RESUMO
Thrombopoietin receptor agonist (TPO-RA) treatment increases the thrombosis rate in immune thrombocytopenia (ITP). We hypothesize that TPO-RAs influence platelet function, global and secondary hemostasis and/or fibrinolysis. A systematic review was performed. If possible, data were compared between responders (relevant increase in platelet count), and non-responders. Twelve observational studies with 305 patients were included (responders (127/150 (85%))). There were indications that TPO-RA treatment enhanced platelet function, with respect to platelet-monocyte aggregates, soluble P-selectin, GPVI expression, and adhesion under flow. Studies addressing global and secondary hemostasis and fibrinolysis were scarce. Overall, no changes were found during TPO-RA treatment, apart from an accelerated clot formation and conflicting data on levels of plasminogen activator inhibitor (PAI)-1. The parameters that increased have previously been associated with thrombosis in other patient groups, and might contribute to the increased rate of thrombosis observed in TPO-RA-treated ITP patients.
Assuntos
Plaquetas/metabolismo , Hemostáticos/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Púrpura Trombocitopênica Idiopática , Receptores de Trombopoetina/agonistas , Plaquetas/patologia , Fibrinólise/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hemostáticos/efeitos adversos , Humanos , Monócitos/metabolismo , Monócitos/patologia , Selectina-P/biossíntese , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Glicoproteínas da Membrana de Plaquetas/biossíntese , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/metabolismo , Púrpura Trombocitopênica Idiopática/patologia , Receptores de Trombopoetina/metabolismo , Trombose/induzido quimicamente , Trombose/metabolismo , Trombose/patologiaRESUMO
BACKGROUND: For patients treated with dual antiplatelet therapy, standardized drug-specific 3-to-7 day cessation is recommended prior to major surgery to reach sufficient platelet function recovery. Here we investigated the hypothesis that supplemental fibrinogen might mitigate the inhibitory effects of antiplatelet therapy. METHODS AND RESULTS: To this end blood from healthy donors was treated in vitro with platelet inhibitors, and in vitro thrombus formation and platelet activation were assessed. Ticagrelor, acetylsalicylic acid, the combination of both, and tirofiban all markedly attenuated the formation of adherent thrombi, when whole blood was perfused through collagen-coated microchannels at physiological shear rates. Addition of fibrinogen restored in vitro thrombus formation in the presence of antiplatelet drugs and heparin. However, platelet activation, as investigated in assays of P-selectin expression and calcium flux, was not altered by fibrinogen supplementation. Most importantly, fibrinogen was able to restore in vitro thrombogenesis in patients on maintenance dual antiplatelet therapy after percutaneous coronary intervention. CONCLUSION: Thus, our in vitro data support the notion that supplementation of fibrinogen influences the perioperative hemostasis in patients undergoing surgery during antiplatelet therapy by promoting thrombogenesis without significantly interfering with platelet activation.
Assuntos
Fibrinogênio/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Trombose/prevenção & controle , Idoso , Aspirina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Feminino , Hemorreologia , Heparina/farmacologia , Hirudinas/farmacologia , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Selectina-P/biossíntese , Selectina-P/genética , Ticagrelor/farmacologia , Tirofibana/farmacologiaRESUMO
Arsenic, an environmental contaminant in drinking water worldwide is well-established to increase cardiovascular diseases (CVDs) in humans. Of these, thrombotic events represent a major adverse effect associated with arsenic exposure, for which an abundance of epidemiological evidence exists. Platelet aggregation constitutes a pivotal step in thrombosis but arsenic alone doesn't induce aggregation and the mechanism underlying arsenic-induced thrombosis still remains unclear. Here we demonstrated that arsenic induces morphological changes of platelets, i.e., contraction and pseudopod projection, the primal events of platelet activation, which can increase platelet reactivity. Arsenite induced prominent platelet shape changes in a dose-dependent manner in freshly isolated human platelets. Of note, arsenite suppressed focal adhesion kinase (FAK) activity, which in turn activated RhoA, leading to altered actin assembly through LIMK activation, and subsequent cofilin inactivation. Arsenic-induced platelet shape change appeared to increase the sensitivity to thrombin and ADP-induced aggregation. Supporting this, latrunculin A, an inhibitor of actin-dynamics abolished it. Taken together, we demonstrated that arsenic induces cytoskeletal changes and shape changes of platelets through FAK-mediated alteration of actin dynamics, which renders platelets reactive to activating stimuli, ultimately contributing to increased thrombosis.
