RESUMO
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.
Assuntos
Lesões das Artérias Carótidas/enzimologia , Trombose das Artérias Carótidas/enzimologia , Células Endoteliais/enzimologia , Fibrinólise , Sirtuínas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/enzimologia , Adulto , Idoso , Animais , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/genética , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/genética , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sirtuínas/genéticaRESUMO
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.
Assuntos
Plaquetas/efeitos dos fármacos , Trombose das Artérias Carótidas/prevenção & controle , Inibidores Enzimáticos/farmacologia , Fibrinolíticos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Pirrolidinas/farmacologia , Timidina Fosforilase/antagonistas & inibidores , Timina/farmacologia , Animais , Aspirina/farmacologia , Plaquetas/enzimologia , Células COS , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/enzimologia , Trombose das Artérias Carótidas/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Terapia Antiplaquetária Dupla , Inibidores Enzimáticos/toxicidade , Feminino , Fibrinolíticos/toxicidade , Hemorragia/induzido quimicamente , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/toxicidade , Ligação Proteica , Pirrolidinas/toxicidade , Transdução de Sinais , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismo , Timina/toxicidade , Domínios de Homologia de src , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Plaquetas/metabolismo , Trombose das Artérias Carótidas/sangue , Hemostasia , Agregação Plaquetária , Receptores Acoplados a Proteínas G/sangue , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/sangue , Animais , Células CHO , Trombose das Artérias Carótidas/prevenção & controle , Cricetulus , Modelos Animais de Doenças , Feminino , Fibrinolíticos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/sangue , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Trombina/sangue , Transdução de Sinais , Trombina/metabolismoRESUMO
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.
Assuntos
Coagulação Sanguínea , Plaquetas/enzimologia , Trombose das Artérias Carótidas/enzimologia , NADPH Oxidases/sangue , Ativação Plaquetária , Embolia Pulmonar/enzimologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/genética , Trombose das Artérias Carótidas/prevenção & controle , GMP Cíclico/sangue , Proteínas Quinases Dependentes de GMP Cíclico/sangue , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Fibrinolíticos/farmacologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Ativação Plaquetária/efeitos dos fármacos , Embolia Pulmonar/sangue , Embolia Pulmonar/genética , Embolia Pulmonar/prevenção & controle , Transdução de Sinais , Superóxidos/sangueRESUMO
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.
Assuntos
Plaquetas/metabolismo , Trombose das Artérias Carótidas/etiologia , Vapor do Cigarro Eletrônico/efeitos adversos , Sistemas Eletrônicos de Liberação de Nicotina , Ativação Plaquetária , Vaping/efeitos adversos , Animais , Trombose das Artérias Carótidas/sangue , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Fosfatidilserinas/sangue , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Vaping/sangueRESUMO
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.
Assuntos
Betacoronavirus , Trombose das Artérias Carótidas/etiologia , Infecções por Coronavirus/complicações , Infarto da Artéria Cerebral Média/etiologia , Pandemias , Pneumonia Viral/complicações , Adulto , Idoso , Anticoagulantes/uso terapêutico , Betacoronavirus/isolamento & purificação , Biomarcadores , Proteína C-Reativa/análise , COVID-19 , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/diagnóstico por imagem , Trombose das Artérias Carótidas/tratamento farmacológico , Angiografia Cerebral , Angiografia por Tomografia Computadorizada , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Emergências , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Hemiplegia/etiologia , Humanos , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque/epidemiologia , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , SARS-CoV-2 , Tromboflebite/complicaçõesRESUMO
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).
Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Trombose das Artérias Carótidas/metabolismo , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Acidente Vascular Cerebral/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Trombose das Artérias Carótidas/sangue , Artéria Carótida Interna/patologia , Micropartículas Derivadas de Células/metabolismo , Feminino , Fibrina/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Ativação Plaquetária , Acidente Vascular Cerebral/sangue , Sulfonamidas/farmacologia , Trombina/metabolismoRESUMO
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.
Assuntos
Plaquetas/fisiologia , Trombose das Artérias Carótidas/sangue , Proteínas de Ligação a DNA/fisiologia , Animais , Trombose das Artérias Carótidas/induzido quimicamente , Cloretos/toxicidade , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Compostos Férricos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária , Trombopoese/fisiologiaRESUMO
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.
Assuntos
Endotélio Vascular/metabolismo , Fator XI/metabolismo , Glicosaminoglicanos/sangue , Animais , Sítios de Ligação , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Cloretos/toxicidade , Fator XI/química , Deficiência do Fator XI/sangue , Compostos Férricos/toxicidade , Heparina/farmacologia , Humanos , Cininogênios/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Papio , Pré-Calicreína/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Eletricidade EstáticaRESUMO
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.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Clorófitas/química , Fibrinolíticos/farmacologia , Polissacarídeos/farmacologia , Sulfatos/farmacologia , Animais , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Humanos , Masculino , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Sulfatos/química , Sulfatos/isolamento & purificaçãoRESUMO
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.
Assuntos
Isquemia Encefálica/cirurgia , Trombose das Artérias Carótidas/cirurgia , Procedimentos Endovasculares , Infarto da Artéria Cerebral Média/cirurgia , Acidente Vascular Cerebral/cirurgia , Trombectomia , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Isquemia Encefálica/sangue , Isquemia Encefálica/diagnóstico por imagem , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/diagnóstico por imagem , Angiografia Cerebral , Angiografia por Tomografia Computadorizada , Constrição Patológica , Feminino , Humanos , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Modelos Logísticos , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Contagem de Plaquetas , Recidiva , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/diagnóstico por imagemRESUMO
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.
