RESUMO
ABSTRACT: Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.
Assuntos
Anticoagulantes , Coagulação Sanguínea , Modelos Animais de Doenças , Sinergismo Farmacológico , Fator XIIa , Fator XIa , Animais , Fator XIa/antagonistas & inibidores , Fator XIa/metabolismo , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Masculino , Fator XIIa/antagonistas & inibidores , Fator XIIa/metabolismo , Trombose das Artérias Carótidas/prevenção & controle , Trombose das Artérias Carótidas/induzido quimicamente , Trombose das Artérias Carótidas/tratamento farmacológico , Camundongos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Hemorragia/induzido quimicamente , Camundongos Endogâmicos C57BL , Tempo de Tromboplastina ParcialRESUMO
OBJECTIVE: It is unclear if stopping treatment with dabigatran, a new oral anticoagulant (NOAC), induces a paradoxical rebound prothrombotic state. We investigated if short-term (1-3 days) dabigatran cessation is associated with a higher thrombus volume than expected from a simple reversal of the anticoagulant effect. METHODS: Ten-week-old C57Bl/6 mice (n = 338) received one of the following oral treatments: phosphate-buffered saline (PBS), dabigatran for 7 days with or without 1 to 4 day cessation, and aspirin in either a single dose or daily for 7 days. Some of the animals that ceased dabigatran for 1 to 3 days received single-dose aspirin. Thereafter, we induced FeCl3 -mediated carotid thrombosis in 130 mice, after which we performed micro computed tomography thrombus imaging. The other 208 mice underwent coagulation assays or platelet function tests. As an explorative pilot study, we reviewed the medical records of 18 consecutive patients with NOAC cessation-related cerebral infarction in a large acute stroke cohort. RESULTS: We observed a ~ 40% higher volume of carotid thrombus after dabigatran cessation at 1 to 3 days than after vehicle treatment and showed that this effect could be prevented by single-dose aspirin pretreatment. Dabigatran cessation unduly increased platelet aggregability for 2 days after drug cessation, an effect mediated through thrombin or arachidonic acid, which effect was significantly attenuated by single-dose aspirin pretreatment. In patients, short-term (≤ 3 days) cessation of NOAC therapy, compared with longer-term (≥ 5 days) cessation, tended to be associated with relatively high stroke severity. INTERPRETATION: We provide the first preclinical evidence that a rebound prothrombotic state follows short-term cessation of dabigatran therapy. ANN NEUROL 2021;89:444-458.
Assuntos
Antitrombinas/efeitos adversos , Trombose das Artérias Carótidas/diagnóstico por imagem , Dabigatrana/efeitos adversos , Desprescrições , Agregação Plaquetária/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/sangue , Trombofilia/sangue , Idoso , Idoso de 80 Anos ou mais , Animais , Antitrombinas/farmacologia , Ácido Araquidônico/sangue , Aspirina/farmacologia , Trombose das Artérias Carótidas/induzido quimicamente , Trombose das Artérias Carótidas/prevenção & controle , Infarto Cerebral/diagnóstico por imagem , Infarto Cerebral/etiologia , Infarto Cerebral/fisiopatologia , Infarto Cerebral/prevenção & controle , Cloretos/toxicidade , Angiografia por Tomografia Computadorizada , Dabigatrana/farmacologia , Inibidores do Fator Xa/efeitos adversos , Feminino , Compostos Férricos/toxicidade , Humanos , AVC Isquêmico/diagnóstico por imagem , AVC Isquêmico/etiologia , AVC Isquêmico/fisiopatologia , AVC Isquêmico/prevenção & controle , Angiografia por Ressonância Magnética , Masculino , Volume Plaquetário Médio , Camundongos , Noxas/toxicidade , Projetos Piloto , Inibidores da Agregação Plaquetária/farmacologia , Contagem de Plaquetas , Pirazóis/efeitos adversos , Piridonas/efeitos adversos , Rivaroxabana/efeitos adversos , Índice de Gravidade de Doença , Síndrome de Abstinência a Substâncias/etiologia , Síndrome de Abstinência a Substâncias/prevenção & controle , Trombina/metabolismo , Trombofilia/etiologia , Trombofilia/prevenção & controle , Microtomografia por Raio-XRESUMO
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: 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: 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: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.