Assuntos
Actinas/metabolismo , Arsenitos/toxicidade , Plaquetas/patologia , Plaquetas/ultraestrutura , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Compostos de Sódio/toxicidade , Difosfato de Adenosina/farmacologia , Adolescente , Adulto , Humanos , Técnicas In Vitro , Quinases Lim/antagonistas & inibidores , Masculino , Selectina-P/biossíntese , Agregação Plaquetária/efeitos dos fármacos , Adulto Jovem , Proteína rhoA de Ligação ao GTPRESUMO
Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 µg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.
Assuntos
Difosfato de Adenosina/farmacologia , Diarilquinolinas/farmacologia , Infecções por HIV/sangue , Fosfatidilinositol 3-Quinases/metabolismo , Ativação Plaquetária , Infecções Pneumocócicas/sangue , Tuberculose/sangue , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Adulto , Antituberculosos/efeitos adversos , Antituberculosos/farmacologia , Sinalização do Cálcio , Diarilquinolinas/efeitos adversos , Relação Dose-Resposta a Droga , Estrenos/farmacologia , Feminino , Infecções por HIV/fisiopatologia , Humanos , Síndrome do QT Longo/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Selectina-P/biossíntese , Selectina-P/genética , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Plasma Rico em Plaquetas , Infecções Pneumocócicas/fisiopatologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirrolidinonas/farmacologia , Trombina/farmacologia , Tuberculose/fisiopatologia , Wortmanina/farmacologia , Adulto JovemRESUMO
Objective- Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 ( ENTPD1 or Cd39), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results- CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39-null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39-deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39-null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions- These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.
Assuntos
Antígenos CD/fisiologia , Apirase/fisiologia , Quimiotaxia de Leucócito/fisiologia , Hemorreologia , Vasculite/enzimologia , Trombose Venosa/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/genética , Apirase/deficiência , Apirase/genética , Plaquetas/fisiologia , Adesão Celular , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Selectina-P/biossíntese , Selectina-P/genética , Receptores Purinérgicos P2Y1/metabolismo , Vasculite/fisiopatologia , Veia Cava Inferior , Trombose Venosa/fisiopatologia , Fator de von Willebrand/biossíntese , Fator de von Willebrand/genéticaRESUMO
Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses.
Assuntos
Células Endoteliais/metabolismo , Janus Quinase 2/biossíntese , Selectina-P/biossíntese , Trombose/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Janus Quinase 2/genética , Camundongos , Camundongos Transgênicos , Selectina-P/genética , Trombose/tratamento farmacológico , Trombose/genética , Trombose/patologiaRESUMO
Platelet transfusions may be associated with certain adverse effects in recipients, potentially caused by the presence of biological response modifiers contained in the platelet concentrates. The aim of this study is to identify the parameters that reflect platelet activation during both the preparation process and the storage of platelet concentrates. A total of 3,949apheresis platelet concentrate samples were studied with regard to parameters related to the donor as well as to the preparation process and their storage. Key glycoproteins characteristic of platelet activation, i.e. soluble CD40L and CD62P, were quantified in platelet concentrate supernatants on completion of their processing and during storage, using Luminex technology. We observed an increase in soluble factors over time. However, the different parameters studied in connection either with the donors or with the donations, such as (i) donor gender, (ii) donor blood group, (iii) time of collection and (iv) type of apheresis separator, do not seem to have any effect on platelet activation or the release of soluble CD40L and CD62P.
