RESUMEN
ABSTRACT: Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.
Asunto(s)
Anticoagulantes , Coagulación Sanguínea , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Factor XIIa , Factor XIa , Animales , Factor XIa/antagonistas & inhibidores , Factor XIa/metabolismo , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Masculino , Factor XIIa/antagonistas & inhibidores , Factor XIIa/metabolismo , Trombosis de las Arterias Carótidas/prevención & control , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Ratones , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Hemorragia/inducido químicamente , Ratones Endogámicos C57BL , Tiempo de Tromboplastina ParcialRESUMEN
BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.
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Indicán/fisiología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/sangre , Quinurenina/fisiología , Terapia Molecular Dirigida , Complicaciones Posoperatorias/enzimología , Insuficiencia Renal Crónica/enzimología , Trombosis/enzimología , Procedimientos Quirúrgicos Vasculares/efectos adversos , Animales , Aorta , Traumatismos de las Arterias Carótidas/complicaciones , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/prevención & control , Medios de Cultivo/farmacología , Inducción Enzimática/efectos de los fármacos , Retroalimentación Fisiológica , Femenino , Células HEK293 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/deficiencia , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Quinurenina/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/prevención & control , Insuficiencia Renal Crónica/tratamiento farmacológico , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/etiología , Trombosis/prevención & control , Triptófano/metabolismo , Uremia/sangreRESUMEN
OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.
Asunto(s)
Plaquetas/efectos de los fármacos , Trombosis de las Arterias Carótidas/prevención & control , Inhibidores Enzimáticos/farmacología , Fibrinolíticos/farmacología , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Pirrolidinas/farmacología , Timidina Fosforilasa/antagonistas & inhibidores , Timina/farmacología , Animales , Aspirina/farmacología , Plaquetas/enzimología , Células COS , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/enzimología , Trombosis de las Arterias Carótidas/genética , Chlorocebus aethiops , Modelos Animales de Enfermedad , Terapia Antiplaquetaria Doble , Inhibidores Enzimáticos/toxicidad , Femenino , Fibrinolíticos/toxicidad , Hemorragia/inducido químicamente , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/toxicidad , Unión Proteica , Pirrolidinas/toxicidad , Transducción de Señal , Timidina Fosforilasa/genética , Timidina Fosforilasa/metabolismo , Timina/toxicidad , Dominios Homologos src , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
OBJECTIVE: It is unclear if stopping treatment with dabigatran, a new oral anticoagulant (NOAC), induces a paradoxical rebound prothrombotic state. We investigated if short-term (1-3 days) dabigatran cessation is associated with a higher thrombus volume than expected from a simple reversal of the anticoagulant effect. METHODS: Ten-week-old C57Bl/6 mice (n = 338) received one of the following oral treatments: phosphate-buffered saline (PBS), dabigatran for 7 days with or without 1 to 4 day cessation, and aspirin in either a single dose or daily for 7 days. Some of the animals that ceased dabigatran for 1 to 3 days received single-dose aspirin. Thereafter, we induced FeCl3 -mediated carotid thrombosis in 130 mice, after which we performed micro computed tomography thrombus imaging. The other 208 mice underwent coagulation assays or platelet function tests. As an explorative pilot study, we reviewed the medical records of 18 consecutive patients with NOAC cessation-related cerebral infarction in a large acute stroke cohort. RESULTS: We observed a ~ 40% higher volume of carotid thrombus after dabigatran cessation at 1 to 3 days than after vehicle treatment and showed that this effect could be prevented by single-dose aspirin pretreatment. Dabigatran cessation unduly increased platelet aggregability for 2 days after drug cessation, an effect mediated through thrombin or arachidonic acid, which effect was significantly attenuated by single-dose aspirin pretreatment. In patients, short-term (≤ 3 days) cessation of NOAC therapy, compared with longer-term (≥ 5 days) cessation, tended to be associated with relatively high stroke severity. INTERPRETATION: We provide the first preclinical evidence that a rebound prothrombotic state follows short-term cessation of dabigatran therapy. ANN NEUROL 2021;89:444-458.