Assuntos
Indicã/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/sangue , Cinurenina/fisiologia , Terapia de Alvo Molecular , Complicações Pós-Operatórias/enzimologia , Insuficiência Renal Crônica/enzimologia , Trombose/enzimologia , Procedimentos Cirúrgicos Vasculares/efeitos adversos , Animais , Aorta , Lesões das Artérias Carótidas/complicações , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/prevenção & controle , Meios de Cultura/farmacologia , Indução Enzimática/efeitos dos fármacos , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/deficiência , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Insuficiência Renal Crônica/tratamento farmacológico , Tromboplastina/metabolismo , Trombose/sangue , Trombose/etiologia , Trombose/prevenção & controle , Triptofano/metabolismo , Uremia/sangueRESUMO
Growing evidence supports a central role of NADPH oxidases (NOXs) in the regulation of platelets, which are circulating cells involved in both hemostasis and thrombosis. Here, the use of Nox1-/- and Nox1+/+ mice as experimental models of human responses demonstrated a critical role of NOX1 in collagen-dependent platelet activation and pathological arterial thrombosis, as tested in vivo by carotid occlusion assays. In contrast, NOX1 does not affect platelet responses to thrombin and normal hemostasis, as assayed in tail bleeding experiments. Therefore, as NOX1 inhibitors are likely to have antiplatelet effects without associated bleeding risks, the NOX1-selective inhibitor 2-acetylphenothiazine (2APT) and a series of its derivatives generated to increase inhibitory potency and drug bioavailability were tested. Among the 2APT derivatives, 1-(10H-phenothiazin-2-yl)vinyl tert-butyl carbonate (2APT-D6) was selected for its high potency. Both 2APT and 2APT-D6 inhibited collagen-dependent platelet aggregation, adhesion, thrombus formation, superoxide anion generation, and surface activation marker expression, while responses to thrombin or adhesion to fibrinogen were not affected. In vivo administration of 2APT or 2APT-D6 led to the inhibition of mouse platelet aggregation, oxygen radical output, and thrombus formation, and carotid occlusion, while tail hemostasis was unaffected. Differently to in vitro experiments, 2APT-D6 and 2APT displayed similar potency in vivo. In summary, NOX1 inhibition with 2APT or its derivative 2APT-D6 is a viable strategy to control collagen-induced platelet activation and reduce thrombosis without deleterious effects on hemostasis. These compounds should, therefore, be considered for the development of novel antiplatelet drugs to fight cardiovascular diseases in humans.
Assuntos
Trombose das Artérias Carótidas/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , NADPH Oxidase 1/antagonistas & inibidores , Fenotiazinas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Animais , Trombose das Artérias Carótidas/prevenção & controle , Células Cultivadas , Colágeno/metabolismo , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Feminino , Fibrinogênio/metabolismo , Hemorragia/etiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fenotiazinas/efeitos adversos , Fenotiazinas/uso terapêutico , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Trombina/metabolismoRESUMO
OBJECTIVE: Clopidogrel is a commonly used P2Y12 inhibitor to treat and prevent arterial thrombotic events. Clopidogrel is a prodrug that requires bioactivation by CYP (cytochrome P450) enzymes to exert antiplatelet activity. Diabetes mellitus is associated with an increased risk of ischemic events, and impaired ability to generate the active metabolite (AM) from clopidogrel. The objective of this study is to identify the mechanism of clopidogrel resistance in a murine model of diet-induced obesity (DIO). Approach and Results: C57BL/6J mice and IL-1R-/- mice were given high-fat diet for 10 weeks to generate a murine model of diet-induced obesity. Platelet aggregation and carotid arterial thrombosis were assessed in response to clopidogrel treatment. Wild-type DIO mice exhibited resistance to antiplatelet and antithrombotic effects of clopidogrel that was associated with reduced hepatic expression of CYP genes and reduced generation of the AM. IL (Interleukin)-1 receptor-deficient DIO (IL1R-/- DIO) mice showed no resistance to clopidogrel. Lack of resistance was accompanied by increased exposure of the clopidogrel AM. This resistance was also absent when wild-type DIO mice were treated with the conjugate of the clopidogrel AM, DT-678. CONCLUSIONS: These findings indicate that antiplatelet effects of clopidogrel may be impaired in the setting of diabetes mellitus due to reduced prodrug bioactivation related to IL-1 receptor signaling. Therapeutic targeting of P2Y12 in patients with diabetes mellitus using the conjugate of clopidogrel AM may lead to improved outcomes.