Assuntos
Plaquetas , Preservação de Sangue , Ligante de CD40/análise , Selectina-P/análise , Ativação Plaquetária , Transfusão de Plaquetas , Plaquetoferese , Doadores de Sangue , Plaquetas/metabolismo , Ligante de CD40/biossíntese , Feminino , Humanos , Masculino , Selectina-P/biossíntese , Fatores de TempoRESUMO
BACKGROUND: Regardless of different sources, methods or devices which are applied for preparation of therapeutic platelets, these products are generally isolated from whole blood by the sedimentation techniques which are based on PRP or buffy coat (BC) separation. As a general fact, platelet preparation and storage are also associated with some deleterious changes that known as platelet storage lesion (PSL). Although these alternations in platelet functional activity are aggravated during storage, whether technical issues within preparation can affect integrin activation and platelet adhesion to fibrinogen were investigated in this study. METHODS: PRP- and BC-platelet concentrates (PCs) were subjected to flowcytometry analysis to examine the expression of platelet activation marker, P-selectin as well as active confirmation of the GPIIb/IIIa (αIIbß3) on day 0, 1, 3 and 5 post-storage. Platelet adhesion to fibrinogen matrix was evaluated by fluorescence microscopy. Glucose concentration and LDH activity were also measured by colorimetric methods. RESULTS: The increasing P-selectin expression during storage was in a reverse correlation with PAC-1 binding (râ¯=â¯-0.67; pâ¯=â¯.001). PRP-PCs showed the higher level of P-selectin expression than BC-PCs, whereas the levels of PAC-1 binding and platelet adhesion to fibrinogen matrix were significantly lower in PRP-PCs. Higher levels of active confirmation of the GPIIb/IIIa in BC-PCs were also associated with greater concentration of glucose in these products. CONCLUSION: We demonstrated the superior capacities of integrin activation and adhesion to fibrinogen for BC-PCs compared to those of PRP-PCs. These findings may provide more advantages for BC method of platelet preparation.
Assuntos
Buffy Coat , Preservação de Sangue , Integrina beta3/biossíntese , Selectina-P/biossíntese , Adesividade Plaquetária , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Masculino , CamundongosRESUMO
BACKGROUND AND AIMS: ß-Caryophyllene (BCP) is a plant-derived FDA approved food additive with anti-inflammatory properties. Some of its beneficial effects in vivo are reported to involve activation of cannabinoid CB2 receptors that are predominantly expressed in immune cells. Here, we evaluated the translational potential of BCP using a well-established model of chronic and binge alcohol-induced liver injury. METHODS: In this study, we investigated the effects of BCP on liver injury induced by chronic plus binge alcohol feeding in mice in vivo by using biochemical assays, real-time PCR and histology analyses. Serum and hepatic BCP levels were also determined by GC/MS. RESULTS: Chronic treatment with BCP alleviated the chronic and binge alcohol-induced liver injury and inflammation by attenuating the pro-inflammatory phenotypic `M1` switch of Kupffer cells and by decreasing the expression of vascular adhesion molecules intercellular adhesion molecule 1, E-Selectin and P-Selectin, as well as the neutrophil infiltration. It also beneficially influenced hepatic metabolic dysregulation (steatosis, protein hyperacetylation and PPAR-α signalling). These protective effects of BCP against alcohol-induced liver injury were attenuated in CB2 receptor knockout mice, indicating that the beneficial effects of this natural product in liver injury involve activation of these receptors. Following acute or chronic administration, BCP was detectable both in the serum and liver tissue homogenates but not in the brain. CONCLUSIONS: Given the safety of BCP in humans, this food additive has a high translational potential in treating or preventing hepatic injury associated with oxidative stress, inflammation and steatosis. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Etanol/toxicidade , Fígado Gorduroso/tratamento farmacológico , Inflamação/tratamento farmacológico , Sesquiterpenos/uso terapêutico , Acetilação/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Selectina E/biossíntese , Etanol/farmacocinética , Fígado Gorduroso/induzido quimicamente , Molécula 1 de Adesão Intercelular/biossíntese , Células de Kupffer/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Selectina-P/biossíntese , PPAR alfa/metabolismo , Sesquiterpenos Policíclicos , Receptor CB2 de Canabinoide/genética , Sesquiterpenos/sangue , Sesquiterpenos/farmacocinéticaRESUMO