Asunto(s)
Antitrombinas/efectos adversos , Trombosis de las Arterias Carótidas/diagnóstico por imagen , Dabigatrán/efectos adversos , Deprescripciones , Agregación Plaquetaria/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/sangre , Trombofilia/sangre , Anciano , Anciano de 80 o más Años , Animales , Antitrombinas/farmacología , Ácido Araquidónico/sangre , Aspirina/farmacología , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/prevención & control , Infarto Cerebral/diagnóstico por imagen , Infarto Cerebral/etiología , Infarto Cerebral/fisiopatología , Infarto Cerebral/prevención & control , Cloruros/toxicidad , Angiografía por Tomografía Computarizada , Dabigatrán/farmacología , Inhibidores del Factor Xa/efectos adversos , Femenino , Compuestos Férricos/toxicidad , Humanos , Accidente Cerebrovascular Isquémico/diagnóstico por imagen , Accidente Cerebrovascular Isquémico/etiología , Accidente Cerebrovascular Isquémico/fisiopatología , Accidente Cerebrovascular Isquémico/prevención & control , Angiografía por Resonancia Magnética , Masculino , Volúmen Plaquetario Medio , Ratones , Noxas/toxicidad , Proyectos Piloto , Inhibidores de Agregación Plaquetaria/farmacología , Recuento de Plaquetas , Pirazoles/efectos adversos , Piridonas/efectos adversos , Rivaroxabán/efectos adversos , Índice de Severidad de la Enfermedad , Síndrome de Abstinencia a Sustancias/etiología , Síndrome de Abstinencia a Sustancias/prevención & control , Trombina/metabolismo , Trombofilia/etiología , Trombofilia/prevención & control , Microtomografía por Rayos XRESUMEN
OBJECTIVE: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. CONCLUSIONS: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.
Asunto(s)
Plaquetas/metabolismo , Trombosis de las Arterias Carótidas/sangre , Hemostasis , Agregación Plaquetaria , Receptores Acoplados a Proteínas G/sangre , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/sangre , Animales , Células CHO , Trombosis de las Arterias Carótidas/prevención & control , Cricetulus , Modelos Animales de Enfermedad , Femenino , Fibrinolíticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/sangre , Humanos , Masculino , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Trombina/sangre , Transducción de Señal , Trombina/metabolismoRESUMEN
OBJECTIVE: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. CONCLUSIONS: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.
Asunto(s)
Coagulación Sanguínea , Plaquetas/enzimología , Trombosis de las Arterias Carótidas/enzimología , NADPH Oxidasas/sangre , Activación Plaquetaria , Embolia Pulmonar/enzimología , Animales , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/genética , Trombosis de las Arterias Carótidas/prevención & control , GMP Cíclico/sangre , Proteínas Quinasas Dependientes de GMP Cíclico/sangre , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Fibrinolíticos/farmacología , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Activación Plaquetaria/efectos de los fármacos , Embolia Pulmonar/sangre , Embolia Pulmonar/genética , Embolia Pulmonar/prevención & control , Transducción de Señal , Superóxidos/sangreRESUMEN
Growing evidence supports a central role of NADPH oxidases (NOXs) in the regulation of platelets, which are circulating cells involved in both hemostasis and thrombosis. Here, the use of Nox1-/- and Nox1+/+ mice as experimental models of human responses demonstrated a critical role of NOX1 in collagen-dependent platelet activation and pathological arterial thrombosis, as tested in vivo by carotid occlusion assays. In contrast, NOX1 does not affect platelet responses to thrombin and normal hemostasis, as assayed in tail bleeding experiments. Therefore, as NOX1 inhibitors are likely to have antiplatelet effects without associated bleeding risks, the NOX1-selective inhibitor 2-acetylphenothiazine (2APT) and a series of its derivatives generated to increase inhibitory potency and drug bioavailability were tested. Among the 2APT derivatives, 1-(10H-phenothiazin-2-yl)vinyl tert-butyl carbonate (2APT-D6) was selected for its high potency. Both 2APT and 2APT-D6 inhibited collagen-dependent platelet aggregation, adhesion, thrombus formation, superoxide anion generation, and surface activation marker expression, while responses to thrombin or adhesion to fibrinogen were not affected. In vivo administration of 2APT or 2APT-D6 led to the inhibition of mouse platelet aggregation, oxygen radical output, and thrombus formation, and carotid occlusion, while tail hemostasis was unaffected. Differently to in vitro experiments, 2APT-D6 and 2APT displayed similar potency in vivo. In summary, NOX1 inhibition with 2APT or its derivative 2APT-D6 is a viable strategy to control collagen-induced platelet activation and reduce thrombosis without deleterious effects on hemostasis. These compounds should, therefore, be considered for the development of novel antiplatelet drugs to fight cardiovascular diseases in humans.