Assuntos
Clopidogrel/farmacocinética , Clopidogrel/uso terapêutico , Resistência a Medicamentos , Obesidade/complicações , Receptores de Interleucina-1/fisiologia , Animais , Trombose das Artérias Carótidas/prevenção & controle , Clopidogrel/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Diabetes Mellitus , Dieta Hiperlipídica , Modelos Animais de Doenças , Fibrinolíticos , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/enzimologia , Obesidade/etiologia , Obesidade/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária , Pró-Fármacos/farmacocinética , Pró-Fármacos/uso terapêutico , Receptores de Interleucina-1/deficiênciaRESUMO
Multiple myeloma confers a high risk for vascular thrombosis, a risk that is increased by treatment with immunomodulatory agents. Strikingly, inclusion of the proteasome inhibitor bortezomib reduces thrombotic risk, yet the molecular basis for this observation remains unknown. Here, we show that bortezomib prolongs thrombosis times in the carotid artery photochemical injury assay in normal mice. Cell-based studies show that bortezomib increases expression of the transcription factor Kruppel-like factor 2 (KLF2) in multiple cell types. Global postnatal overexpression of KLF2 (GL-K2-TG) increased time to thrombosis, and global postnatal deletion of KLF2 (GL-K2-KO) conferred an antiparallel effect. Finally, studies in GL-K2-KO mice showed that the thromboprotective effect of bortezomib is KLF2 dependent. These findings identify a transcriptional basis for the antithrombotic effects of bortezomib.
Assuntos
Ácidos Borônicos/farmacologia , Trombose das Artérias Carótidas/prevenção & controle , Citoproteção/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Pirazinas/farmacologia , Animais , Bortezomib , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/genética , Trombose das Artérias Carótidas/patologia , Células Cultivadas , Oclusão Coronária/genética , Oclusão Coronária/patologia , Oclusão Coronária/prevenção & controle , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tempo de Coagulação do Sangue TotalRESUMO
Carotid endarterectomy is a commonly performed procedure for prevention of stroke related to carotid stenosis. Intraoperative sonography is used to identify potentially correctable technical defects during carotid endarterectomy. The main risk of endarterectomy is perioperative stroke, and great effort has been put into trying to reduce this risk through various surgical techniques and evaluation of the surgical bed. Postoperative carotid thrombosis, or thombo-embolization from the arterectomy site, remains a common cause of perioperative stroke and is often related to technical defects in the arterial reconstruction procedure. Re-exploration and repair of any imperfections have the potential to improve outcomes. Intraoperative imaging can identify potentially occult lesions, provide the option for correction, and thus reduce chance of stroke. Familiarity with the spectrum of intraoperative sonographic findings helps correctly identify residual intimal dissection flaps, plaque, thrombi, and stenosis, which may require immediate surgical revision. Our objective is to illustrate the spectrum of intraoperative findings and their importance.
Assuntos
Trombose das Artérias Carótidas/diagnóstico por imagem , Trombose das Artérias Carótidas/etiologia , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas/efeitos adversos , Ultrassonografia Doppler Dupla/métodos , Trombose das Artérias Carótidas/prevenção & controle , Endarterectomia das Carótidas/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Cirurgia Assistida por Computador/métodos , Resultado do TratamentoRESUMO
The most serious complication of carotid artery stenting (CAS) is acute carotid artery stent thrombosis (ACAST). ACAST is a very rare complication, but it may lead to dramatic and catastrophic consequences. The most important cause is inadequate or ineffective antiaggregant therapy. It is very important to identify, before CAS, those patients who might be candidates for ACAST and to start antiplatelet therapy for them. Testing patients who are candidates for CAS for acetylsalicylic acid and clopidogrel resistance may prevent this complication.
Assuntos
Aspirina/uso terapêutico , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/prevenção & controle , Inibidores da Agregação Plaquetária/uso terapêutico , Stents/efeitos adversos , Ticlopidina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Trombose das Artérias Carótidas/complicações , Angiografia Cerebral , Clopidogrel , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ticlopidina/uso terapêuticoRESUMO
Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl(3)-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein α(IIb)/ß(3), in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A(2A) or A(2B) adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.
