RESUMEN
AIMS: Arterial thrombosis as a result of plaque rupture or erosion is a key event in acute cardiovascular events. Sirtuin 5 (SIRT5) belongs to the lifespan-regulating sirtuin superfamily and has been implicated in acute ischaemic stroke and cardiac hypertrophy. This project aims at investigating the role of SIRT5 in arterial thrombus formation. METHODS AND RESULTS: Sirt5 transgenic (Sirt5Tg/0) and knock-out (Sirt5-/-) mice underwent photochemically induced carotid endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) were treated with SIRT5 silencing-RNA (si-SIRT5) as well as peripheral blood mononuclear cells from acute coronary syndrome (ACS) patients and non-ACS controls (case-control study, total n = 171) were used to increase the translational relevance of our data. Compared to wild-type controls, Sirt5Tg/0 mice displayed accelerated arterial thrombus formation following endothelial-specific damage. Conversely, in Sirt5-/- mice, arterial thrombosis was blunted. Platelet function was unaltered, as assessed by ex vivo collagen-induced aggregometry. Similarly, activation of the coagulation cascade as assessed by vascular and plasma tissue factor (TF) and TF pathway inhibitor expression was unaltered. Increased thrombus embolization episodes and circulating D-dimer levels suggested augmented activation of the fibrinolytic system in Sirt5-/- mice. Accordingly, Sirt5-/- mice showed reduced plasma and vascular expression of the fibrinolysis inhibitor plasminogen activator inhibitor (PAI)-1. In HAECs, SIRT5-silencing inhibited PAI-1 gene and protein expression in response to TNF-α. This effect was mediated by increased AMPK activation and reduced phosphorylation of the MAP kinase ERK 1/2, but not JNK and p38 as shown both in vivo and in vitro. Lastly, both PAI-1 and SIRT5 gene expressions are increased in ACS patients compared to non-ACS controls after adjustment for cardiovascular risk factors, while PAI-1 expression increased across tertiles of SIRT5. CONCLUSION: SIRT5 promotes arterial thrombosis by modulating fibrinolysis through endothelial PAI-1 expression. Hence, SIRT5 may be an interesting therapeutic target in the context of atherothrombotic events.
Asunto(s)
Traumatismos de las Arterias Carótidas/enzimología , Trombosis de las Arterias Carótidas/enzimología , Células Endoteliales/enzimología , Fibrinólisis , Sirtuinas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Síndrome Coronario Agudo/sangre , Síndrome Coronario Agudo/enzimología , Adulto , Anciano , Animales , Traumatismos de las Arterias Carótidas/sangre , Traumatismos de las Arterias Carótidas/genética , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/genética , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Fosforilación , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Sirtuinas/genéticaRESUMEN
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: 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
BACKGROUND: Smoking is the main preventable cause of death in the United States and worldwide and is associated with serious cardiovascular health consequences, including thrombotic diseases. Recently, electronic cigarettes (e-cigarettes) and, in particular JUUL, have attained wide popularity among smokers, nonsmokers, pregnant females, and even the youth, which is alarming. Interestingly, there is/are no information/studies regarding the effect of JUUL on cardiovascular diseases, specifically in the context of modulation of platelet activation. Thus, it is important to discern the cardiovascular disease health risks associated with JUUL. METHODS AND RESULTS: We used a passive e-vape vapor inhalation system where C57BL/6J mice (10-12 weeks old) were exposed to JUUL e-cigarette vape. Menthol flavored JUUL pods containing 5% nicotine by weight were used as the e-liquid. Mice were exposed to a total of 70 puffs daily for 2 weeks; 3-second puff duration, and 25-second puff interval. The effects of JUUL relative to clean air were analyzed, on mouse platelet function in vitro (eg, aggregation) and in vivo (eg, FeCl3-induced carotid artery injury thrombosis model). Our results indicate that short-term exposure to JUUL e-cigarette causes hyperactivation of platelets and shortens the thrombus occlusion as well as hemostasis/bleeding times, relative to clean air (medians of 14 vs. 200 seconds, P < .01 and 35 vs. 295 seconds, P < .001, respectively). CONCLUSION: Our findings document-for the first time-that short-term exposure to the JUUL e-cigarette increases the risk of thrombotic events, in part by modulating platelet function, such as aggregation and secretion, in mice.