Asunto(s)
Trombosis de las Arterias Carótidas/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , NADPH Oxidasa 1/antagonistas & inhibidores , Fenotiazinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Animales , Trombosis de las Arterias Carótidas/prevención & control , Células Cultivadas , Colágeno/metabolismo , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/uso terapéutico , Femenino , Fibrinógeno/metabolismo , Hemorragia/etiología , Humanos , Ratones , Ratones Endogámicos C57BL , Fenotiazinas/efectos adversos , Fenotiazinas/uso terapéutico , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Trombina/metabolismoRESUMEN
OBJECTIVE: Clopidogrel is a commonly used P2Y12 inhibitor to treat and prevent arterial thrombotic events. Clopidogrel is a prodrug that requires bioactivation by CYP (cytochrome P450) enzymes to exert antiplatelet activity. Diabetes mellitus is associated with an increased risk of ischemic events, and impaired ability to generate the active metabolite (AM) from clopidogrel. The objective of this study is to identify the mechanism of clopidogrel resistance in a murine model of diet-induced obesity (DIO). Approach and Results: C57BL/6J mice and IL-1R-/- mice were given high-fat diet for 10 weeks to generate a murine model of diet-induced obesity. Platelet aggregation and carotid arterial thrombosis were assessed in response to clopidogrel treatment. Wild-type DIO mice exhibited resistance to antiplatelet and antithrombotic effects of clopidogrel that was associated with reduced hepatic expression of CYP genes and reduced generation of the AM. IL (Interleukin)-1 receptor-deficient DIO (IL1R-/- DIO) mice showed no resistance to clopidogrel. Lack of resistance was accompanied by increased exposure of the clopidogrel AM. This resistance was also absent when wild-type DIO mice were treated with the conjugate of the clopidogrel AM, DT-678. CONCLUSIONS: These findings indicate that antiplatelet effects of clopidogrel may be impaired in the setting of diabetes mellitus due to reduced prodrug bioactivation related to IL-1 receptor signaling. Therapeutic targeting of P2Y12 in patients with diabetes mellitus using the conjugate of clopidogrel AM may lead to improved outcomes.