Assuntos
Antígenos CD/fisiologia , Apirase/fisiologia , Trombose das Artérias Carótidas/prevenção & controle , Adenosina/fisiologia , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Trombose das Artérias Carótidas/induzido quimicamente , Trombose das Artérias Carótidas/patologia , Células Cultivadas , Cloretos , Compostos Férricos , Camundongos , Camundongos Transgênicos , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Antithrombotic activities of enoxifol, a new antioxidant with antiaggregant activity demonstrated in vitro and in vivo, and antioxidant mexidol were compared on the rat model of arterial thrombosis induced by application of 50% ferric chloride. Acetylsalicylic acid (antiaggregant) served as the reference drug. All drugs exhibited dose-dependent antithrombotic activity. Enoxifol was more effective than mexidol, both drugs being more active than the reference drug (acetylsalicylic acid). Taking into account the pathogenesis of the thrombosis in this experimental model, we can hypothesize that the pronounced antithrombotic effect of enoxifol was due to its antiaggregant and antioxidant activities.
Assuntos
Anticoagulantes/farmacologia , Antioxidantes/farmacologia , Benzimidazóis/farmacologia , Picolinas/farmacologia , Animais , Animais não Endogâmicos , Aspirina/farmacologia , Trombose das Artérias Carótidas/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Masculino , Agregação Plaquetária/efeitos dos fármacos , RatosRESUMO
OBJECTIVE: Plant-derived α-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown. METHODS AND RESULTS: Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-κB activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells. CONCLUSIONS: ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.
Assuntos
Cardiotônicos/administração & dosagem , Trombose das Artérias Carótidas/prevenção & controle , Ativação Plaquetária/efeitos dos fármacos , Tromboplastina/metabolismo , Ácido alfa-Linolênico/administração & dosagem , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/metabolismo , Células Cultivadas , Suplementos Nutricionais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Tromboplastina/genética , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Heparin-like glycans with diverse disaccharide composition and high anticoagulant activity have been described in several families of marine mollusks. The present work focused on the structural characterization of a new heparan sulfate (HS)-like polymer isolated from the mollusk Nodipecten nodosus (Linnaeus, 1758) and on its anticoagulant and antithrombotic properties. Total glycans were extracted from the mollusk and fractionated by ethanol precipitation. The main component (>90%) was identified as HS-like glycosaminoglycan, representing approximately 4.6 mg g(-1) of dry tissue. The mollusk HS resists degradation with heparinase I but is cleaved by nitrous acid. Analysis of the mollusk glycan by one-dimensional (1)H, two-dimensional correlated spectroscopy, and heteronuclear single quantum coherence nuclear magnetic resonance revealed characteristic signals of glucuronic acid and glucosamine residues. Signals corresponding to anomeric protons of nonsulfated, 3- or 2-sulfated glucuronic acid as well as N-sulfated and/or 6-sulfated glucosamine were also observed. The mollusk HS has an anticoagulant activity of 36 IU mg(-1), 5-fold lower than porcine heparin (180 IU mg(-1)), as measured by the activated partial thromboplastin time assay. It also inhibits factor Xa (IC(50) = 0.835 microg ml(-1)) and thrombin (IC(50) = 9.3 microg ml(-1)) in the presence of antithrombin. In vivo assays demonstrated that at the dose of 1 mg kg(-1), the mollusk HS inhibited thrombus growth in photochemically injured arteries. No bleeding effect, factor XIIa-mediated kallikrein activity, or toxic effect on fibroblast cells was induced by the invertebrate HS at the antithrombotic dose.