Asunto(s)
Plaquetas/metabolismo , Trombosis de las Arterias Carótidas/etiología , Cigarrillo Electrónico a Vapor/efectos adversos , Sistemas Electrónicos de Liberación de Nicotina , Activación Plaquetaria , Vapeo/efectos adversos , Animales , Trombosis de las Arterias Carótidas/sangre , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Fosfatidilserinas/sangre , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal , Vapeo/sangreRESUMEN
Coronavirus disease 2019 (COVID-19) is a pandemic disease currently affecting millions of people worldwide. Its neurological implications are poorly understood, and further study is urgently required. A hypercoagulable state has been reported in patients with severe COVID-19, but nothing is known about coagulopathy in patients with milder disease. We describe cases of patients in New York City presenting with stroke secondary to large vessel thrombosis without occlusion, incidentally found to have COVID-19 with only mild respiratory symptoms. This is in contrast to the venous thrombosis and microangiopathy that has been reported in patients with severe COVID-19. Our cases suggest that even in the absence of severe disease, patients with COVID-19 may be at increased risk of thrombus formation leading to stroke, perhaps resulting from viral involvement of the endothelium. Further systematic study is needed because this may have implications for primary and secondary stroke prevention in patients with COVID-19.
Asunto(s)
Betacoronavirus , Trombosis de las Arterias Carótidas/etiología , Infecciones por Coronavirus/complicaciones , Infarto de la Arteria Cerebral Media/etiología , Pandemias , Neumonía Viral/complicaciones , Adulto , Anciano , Anticoagulantes/uso terapéutico , Betacoronavirus/aislamiento & purificación , Biomarcadores , Proteína C-Reactiva/análisis , COVID-19 , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/diagnóstico por imagen , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Angiografía Cerebral , Angiografía por Tomografía Computarizada , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Urgencias Médicas , Femenino , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Hemiplejía/etiología , Humanos , Infarto de la Arteria Cerebral Media/sangre , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Angiografía por Resonancia Magnética , Masculino , Persona de Mediana Edad , Ciudad de Nueva York/epidemiología , Neumonía Viral/sangre , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , SARS-CoV-2 , Tromboflebitis/complicacionesRESUMEN
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).
Asunto(s)
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
OBJECTIVE: Retinoid X receptors (RXR) are a family of nuclear receptors that play critical roles in the regulation of numerous fundamental biological processes including cell proliferation, differentiation, and death. Earlier studies suggested that treatment with RXR agonists attenuates platelet activation in all adults (male and femal) and mice; however, the underlying molecular mechanisms have remained insufficiently understood. To elaborate further on this issue, we characterized megakaryocyte and platelet-specific RXR knockout mice to study platelet function in vitro and arterial thrombosis in vivo. APPROACH AND RESULTS: First, we identified RXRß as the dominant RXR receptor in mouse platelets, prompting us to generate a megakaryocyte and platelet-specific PF4Cre ;RXRßflox/flox mouse. Second, we studied activation, spreading, and aggregation of platelets from C57Bl/6 wild-type mice (WT), PF4Cre+ ;RXRßflox/flox mice, and PF4Cre- ;RXRßflox/flox littermate controls in the presence or absence of RXR ligands, that is, 9-cis-retinoic acid (9cRA) and methoprene acid (MA). We found that in vitro treatment with RXR ligands attenuates spreading and aggregation of platelets and increases proplatelet particle formation from megakaryocytes (MK). However, these effects are also observed in RXRß-deficient platelets and MKs and are thus independent of RXRß. Third, we investigated arterial thrombus formation in an iron chloride (FeCl3)-induced vascular injury model in vivo, which is also not affected by the absence of RXRß in platelets. CONCLUSIONS: Absence of the most abundant RXR receptor in mouse platelets, RXRß, does not affect platelet function in vitro and thrombus formation in vivo. Furthermore, RXR agonists' mediated effects on platelet function are independent of RXRß expression. Hence, our data do not support a significant contribution of RXRß to arterial thrombosis in mice.