Asunto(s)
Clopidogrel/farmacocinética , Clopidogrel/uso terapéutico , Resistencia a Medicamentos , Obesidad/complicaciones , Receptores de Interleucina-1/fisiología , Animales , Trombosis de las Arterias Carótidas/prevención & control , Clopidogrel/farmacología , Sistema Enzimático del Citocromo P-450/genética , Diabetes Mellitus , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Fibrinolíticos , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/enzimología , Obesidad/etiología , Obesidad/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria , Profármacos/farmacocinética , Profármacos/uso terapéutico , Receptores de Interleucina-1/deficienciaRESUMEN
The present study provides first evidence on the role of plasma gelsolin in protecting pulmonary thromboembolism and thrombosis in a mouse model. Gelsolin is the most abundant actin depolymerizing protein in plasma and its significantly depleted values have been reported in metabolic disorders including cardiovascular diseases and myocardial infarction. Though gelsolin replacement therapy (GRT) has been shown to be effective in some animal models, no such study has been reported for thrombotic diseases that are acutely in need of bio-therapeutics for immediate and lasting relief. Here, using mice model and recombinant human gelsolin (rhuGSN), we demonstrate the antithrombotic effect of gelsolin in ferric chloride induced thrombosis in carotid artery and thrombin induced acute pulmonary thromboembolism. In thrombosis model, arterial occlusion time was significantly enhanced upon subcutaneous (SC) treatment with 8 mg of gelsolin per mice viz. 15.83 minutes vs. 8 minutes in the placebo group. Pertinently, histopathological examination showed channel formation within the thrombi in the carotid artery following injection of gelsolin. Fluorescence molecular tomography imaging further confirmed that administration of gelsolin reduced thrombus formation following carotid artery injury. In thrombin-induced acute pulmonary thromboembolism, mice pretreated with aspirin or gelsolin showed 100 and 83.33% recovery, respectively. In contrast, complete mortality of mice was observed in vehicle treated group within 5 minutes of thrombin injection. Overall, our studies provide conclusive evidence on the thrombo-protective role of plasma gelsolin in mice model of pulmonary thromboembolism and thrombosis.
Asunto(s)
Trombosis de las Arterias Carótidas/prevención & control , Arteria Carótida Común/efectos de los fármacos , Gelsolina/farmacología , Embolia Pulmonar/prevención & control , Proteínas Recombinantes/farmacología , Trombosis/prevención & control , Enfermedad Aguda , Animales , Trombosis de las Arterias Carótidas/diagnóstico por imagen , Trombosis de las Arterias Carótidas/fisiopatología , Arteria Carótida Común/diagnóstico por imagen , Arteria Carótida Común/fisiopatología , Modelos Animales de Enfermedad , Femenino , Colorantes Fluorescentes/química , Gelsolina/genética , Humanos , Ratones Endogámicos BALB C , Sustancias Protectoras/farmacología , Embolia Pulmonar/diagnóstico por imagen , Embolia Pulmonar/fisiopatología , Trombosis/diagnóstico por imagen , Trombosis/fisiopatología , Tomografía/métodosRESUMEN
Decellularized vascular scaffolds are promising materials for vessel replacements. However, despite the natural origin of decellularized vessels, issues such as biomechanical incompatibility, immunogenicity risks and the hazards of thrombus formation, still need to be addressed. In this study, we coated decellularized vessels obtained from porcine carotid arteries with poly (ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) with the purpose of improving endothelial coverage and minimizing platelet attachment while enhancing the mechanical properties of the decellularized vascular scaffolds. The polymer facilitated binding of endothelial cells (ECs) with high affinity and also induced endothelial cell capillary tube formation. In addition, platelets showed reduced adhesion on the polymer under flow conditions. Moreover, the coating of the decellularized arteries improved biomechanical properties by increasing its tensile strength and load. In addition, after 5 days in culture, ECs seeded on the luminal surface of 8g7-coated decellularized arteries showed good regeneration of the endothelium. Overall, this study shows that polymer coating of decellularized vessels provides a new strategy to improve re-endothelialization of vascular grafts, maintaining or enhancing mechanical properties while reducing the risk of thrombogenesis. These results could have potential applications in improving tissue-engineered vascular grafts for cardiovascular therapies with small caliber vessels.