Assuntos
Anticoagulantes/química , Anticoagulantes/metabolismo , Artérias , Trombose das Artérias Carótidas/prevenção & controle , Endotélio Vascular , Matriz Extracelular/química , Proteoglicanas de Heparan Sulfato/metabolismo , Proteoglicanas de Heparan Sulfato/uso terapêutico , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/uso terapêutico , Antitrombinas/metabolismo , Artérias/efeitos dos fármacos , Artérias/patologia , Artérias/efeitos da radiação , Bivalves/metabolismo , Configuração de Carboidratos , Linhagem Celular , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/efeitos da radiação , Matriz Extracelular/metabolismo , Fator Xa/metabolismo , Inibidores do Fator Xa , Feminino , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/isolamento & purificação , Heparina/metabolismo , Heparina/uso terapêutico , Cofator II da Heparina/metabolismo , Heparina Liase/metabolismo , Humanos , Masculino , Ácido Nitroso/metabolismo , Ratos , Análise Espectral/métodos , Suínos , Trombina/antagonistas & inibidores , Trombina/metabolismoRESUMO
BACKGROUND: In patients with carotid artery disease, carotid endarterectomy (CEA) and carotid stenting (CAS) are treatment options. Controversy exists as to the relative efficacy of the 2 techniques in preventing late events. METHODS AND RESULTS: The Reduction of Atherothrombosis for Continued Health (REACH) Registry recruited > 68,000 outpatients ≥ 45 years of age with established atherothrombotic disease or ≥ 3 risk factors for atherothrombosis. Patients with CAS or CEA were chosen and followed up prospectively for the occurrence of cardiovascular events. Propensity score matching was performed to assemble a cohort of patients in whom all baseline covariates would be well balanced. Primary outcome was defined as death or stroke at the 2-year follow-up. Secondary outcome was stroke or transient ischemic attack. Tertiary outcome was a composite of death, myocardial infarction, or stroke and the individual outcomes. Of the 68 236 patients with atherothrombosis, 3412 patients (5%) had a history of carotid artery revascularization (70% asymptomatic carotid stenosis), 1025 (30%) with CAS and 2387 (70%) with CEA. Propensity score analyses matched 836 CAS patients with 836 CEA patients. At the end of 2 years of follow-up, in the propensity score-matched cohort, CAS was associated with a risk similar to CEA for the primary (hazard ratio [HR], 0.85; 95% confidence interval [CI], 0.57 to 1.26), secondary (HR, 1.20; 95% CI, 0.73 to 1.96), and tertiary (HR, 0.72; 95% CI, 0.51 to 1.01) composite outcome, death (HR, 0.63; 95% CI, 0.40 to 1.00), and stroke (HR, 1.48; 95% CI, 0.79 to 2.80). CONCLUSIONS: In a real-world cohort of patients with a history of carotid artery revascularization, CAS was comparable to CEA for late outcomes.
Assuntos
Doenças das Artérias Carótidas/terapia , Endarterectomia das Carótidas , Pontuação de Propensão , Stents , Idoso , Idoso de 80 Anos ou mais , Doenças das Artérias Carótidas/complicações , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/prevenção & controle , Estudos de Coortes , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sistema de Registros , Resultado do TratamentoRESUMO
ADAMTS13, a metalloprotease primarily synthesized in liver and endothelial cells, cleaves von Willebrand factor (VWF) at the central A2 domain, thereby reducing the sizes of circulating VWF multimers. Genetic or acquired deficiency of plasma ADAMTS13 activity leads to a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). To date, plasma infusion or exchange is the only proven effective therapy for TTP. In search for a better therapy, an autologous transplantation of hematopoietic progenitor cells transduced ex vivo with a self-inactivating lentiviral vector encoding a full-length murine Adamts13 and an enhanced green fluorescent protein (GFP) reporter gene was performed in Adamts13(-/-) mice after irradiation. All recipient mice showed detectable ADAMTS13 antigen and proteolytic activity in plasma despite only low levels of bone marrow chimerism. The levels of plasma ADAMTS13 were sufficient to eliminate the ultralarge VWF multimers and offered systemic protection against ferric chloride-induced arterial thrombosis. The data suggest that hematopoietic progenitor cells can be genetically modified ex vivo and transplanted in an autologous model to provide adequate levels of functional ADAMTS13 metalloprotease. This success may provide the basis for development of a novel therapeutic strategy to cure hereditary TTP in humans.