Asunto(s)
Plaquetas/fisiología , Trombosis de las Arterias Carótidas/sangre , Proteínas de Unión al ADN/fisiología , Animales , Trombosis de las Arterias Carótidas/inducido químicamente , Cloruros/toxicidad , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Compuestos Férricos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Plaquetaria , Trombopoyesis/fisiologíaRESUMEN
BACKGROUND: The homologous plasma proteins prekallikrein and factor XI (FXI) circulate as complexes with high molecular weight kininogen. Although evidence supports an interaction between the prekallikrein-kininogen complexes and vascular endothelium, there is conflicting information regarding FXI binding to endothelium. OBJECTIVE: To study the interaction between FXI and blood vessels in mice. METHODS: C57Bl/6 wild-type or F11-/- mice in which variants of FXI were expressed by hydrodynamic tail vein injection, received intravenous infusions of saline, heparin, polyphosphates, protamine, or enzymes that digest glycosaminoglycans (GAGs). Blood was collected after infusion and plasma was analyzed by western blot for FXI. RESULTS AND CONCLUSIONS: Plasma FXI increased 5- to 10-fold in wild-type mice after infusion of heparin, polyphosphates, protamine, or GAG-digesting enzymes, but not saline. Similar treatments resulted in a much smaller change in plasma FXI levels in rats, and infusions of large boluses of heparin did not change FXI levels appreciably in baboons or humans. The releasable FXI fraction was reconstituted in F11-/- mice by expressing murine FXI, but not human FXI. We identified a cluster of basic residues on the apple 4 domain of mouse FXI that is not present in other species. Replacing the basic residues with alanine prevented the interaction of mouse FXI with blood vessels, whereas introducing the basic residues into human FXI allowed it to bind to blood vessels. Most FXI in mice is noncovalently associated with GAGs on blood vessel endothelium and does not circulate in plasma.
Asunto(s)
Endotelio Vascular/metabolismo , Factor XI/metabolismo , Glicosaminoglicanos/sangre , Animales , Sitios de Unión , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/inducido químicamente , Cloruros/toxicidad , Factor XI/química , Deficiencia del Factor XI/sangre , Compuestos Férricos/toxicidad , Heparina/farmacología , Humanos , Quininógenos/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Papio , Precalicreína/metabolismo , Unión Proteica , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad de la Especie , Electricidad EstáticaRESUMEN
Sulfated polysaccharides from marine algae have high potential as promising candidates for marine drug development. In this study, a homogeneous sulfated polysaccharide from the marine green alga Monostroma nitidum, designated MS-1, was isolated using water extraction and anion-exchange and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that MS-1 mainly consisted of â3)-α-l-Rhap-(1â and â2)-α-l-Rhap-(1â residues, with additional branches consisting of 4-linked ß-d-xylose, 4-/6-linked d-glucose, terminal ß-d-glucuronic acid, and 3-/2-linked α-l-rhamnose. Sulfate ester groups substituted mainly at C-2/C-4 of â3)-α-l-Rhap-(1â and C-4 of â2)-α-l-Rhap-(1â residues, slightly at C-2 of terminal ß-d-glucuronic residues. MS-1 exhibited strong anticoagulant activity in vitro and in vivo as evaluated by the activated partial thromboplastin time and thrombin time assays, and significantly decreased platelet aggregation. The anticoagulant activity mechanism of MS-1 was mainly attributed to strong potentiation thrombin by heparin cofactor-II, and it also hastened thrombin and coagulation factor Xa inhibitions by potentiating antithrombin-III. MS-1 possessed markedly thrombolytic activity evaluated by plasminogen activator inhibitior-1, fibrin degradation products, and D-dimer levels using rats plasma, and recanalization rate by FeCl3-induced carotid artery thrombosis in mice. MS-1 exhibited strong antithrombotic activity in vitro and in vivo evaluated by the wet weighs and lengths of thrombus, and thrombus occlusion time by electrically-induced carotid artery thrombosis in rats. These results suggested that MS-1 could be a promising marine drug for prevention and therapy of thromboembolic disease.
