platelet degranulation and integrin αIIbß3 activation. A TSLPR deficiency caused defective platelet aggregation, defective platelet secretion and markedly blunted thrombus growth in perfusion chambers at both low and high shear rates. TSLPR KO mice exhibited defective carotid artery thrombus formation after exposure to FeCl3. TSLP increased Akt phosphorylation, an effect that was abrogated by the PI3K inhibitors wortmannin and LY294002. The PI3K inhibitors further diminished TSLP-induced platelet activation. TSLP-mediated platelet degranulation, integrin αIIbß3 activation and Akt phosphorylation were blunted in platelets that lacked the TSLP receptor. CONCLUSION: This study demonstrated that the functional TSLPR was surface-expressed on murine platelets. The inflammatory cytokine TSLP triggered platelet activation and thrombus formation via TSLP-dependent PI3K/Akt signaling, which suggests an important role for TSLP in linking vascular inflammation and thrombo-occlusive diseases.
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Plaquetas/metabolismo , Citocinas/farmacología , Inmunoglobulinas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Receptores de Citocinas/metabolismo , Transducción de Señal/efectos de los fármacos , Androstadienos/farmacología , Animales , Plaquetas/efectos de los fármacos , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/metabolismo , Trombosis de las Arterias Carótidas/patología , Cloruros/toxicidad , Cromonas/farmacología , Modelos Animales de Enfermedad , Compuestos Férricos/toxicidad , Humanos , Inmunoglobulinas/deficiencia , Inmunoglobulinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Selectina-P/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Citocinas/deficiencia , Receptores de Citocinas/genética , Wortmanina , Linfopoyetina del Estroma TímicoRESUMEN
The precise mechanism for reduced thrombosis in prekallikrein null mice (Klkb1(-/-)) is unknown. Klkb1(-/-) mice have delayed carotid artery occlusion times on the rose bengal and ferric chloride thrombosis models. Klkb1(-/-) plasmas have long-activated partial thromboplastin times and defective contact activation-induced thrombin generation that partially corrects upon prolonged incubation. However, in contact activation-induced pulmonary thromboembolism by collagen/epinephrine or long-chain polyphosphate, Klkb1(-/-) mice, unlike F12(-/-) mice, do not have survival advantage. Klkb1(-/-) mice have reduced plasma BK levels and renal B2R mRNA. They also have increased expression of the renal receptor Mas and plasma prostacyclin. Increased prostacyclin is associated with elevated aortic vasculoprotective transcription factors Sirt1 and KLF4. Treatment of Klkb1(-/-) mice with the Mas antagonist A-779, COX-2 inhibitor nimesulide, or Sirt1 inhibitor splitomicin lowers plasma prostacyclin and normalizes arterial thrombosis times. Treatment of normal mice with the Mas agonist AVE0991 reduces thrombosis. Klkb1(-/-) mice have reduced aortic tissue factor (TF) mRNA, antigen, and activity. In sum, Klkb1(-/-) mice have a novel mechanism for thrombosis protection in addition to reduced contact activation. This pathway arises when bradykinin delivery to vasculature is compromised and mediated by increased receptor Mas, prostacyclin, Sirt1, and KLF4, leading to reduced vascular TF.
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Trombosis de las Arterias Carótidas , Epoprostenol , Factores de Transcripción de Tipo Kruppel , Precalicreína , Proteínas Proto-Oncogénicas , Receptores Acoplados a Proteínas G , Tromboplastina , Angiotensina II/análogos & derivados , Angiotensina II/farmacología , Animales , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/genética , Trombosis de las Arterias Carótidas/metabolismo , Trombosis de las Arterias Carótidas/patología , Epoprostenol/biosíntesis , Epoprostenol/genética , Imidazoles/farmacología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Noqueados , Naftalenos/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Tiempo de Tromboplastina Parcial , Fragmentos de Péptidos/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Pironas/farmacología , ARN Mensajero , Receptor de Bradiquinina B2/biosíntesis , Receptor de Bradiquinina B2/genética , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/biosíntesis , Sirtuina 1/genética , Sulfonamidas/farmacología , Sinaptotagminas/biosíntesis , Sinaptotagminas/genética , Tromboplastina/antagonistas & inhibidores , Tromboplastina/biosíntesis , Tromboplastina/genéticaRESUMEN
BACKGROUND: Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models. METHODS AND RESULTS: The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30-90 minutes, >15-fold at 240-285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic-computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator-treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator-treated animals, but enduring high thrombus activity in control rats. CONCLUSIONS: We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation.