Assuntos
Proteínas ADAM/deficiência , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Púrpura Trombocitopênica Trombótica/terapia , Proteínas ADAM/genética , Proteína ADAMTS13 , Animais , Western Blotting , Trombose das Artérias Carótidas/prevenção & controle , Citometria de Fluxo , Vetores Genéticos , Humanos , Imuno-Histoquímica , Lentivirus/genética , Camundongos , Reação em Cadeia da Polimerase , Púrpura Trombocitopênica Trombótica/genética , Transdução Genética , Fator de von Willebrand/análiseRESUMO
Inhibition of platelet aggregation is indispensable for the treatment of acute arterial thrombotic episodes. We have previously reported the synthesis of a highly constrained cyclic peptide, that incorporates the -CDC- sequence, (S,S) PSRCDCR-NH(2), which potently inhibits aggregation and fibrinogen binding to human platelets in vitro. We have tested the safety and efficacy of the peptide on the electrically induced carotid artery thrombosis experimental rabbit model. The peptide's effects on carotid blood flow, thrombus weight, in vitro and ex vivo platelet aggregation, and bleeding and hemostatic parameters were evaluated. The peptide was administered via the femoral vein. Carotid blood flow was continuously monitored for 90 min after electrical thrombus formation. The peptide, at 12 mg/kg, prevented total artery occlusion and significantly preserved carotid artery's patency compared with placebo and eptifibatide. Furthermore, (S,S) PSRCDCR-NH(2) administration at 12 mg/kg reduced thrombus weight, whereas it inhibited ex vivo ADP, arachidonic acid (AA) and collagen-induced platelet aggregation. Moreover (S,S) PSRCDCR-NH(2) at 12 mg/kg presented significantly higher inhibitory effects on AA and collagen-induced ex vivo platelet aggregation compared to eptifibatide. The peptide at any dose did not affect the coagulation cascade, the bleeding times or the hemostatic response of the animals. Thus highly constrained cyclic peptides like (S,S) PSRCDCR-NH(2) that incorporate the -CDC- motif and fulfil certain conformational criteria represent novel compounds that potently inhibit thrombus formation, ex vivo platelet aggregation and carotid artery occlusion superiorly to other non-RGD peptides, such as YMESRADR, without causing hemorrhagic complications in a rabbit model of arterial thrombosis.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Trombose das Artérias Carótidas , Peptídeos Cíclicos/uso terapêutico , Inibidores da Agregação Plaquetária/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Trombose , Difosfato de Adenosina/antagonistas & inibidores , Difosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/farmacologia , Tempo de Sangramento , Artérias Carótidas/patologia , Trombose das Artérias Carótidas/tratamento farmacológico , Trombose das Artérias Carótidas/patologia , Trombose das Artérias Carótidas/prevenção & controle , Colágeno/antagonistas & inibidores , Colágeno/farmacologia , Modelos Animais de Doenças , Estimulação Elétrica , Eptifibatida , Humanos , Masculino , Dados de Sequência Molecular , Peptídeos/farmacologia , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/síntese química , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/síntese química , Coelhos , Trombose/tratamento farmacológico , Trombose/prevenção & controleRESUMO
BACKGROUND: The phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation. METHODS AND RESULTS: Guggulsterone inhibited TNF-alpha-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-alpha-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-alpha-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner. CONCLUSIONS: Guggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.
Assuntos
Anti-Inflamatórios/farmacologia , Trombose das Artérias Carótidas/prevenção & controle , Fitosteróis/farmacologia , Pregnenodionas/farmacologia , Tromboplastina/efeitos dos fármacos , Animais , Western Blotting , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 4/efeitos dos fármacos , MAP Quinase Quinase 4/metabolismo , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tromboplastina/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: In patients with coronary artery disease and reduced ejection fraction, amiodarone reduces mortality by decreasing sudden death. Because the latter may be triggered by coronary artery thrombosis as much as ventricular arrhythmias, amiodarone might interfere with tissue factor (TF) expression and thrombus formation. METHODS AND RESULTS: Clinically relevant plasma concentrations of amiodarone reduced TF activity and impaired carotid artery thrombus formation in a mouse photochemical injury model in vivo. PTT, aPTT, and tail bleeding time were not affected; platelet number was slightly decreased. In human endothelial and vascular smooth muscle cells, amiodarone inhibited tumor necrosis factor (TNF)-alpha and thrombin-induced TF expression as well as surface activity. Amiodarone lacking iodine and the main metabolite of amiodarone, N-monodesethylamiodarone, inhibited TF expression. Amiodarone did not affect mitogen-activated protein kinase activation, TF mRNA expression, and TF protein degradation. Metabolic labeling confirmed that amiodarone inhibited TF protein translation. CONCLUSIONS: Amiodarone impairs thrombus formation in vivo; in line with this, it inhibits TF protein expression and surface activity in human vascular cells. These pleiotropic actions occur within the range of amiodarone concentrations measured in patients, and thus may account at least in part for its beneficial effects in patients with coronary artery disease.