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Arterias Carótidas/fisiología , Trombosis de las Arterias Carótidas/prevención & control , Células Endoteliales/fisiología , Metilmetacrilatos/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/química , Biopolímeros , Plaquetas/fisiología , Prótesis Vascular , Arterias Carótidas/ultraestructura , Endotelio Vascular/fisiología , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , PorcinosRESUMEN
Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin ß3, but the underlying effects of Dok-1 on αIIbß3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in αIIbß3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of αIIbß3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p < 0.05 vs WT), increased PLCγ2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p < 0.01 vs WT), demonstrating that Dok-1 negatively regulates αIIbß3 "outside-in" signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p < 0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating αIIbß3 outside-in signalling.
Asunto(s)
Proteínas de Unión al ADN/sangre , Fosfoproteínas/sangre , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas de Unión al ARN/sangre , Trombosis/prevención & control , Animales , Tiempo de Sangría , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/genética , Trombosis de las Arterias Carótidas/prevención & control , Retracción del Coagulo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Fibrinógeno/metabolismo , Hemostasis , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Selectina-P/sangre , Fosfolipasa C gamma/sangre , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Activación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Transducción de Señal , Trombosis/sangre , Trombosis/genéticaRESUMEN
INTRODUCTION: Current treatment guidelines for acute ischemic stroke do not recommend thrombolytic therapy in children and adolescents as data are still very scarce. CASE REPORT: We report the case of a 15-year-old boy who suddenly developed severe left-sided weakness and speech difficulty while stooling. Upon arrival at our Emergency Department, the National Institute of Health Stroke Scale (NIHSS) score was 18. Urgent neurovascular ultrasound showed a distal occlusion of the right internal carotid artery and occlusion at the origin of the middle cerebral artery (MCA) and the anterior cerebral artery. He was treated 2 hours after symptom onset with intravenous recombinant tissue plasminogen activator without any complication. At the end of thrombolysis, a complete recanalization was shown by transcranial Doppler sonography, although a brain magnetic resonance imaging disclosed an acute right middle cerebral artery stroke. At discharge, the boy had mild weakness on his left leg and slight left facial palsy: the NIHSS score was 2. To our knowledge, this is the first intravenous thrombolytic treatment ever reported in an adolescent in Italy. CONCLUSIONS: Despite the lack of evidence regarding the safety and the efficacy of recombinant tissue plasminogen activator in pediatric stroke, this treatment option should be considered, especially in adolescents presenting within 3 hours from symptom onset in centers with consolidated experience in adult thrombolysis.
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Isquemia Encefálica/prevención & control , Trombosis de las Arterias Carótidas/prevención & control , Infarto de la Arteria Cerebral Anterior/prevención & control , Infarto de la Arteria Cerebral Media/prevención & control , Terapia Trombolítica , Activador de Tejido Plasminógeno/uso terapéutico , Administración Intravenosa , Adolescente , Humanos , Masculino , Proteínas Recombinantes , Activador de Tejido Plasminógeno/administración & dosificación , Resultado del TratamientoRESUMEN
The most serious complication of carotid artery stenting (CAS) is acute carotid artery stent thrombosis (ACAST). ACAST is a very rare complication, but it may lead to dramatic and catastrophic consequences. The most important cause is inadequate or ineffective antiaggregant therapy. It is very important to identify, before CAS, those patients who might be candidates for ACAST and to start antiplatelet therapy for them. Testing patients who are candidates for CAS for acetylsalicylic acid and clopidogrel resistance may prevent this complication.
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Aspirina/uso terapéutico , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/prevención & control , Inhibidores de Agregación Plaquetaria/uso terapéutico , Stents/efectos adversos , Ticlopidina/análogos & derivados , Anciano , Anciano de 80 o más Años , Trombosis de las Arterias Carótidas/complicaciones , Angiografía Cerebral , Clopidogrel , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ticlopidina/uso terapéuticoRESUMEN
Platelet-derived microparticles (PDMPs) and adiponectin play an important role in the development of atherothrombosis. We investigated the effect of febuxostat on circulating PDMP levels and adiponectin in hyperuricemic patients. Levels of PDMP and biomarkers were measured using an ELISA at baseline and after 2 and 6 months of treatment. Plasma levels of PDMPs and biomarkers were higher, while those of adiponectin were lower in hyperuricemic patients than in normouricemic controls. Uric acid and interleukin (IL)-6 levels decreased significantly in hyperuricemic patients after 2 months of febuxostat treatment. However, PDMP and biomarkers decreased significantly in hyperuricemic patients after only 6 months of febuxostat treatment and adiponectin increased significantly. These results suggest that the effects of febuxostat for PDMPs seen may be the effect on xanthine oxidase but not the decrease of uric acid, and febuxostat may be beneficial for primary prevention of atherothrombosis in hyperuricemic patients.