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Trombosis de las Arterias Carótidas/diagnóstico , Fibrina , Trombosis Intracraneal/diagnóstico , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Animales , Trombosis de las Arterias Carótidas/metabolismo , Proteínas Portadoras/farmacocinética , Modelos Animales de Enfermedad , Fibrina/farmacocinética , Trombosis Intracraneal/metabolismo , Masculino , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Distribución Tisular , Tomografía Computarizada por Rayos XRESUMEN
There is considerable interest in defining new agents or targets for antithrombotic purposes. The 5-HT2A receptor is a G-protein coupled receptor (GPCR) expressed on many cell types, and a known therapeutic target for many disease states. This serotonin receptor is also known to regulate platelet function. Thus, in our FDA-approved drug repurposing efforts, we investigated the antiplatelet activity of cyproheptadine and pizotifen, two antidepressant 5-HT2A Receptor antagonists. Our results revealed that cyproheptadine and pizotifen reversed serotonin-enhanced ADP-induced platelet aggregation in vitro and ex vivo. And the inhibitory effects of these two agents were found to be similar to that of EMD 281014, a 5-HT2A Receptor antagonist under development. In separate experiments, our studies revealed that these 5-HT2A receptor antagonists have the capacity to reduce serotonin-enhanced ADP-induced elevation in intracellular calcium levels and tyrosine phosphorylation. Using flow cytometry, we also observed that cyproheptadine, pizotifen, and EMD 281014 inhibited serotonin-enhanced ADP-induced phosphatidylserine (PS) exposure, P-selectin expression, and glycoprotein IIb-IIIa activation. Furthermore, using a carotid artery thrombosis model, these agents prolonged the time for thrombotic occlusion in mice in vivo. Finally, the tail-bleeding time was investigated to assess the effect of cyproheptadine and pizotifen on hemostasis. Our findings indicated prolonged bleeding time in both cyproheptadine- and pizotifen-treated mice. Notably, the increases in occlusion and bleeding times associated with these two agents were comparable to that of EMD 281014, and to clopidogrel, a commonly used antiplatelet drug, again, in a fashion comparable to clopidogrel and EMD 281014. Collectively, our data indicate that the antidepressant 5-HT2A antagonists, cyproheptadine and pizotifen do exert antiplatelet and thromboprotective effects, but similar to clopidogrel and EMD 281014, their use may interfere with normal hemostasis.
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Antidepresivos/farmacología , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Ciproheptadina/farmacología , Pizotilina/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/fisiología , Serotonina/farmacología , Adenosina Difosfato/farmacología , Animales , Western Blotting , Calcio/metabolismo , Trombosis de las Arterias Carótidas/metabolismo , Trombosis de las Arterias Carótidas/patología , Clopidogrel , Citometría de Flujo , Hemorragia/tratamiento farmacológico , Hemorragia/metabolismo , Hemorragia/patología , Humanos , Inmunoprecipitación , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Piperazinas/farmacología , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria , Receptor de Serotonina 5-HT2A/química , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision-making on possible therapeutic options in atherosclerosis, cancer and thrombus-related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel (111)In-labeled fibrin-binding peptide (FibPep) to visualize thrombi in mice noninvasively using single-photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin-binding properties were assessed in vitro. FibPep showed enhanced binding compared with NCFibPep to both fibrin and blood clots. FibPep bound to fibrin with a dissociation constant (K(d)) of 0.8 µ m, whereas NCFibPep displayed at least a 100-fold lower affinity towards fibrin. A FeCl3 -injury carotid artery thrombosis mouse model was used to evaluate the peptides in vivo. FibPep and NCFibPep displayed rapid blood clearance and were eliminated via the renal pathway. In vivo SPECT imaging using FibPep allowed clear visualization of thrombi. Ex vivo biodistribution showed significantly increased uptake of FibPep in the thrombus-containing carotid in comparison to the noninjured carotid (5.7 ± 0.7 and 0.6 ± 0.4% injected dose per gram (%ID g(-1)), respectively; p < 0.01; n = 4), whereas nonspecific NCFibPep did not (0.4 ± 0.2 and 0.3 ± 0.0%ID g(-1), respectively; n = 4). In conclusion, FibPep displayed high affinity towards fibrin in vitro and rapid blood clearance in vivo, and allowed sensitive detection of thrombi using SPECT imaging. Therefore, this particular imaging approach may provide a new tool to diagnose and monitor diseases such as atherosclerosis and cancer.