Assuntos
Plaquetas/fisiologia , Microbioma Gastrointestinal/fisiologia , Fígado/enzimologia , Metilaminas/metabolismo , Oxigenases/fisiologia , Trombofilia/enzimologia , Animais , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Artéria Carótida Primitiva , Cloretos/toxicidade , Compostos Férricos/toxicidade , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/farmacologia , Oxigenases/antagonistas & inibidores , Oxigenases/genética , Plasma Rico em Plaquetas , Ribotipagem , Risco , Trombofilia/microbiologia , TransgenesRESUMO
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.
Assuntos
Trombose das Artérias Carótidas/sangue , Endopeptidases/química , Endopeptidases/farmacocinética , Fibrinolíticos/química , Fibrinolíticos/farmacocinética , Polietilenoglicóis/química , Animais , Trombose das Artérias Carótidas/tratamento farmacológico , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/patologia , Modelos Animais de Doenças , Masculino , Estrutura Molecular , Peso Molecular , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Coagulação Sanguínea , Trombose Intracraniana/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/genética , Trombose das Artérias Carótidas/metabolismo , Hemorragia Cerebral/sangue , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Técnicas de Introdução de Genes , Humanos , Trombose Intracraniana/sangue , Trombose Intracraniana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Trombose das Artérias Carótidas , Fator XIa/antagonistas & inibidores , Embolia Intracraniana , Animais , Coagulação Sanguínea/fisiologia , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/complicações , Trombose das Artérias Carótidas/diagnóstico por imagem , Trombose das Artérias Carótidas/tratamento farmacológico , Modelos Animais de Doenças , Desenho de Fármacos , Injeções Intravenosas , Embolia Intracraniana/sangue , Embolia Intracraniana/diagnóstico por imagem , Embolia Intracraniana/etiologia , Embolia Intracraniana/prevenção & controle , Masculino , Coelhos , Ultrassonografia Doppler Transcraniana/métodosRESUMO
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.
Assuntos
Glicemia/metabolismo , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/complicações , Hiperglicemia/sangue , Hiperglicemia/etiologia , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/complicações , Monitorização Fisiológica/instrumentação , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/complicações , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Imagem de Difusão por Ressonância Magnética , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Angiografia por Ressonância Magnética , Masculino , Estudos ProspectivosRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/sangue , Fosfoproteínas/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas de Ligação a RNA/sangue , Trombose/prevenção & controle , Animais , Tempo de Sangramento , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/genética , Trombose das Artérias Carótidas/prevenção & controle , Retração do Coágulo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Fibrinogênio/metabolismo , Hemostasia , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Selectina-P/sangue , Fosfolipase C gama/sangue , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Trombose/sangue , Trombose/genéticaRESUMO
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.
Assuntos
Plaquetas/ultraestrutura , Acetazolamida/farmacologia , Actomiosina/metabolismo , Amidas/farmacologia , Animais , Anoctaminas , Transtornos da Coagulação Sanguínea/sangue , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Trombose das Artérias Carótidas/tratamento farmacológico , Adesão Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Micropartículas Derivadas de Células , Cloretos/metabolismo , Colágeno , Citocalasina D/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Camundongos , Microtúbulos/efeitos dos fármacos , Proteínas de Transferência de Fosfolipídeos/deficiência , Proteínas de Transferência de Fosfolipídeos/fisiologia , Piridinas/farmacologia , Sódio/metabolismo , Trombina/biossíntese , Trombose/prevenção & controle , Água/metabolismoRESUMO
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.
Assuntos
Adiponectina/agonistas , Trombose das Artérias Carótidas/prevenção & controle , Micropartículas Derivadas de Células/efeitos dos fármacos , Febuxostat/uso terapêutico , Supressores da Gota/uso terapêutico , Hiperuricemia/tratamento farmacológico , Adiponectina/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/patologia , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/patologia , Estudos de Casos e Controles , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/patologia , Feminino , Humanos , Hiperuricemia/sangue , Hiperuricemia/complicações , Hiperuricemia/patologia , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Ácido Úrico/antagonistas & inibidores , Ácido Úrico/sangue , Xantina Oxidase/antagonistas & inibidores , Xantina Oxidase/sangueRESUMO
Cigarette smoking is a major risk factor for acute coronary thrombosis. In fact, both active/first-hand smoke and passive/second-hand smoke exposure are known to increase the risk of coronary thrombosis. Although recently a new risk has been identified and termed third-hand smoke (THS), which is the residual tobacco smoke contaminant that remains after a cigarette is extinguished, it remains to be determined whether it can also enhance the risk of thrombogenesis, much like first-hand smoke and second-hand smoke. Therefore, the present studies investigated the impact of THS exposure in the context of platelet biology and related disease states. It was found that THS-exposed mice exhibited an enhanced platelet aggregation and secretion responses as well as enhanced integrin GPIIb-IIIa activation. Furthermore, it was found that THS exposure shortens the tail bleeding time and the occlusion time in a model of thrombosis. Thus, our data demonstrate for the first time (at least in mice) that THS exposure increases the risk of thrombosis-based disease states, which is attributed, at least in part, to their hyperactive platelets.