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Adiponectina/agonistas , Trombosis de las Arterias Carótidas/prevención & control , Micropartículas Derivadas de Células/efectos de los fármacos , Febuxostat/uso terapéutico , Supresores de la Gota/uso terapéutico , Hiperuricemia/tratamiento farmacológico , Adiponectina/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Plaquetas/patología , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/patología , Estudios de Casos y Controles , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/patología , Femenino , Humanos , Hiperuricemia/sangre , Hiperuricemia/complicaciones , Hiperuricemia/patología , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Ácido Úrico/antagonistas & inhibidores , Ácido Úrico/sangre , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/sangreRESUMEN
Carotid endarterectomy is a commonly performed procedure for prevention of stroke related to carotid stenosis. Intraoperative sonography is used to identify potentially correctable technical defects during carotid endarterectomy. The main risk of endarterectomy is perioperative stroke, and great effort has been put into trying to reduce this risk through various surgical techniques and evaluation of the surgical bed. Postoperative carotid thrombosis, or thombo-embolization from the arterectomy site, remains a common cause of perioperative stroke and is often related to technical defects in the arterial reconstruction procedure. Re-exploration and repair of any imperfections have the potential to improve outcomes. Intraoperative imaging can identify potentially occult lesions, provide the option for correction, and thus reduce chance of stroke. Familiarity with the spectrum of intraoperative sonographic findings helps correctly identify residual intimal dissection flaps, plaque, thrombi, and stenosis, which may require immediate surgical revision. Our objective is to illustrate the spectrum of intraoperative findings and their importance.
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Trombosis de las Arterias Carótidas/diagnóstico por imagen , Trombosis de las Arterias Carótidas/etiología , Estenosis Carotídea/diagnóstico por imagen , Estenosis Carotídea/cirugía , Endarterectomía Carotidea/efectos adversos , Ultrasonografía Doppler Dúplex/métodos , Trombosis de las Arterias Carótidas/prevención & control , Endarterectomía Carotidea/métodos , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Cirugía Asistida por Computador/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin. METHODS AND FINDINGS: Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2-15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2ß1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (K(D) 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease. CONCLUSION: Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.
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Proteínas de Insectos/metabolismo , Inhibidores de Agregación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glándulas Salivales/química , Simuliidae/metabolismo , Secuencia de Aminoácidos , Animales , Trombosis de las Arterias Carótidas/prevención & control , Colágeno Tipo III/metabolismo , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Adhesividad Plaquetaria , Agregación Plaquetaria , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
Multiple myeloma confers a high risk for vascular thrombosis, a risk that is increased by treatment with immunomodulatory agents. Strikingly, inclusion of the proteasome inhibitor bortezomib reduces thrombotic risk, yet the molecular basis for this observation remains unknown. Here, we show that bortezomib prolongs thrombosis times in the carotid artery photochemical injury assay in normal mice. Cell-based studies show that bortezomib increases expression of the transcription factor Kruppel-like factor 2 (KLF2) in multiple cell types. Global postnatal overexpression of KLF2 (GL-K2-TG) increased time to thrombosis, and global postnatal deletion of KLF2 (GL-K2-KO) conferred an antiparallel effect. Finally, studies in GL-K2-KO mice showed that the thromboprotective effect of bortezomib is KLF2 dependent. These findings identify a transcriptional basis for the antithrombotic effects of bortezomib.