Asunto(s)
Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Chlorophyta/química , Fibrinolíticos/farmacología , Polisacáridos/farmacología , Sulfatos/farmacología , Animales , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/inducido químicamente , Humanos , Masculino , Ratones , Agregación Plaquetaria/efectos de los fármacos , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Sulfatos/química , Sulfatos/aislamiento & purificaciónRESUMEN
Background and Purpose- Sustained successful reperfusion is an important prognostic factor for good clinical outcome in acute ischemic stroke. We aimed to identify the prevalence, clinical impact, and predictors of early reocclusion after initially successful thrombectomies within a prospective cohort. Methods- A total of 711 stroke patients with successful reperfusion (modified Thrombolysis in Cerebral Infarction, 2b/3) followed with magnetic resonance or computed tomographic angiography at 24 to 48 hours were included. Multivariable logistic regression analysis was used to evaluate associated factors and clinical impact. Results are displayed as adjusted odds ratio (aOR) and 95% CI. Improvement in accuracy of additional imaging findings on angiography control runs after the intervention was evaluated by area under the curve. Results- Early reocclusion was observed in 16 of 711 successfully reperfused patients (2.3%; 95% CI, 1.1-3.3; median delay: 20 hours). Suggestive predictors were higher platelets on admission (aOR, 1.01; 95% CI, 1.01-1.02), prestroke functional dependence (aOR, 7.12; 95% CI, 1.49-34.03), and stroke of undetermined or other specified pathogenesis in the TOAST classification (aOR, 7.19; 95% CI, 1.10-47.05 and aOR, 36.50; 95% CI, 4.47-298.11, respectively). When implementing residual embolic fragments or stenosis at the thrombectomy site into the logistic regression model, discrimination between patients with and without reocclusion improved significantly (area under the curve, 0.955 versus 0.854; P=0.023). Early reocclusion was an independent predictor of unfavorable outcome at 90 days (aOR for modified Rankin Scale ≤2, 0.13; 95% CI, 0.03-0.57). Conclusions- Early reocclusion within 48 hours after successful mechanical thrombectomy is rare but associated with poor outcome. Patients with high platelets on admission and residual embolic fragments or stenosis at the thrombectomy site are at high risk for reocclusion, which may be prevented or corrected after carefully re-evaluating the last angiographic run.
Asunto(s)
Isquemia Encefálica/cirugía , Trombosis de las Arterias Carótidas/cirugía , Procedimientos Endovasculares , Infarto de la Arteria Cerebral Media/cirugía , Accidente Cerebrovascular/cirugía , Trombectomía , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Isquemia Encefálica/sangre , Isquemia Encefálica/diagnóstico por imagen , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/diagnóstico por imagen , Angiografía Cerebral , Angiografía por Tomografía Computarizada , Constricción Patológica , Femenino , Humanos , Infarto de la Arteria Cerebral Media/sangre , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Modelos Logísticos , Angiografía por Resonancia Magnética , Masculino , Persona de Mediana Edad , Análisis Multivariante , Recuento de Plaquetas , Recurrencia , Factores de Riesgo , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/diagnóstico por imagenRESUMEN
Essentials Microbe-dependent production of trimethylamine N-oxide (TMAO) contributes to thrombosis risk. The impact of host flavin monooxygenase 3 (FMO3) modulation on platelet function is unknown. Genetic manipulation of FMO3 in mice alters systemic TMAO levels and thrombosis potential. Genetic manipulation of FMO3 is associated with alteration of gut microbial community structure. SUMMARY: Background Gut microbes play a critical role in the production of trimethylamine N-oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)-containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess. Objectives FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential in vivo has not yet been explored. Methods The impact of FMO3 suppression (antisense oligonucleotide-targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl3 -induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses. Results Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation in vivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and in vivo thrombosis potential. Conclusions The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet-dependent and gut microbiota-dependent changes in both platelet responsiveness and thrombosis potential in vivo.
Asunto(s)
Plaquetas/fisiología , Microbioma Gastrointestinal/fisiología , Hígado/enzimología , Metilaminas/metabolismo , Oxigenasas/fisiología , Trombofilia/enzimología , Animales , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/inducido químicamente , Arteria Carótida Común , Cloruros/toxicidad , Compuestos Férricos/toxicidad , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/farmacología , Oxigenasas/antagonistas & inhibidores , Oxigenasas/genética , Plasma Rico en Plaquetas , Ribotipificación , Riesgo , Trombofilia/microbiología , TransgenesRESUMEN
The present study aimed to prepare injectable Lumbrokinase (LK) with long circulation time in addition to enhanced anti-thrombotic efficacy. Following preparation, the pharmacokinetic and antithrombotic effect of the drug in a rat carotid artery thrombosis model was evaluated. The drug was prepared by conjugation of LK with mPEGSC20000 as previously reported. The pharmacokinetics of the mPEGSC20000LK were then examined and the antithrombotic activity in an arteryvein bypass thrombosis rat model was evaluated. Finally, the parameters of fibrinolysis including thromboxane B2, prostaglandin F1α, tissue plasminogen activator and plasminogen activator inhibitor1 were compared between native LK and mPEGSC20000LK in a FeCl3induced carotid artery thrombosis rat model. mPEGSC20000LK was successfully prepared by PEGylation of LK with mPEG20000SC in optimal conditions. Pharmacokinetic analysis demonstrated that the biological halflife of the mPEG20000SCLK increased by 2.2fold compared with native LK. In vivo antithrombotic analysis indicated that mPEG20000SCLK exhibited a greater antithrombotic effect in arteryvein bypass thrombosis and FeCl3-induced carotid artery thrombosis models compared with native LK. In conclusion, injectable PEGylated LK with prolonged half-life and enhanced antithrombotic effect is a potential antithrombotic agent for longacting treatment of the thrombus diseases.
Asunto(s)
Trombosis de las Arterias Carótidas/sangre , Endopeptidasas/química , Endopeptidasas/farmacocinética , Fibrinolíticos/química , Fibrinolíticos/farmacocinética , Polietilenglicoles/química , Animales , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Masculino , Estructura Molecular , Peso Molecular , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
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.
Asunto(s)
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
Factor XI (FXI) is an integral component of the intrinsic pathway of the coagulation cascade and plays a critical role in thrombus formation. Because its role in the pathogenesis of cerebral microembolic signals (MES) is unclear, this study used a potent and selective small molecule inhibitor of FXIa, compound 1, to assess the effect of FXI blockade in our recently established preclinical model of cerebral MES induced by FeCl3 injury of the carotid artery in male New Zealand White rabbits. Ascending doses of compound 1 were evaluated simultaneously for both carotid arterial thrombosis by a Doppler flowmeter and MES in the middle cerebral artery by a transcranial Doppler. Plasma drug exposure and pharmacodynamic responses to compound 1 treatment were also assessed. The effective dose for 50% inhibition (ED50) of thrombus formation was 0.003 mg/kg/h compound 1, i.v. for the integrated blood flow, 0.004 mg/kg/h for reduction in thrombus weight, and 0.106 mg/kg/h for prevention of MES. The highest dose, 3 mg/kg/h compound 1, achieved complete inhibition in both thrombus formation and MES. In addition, we assessed the potential bleeding liability of compound 1 (5 mg/kg/h, i.v., >1250-fold ED50 levels in arterial thrombosis) in rabbits using a cuticle bleeding model, and observed about 2-fold (not statistically significant) prolongation in bleeding time. Our study demonstrates that compound 1 produced a robust and dose-dependent inhibition of both arterial thrombosis and MES, suggesting that FXIa blockade may represent a novel therapeutic strategy for the reduction in MES in patients at risk for ischemic stroke.