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Ácidos Borónicos/farmacología , Trombosis de las Arterias Carótidas/prevención & control , Citoprotección/genética , Factores de Transcripción de Tipo Kruppel/fisiología , Pirazinas/farmacología , Animales , Bortezomib , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/genética , Trombosis de las Arterias Carótidas/patología , Células Cultivadas , Oclusión Coronaria/genética , Oclusión Coronaria/patología , Oclusión Coronaria/prevención & control , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tiempo de Coagulación de la Sangre TotalRESUMEN
Antithrombotic activities of enoxifol, a new antioxidant with antiaggregant activity demonstrated in vitro and in vivo, and antioxidant mexidol were compared on the rat model of arterial thrombosis induced by application of 50% ferric chloride. Acetylsalicylic acid (antiaggregant) served as the reference drug. All drugs exhibited dose-dependent antithrombotic activity. Enoxifol was more effective than mexidol, both drugs being more active than the reference drug (acetylsalicylic acid). Taking into account the pathogenesis of the thrombosis in this experimental model, we can hypothesize that the pronounced antithrombotic effect of enoxifol was due to its antiaggregant and antioxidant activities.
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Anticoagulantes/farmacología , Antioxidantes/farmacología , Bencimidazoles/farmacología , Picolinas/farmacología , Animales , Animales no Consanguíneos , Aspirina/farmacología , Trombosis de las Arterias Carótidas/prevención & control , Evaluación Preclínica de Medicamentos , Masculino , Agregación Plaquetaria/efectos de los fármacos , RatasRESUMEN
Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.
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Enfermedades Cardiovasculares/prevención & control , Hidrolasas/antagonistas & inhibidores , Ornitina/análogos & derivados , Animales , Autoanticuerpos/sangre , Células de la Médula Ósea/fisiología , Enfermedades Cardiovasculares/enzimología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/inmunología , Trombosis de las Arterias Carótidas/prevención & control , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Endotelio/patología , Femenino , Humanos , Inyecciones Subcutáneas , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/enzimología , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Ornitina/administración & dosificación , Fenotipo , Desiminasas de la Arginina Proteica , Células Madre/fisiología , Vasodilatación/efectos de los fármacosRESUMEN
A combination of low-dose aspirin (ASA) and a phosphodiesterase inhibitor has been clinically tried for the secondary prevention of atherothrombotic diseases. The in vivo antithrombotic property of ibudilast (CAS 50847-11-5), a phosphodiesterase 4 (PDE4) inhibitor, was evaluated in a photochemically-induced guinea pig carotid artery thrombosis model in combination with low-dose ASA. The time required to decrease the carotid artery blood flow to the reading "zero" was defined as the time to occlusion (TTO) of the artery through thrombogenesis. Each independent use of ASA (300 mg/kg, p.o.) and ibudilast (3 and 10 mg/kg, p.o.) significantly prolonged the TTO, and ASA (300 mg/kg) significantly increased bleeding time (BT) and gastric mucosal injury. A selective PDE4 inhibitor rolipram (1 and 5 mg/kg, p.o.) tended to prolong the TTO without extending BT. ASA (100 mg/kg) plus ibudilast (3 mg/kg) and ASA (100 mg/kg) plus rolipram (5 mg/kg) markedly prolonged the TTO compared with each agent alone. Interestingly, ASA (100 mg/kg) plus ibudilast (3 mg/kg) caused a longer TTO than ASA (300 mg/kg) alone, without significant extension of BT and gastric mucosal injury as observed in ASA (300 mg/kg). These results indicate that the combination of low-dose ASA and ibudilast has a more potent antithrombotic effect than ASA alone without increasing bleeding tendency and gastric mucosal injury. The potent in vivo antithrombotic effect of this combination may be brought about by an action that is associated with PDE4 inhibition of ibudilast.