Asunto(s)
Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Trombosis de las Arterias Carótidas , Factor XIa/antagonistas & inhibidores , Embolia Intracraneal , Animales , Coagulación Sanguínea/fisiología , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/complicaciones , Trombosis de las Arterias Carótidas/diagnóstico por imagen , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Modelos Animales de Enfermedad , Diseño de Fármacos , Inyecciones Intravenosas , Embolia Intracraneal/sangre , Embolia Intracraneal/diagnóstico por imagen , Embolia Intracraneal/etiología , Embolia Intracraneal/prevención & control , Masculino , Conejos , Ultrasonografía Doppler Transcraneal/métodosRESUMEN
BACKGROUND: The aim of the present study was to clarify the effect of glucose profiles after stroke, which was assessed by a continuous glucose monitoring (CGM) device. METHODS: Acute ischemic stroke patients within 24 h of onset were prospectively studied. CGM was performed for 72 h after admission. CGM parameters were evaluated as follows: (1) mean glucose level, (2) area under the curve (AUC) for glucose level >140 mg/dl and (3) SD of the glucose level. Infarct volume was measured at admission and 24 and 72 h after admission using diffusion-weighted imaging. CGM data and infarct volume growth were compared at 24 and 72 h. RESULTS: Seventy-eight patients were enrolled in the present study. Spearman's rank correlation coefficients showed that both the mean glucose level (r = 0.433, p < 0.001 for 24 h; r = 0.308, p = 0.006 for 72 h) and AUC >140 mg/dl (r = 0.417, p < 0.001 for 24 h; r = 0.277, p = 0.014 for 72 h) were significantly correlated with acute infarct volume growth. The SD of the glucose level was associated with infarct volume growth at 24 h (r = 0.303, p = 0.007), but not 72 h (r = 0.195, p = 0.088). CONCLUSION: Post-stroke hyperglycemia was associated with infarct volume growth during the acute phase of ischemic stroke.
Asunto(s)
Glucemia/metabolismo , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/complicaciones , Hiperglucemia/sangre , Hiperglucemia/etiología , Infarto de la Arteria Cerebral Media/sangre , Infarto de la Arteria Cerebral Media/complicaciones , Monitoreo Fisiológico/instrumentación , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/complicaciones , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Imagen de Difusión por Resonancia Magnética , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Angiografía por Resonancia Magnética , Masculino , Estudios ProspectivosRESUMEN
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
BACKGROUND: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. METHODS AND RESULTS: Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. CONCLUSIONS: Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.
Asunto(s)
Plaquetas/ultraestructura , Acetazolamida/farmacología , Actomiosina/metabolismo , Amidas/farmacología , Animales , Anoctaminas , Trastornos de la Coagulación Sanguínea/sangre , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Adhesión Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Forma de la Célula/efectos de los fármacos , Forma de la Célula/fisiología , Tamaño de la Célula/efectos de los fármacos , Micropartículas Derivadas de Células , Cloruros/metabolismo , Colágeno , Citocalasina D/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Ratones , Microtúbulos/efectos de los fármacos , Proteínas de Transferencia de Fosfolípidos/deficiencia , Proteínas de Transferencia de Fosfolípidos/fisiología , Piridinas/farmacología , Sodio/metabolismo , Trombina/biosíntesis , Trombosis/prevención & control , Agua/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
Asunto(s)
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
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.