BACKGROUND: Platelets (PLTs) are prone to activation and the release of biologic response modifiers (BRMs) under storage conditions. The transfusion inflammatory reaction in the vascular compartment involves endothelial cell activation due to cell-cell interactions and BRMs infused with the blood products. Endocan/ESM-1 is a proteoglycan secreted by endothelial cells under the control of proinflammatory cytokines. We aimed to measure endocan activity in supernatants of PLT components (PCs), implicated in serious adverse reactions (SARs) or not (no.AR), sampled at different stages during storage. STUDY DESIGN AND METHODS: PLT function, by quantification of soluble CD62P, and their ability to produce endocan were assessed. Functional testing of PC supernatants was performed on EA.hy926 endothelial cells in vitro by exposing them to PC supernatants from each group (no.AR or SARs); EA.hy926 activation was evaluated by their production of interleukin (IL)-6 and endocan. RESULTS: PLT endocan secretion was not induced in response to PLT surface molecule agonists, and no significant correlation was observed between sCD62P and endocan concentration after PLT activation. However, we observed a significant increase in the secretion of IL-6 and endocan after EA.hy926 activation by all PC supernatants. IL-6 and endocan secretion were significantly higher for cells stimulated with SAR than those stimulated with no.AR PC supernatants, as well as cell apoptosis. CONCLUSION: The correlation between the secretion of endocan and that of IL-6 by endothelial cells suggests that endocan can be used as a predictive marker of inflammation for the quality assessment of transfusion grade PLTs.
Assuntos
Plaquetas/metabolismo , Células Endoteliais/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Selectina-P/biossíntese , Proteoglicanas/metabolismo , Biomarcadores/metabolismo , Plaquetas/citologia , Técnicas de Cocultura , Células Endoteliais/citologia , Humanos , Interleucina-6/biossíntese , Transfusão de PlaquetasRESUMO
OBJECTIVES: The objective of this study was to test platelet function pre- and peri-operatively in clopidogrel-treated patients undergoing transurethral resection of the prostate. METHODS: This was a pilot study involving 20 male patients treated with clopidogrel (75 mg/day) for the secondary prevention of cardiovascular disease and scheduled for elective transurethral resection of the prostate. Platelet function testing with light transmittance aggregometry in platelet-rich plasma of four samples (T0, T1, T2, and T3 drawn on the same day, 3 and 7 days of clopidogrel cessation and 24-h post-operatively, respectively) was performed and evaluated in each patient. P-selectin membrane expression was evaluated using monoclonal antibodies. RESULTS: The platelet response to adenosine diphosphate 5 µΜ and 20 µΜ at T0 were 42 ± 15 and 60 ± 14%, respectively. After discontinuation of clopidogrel, corresponding maximum aggregation values at T1 were 60 ± 16 and 74 ± 14%, and increased to 69 ± 16 and 79 ± 18% at T2. No significant difference in platelet aggregation values were noted between T1 and T2, while similar aggregation values were recorded at T3. CONCLUSIONS: Our findings indicate that in patients undergoing transurethral resection of the prostate, platelet activation is similar 3 and 7 days from clopidogrel cessation. These results may be of relevance in subjects at increased thrombotic risk prior to a surgical procedure carrying a high-bleeding risk.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Hiperplasia Prostática/cirurgia , Ticlopidina/análogos & derivados , Ressecção Transuretral da Próstata/métodos , Idoso , Doenças Cardiovasculares/sangue , Clopidogrel , Humanos , Masculino , Selectina-P/biossíntese , Projetos Piloto , Agregação Plaquetária , Inibidores da Agregação Plaquetária/administração & dosagem , Testes de Função Plaquetária , Medicina de Precisão/métodos , Hiperplasia Prostática/sangue , Ticlopidina/administração & dosagemRESUMO
BACKGROUND AND PURPOSE: The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. METHODS: We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia-ischemia- and transient hypoxia-ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia-ischemia-injured sickle mice. RESULTS: Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia-ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia-ischemia. Yet, post-transient hypoxia-ischemia treatment with tPA reduced thrombosis and mortality in SS mice. CONCLUSIONS: Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy.
Assuntos
Anemia Falciforme/complicações , Fibrinolíticos/uso terapêutico , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/uso terapêutico , Anemia Falciforme/diagnóstico por imagem , Animais , Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/fisiopatologia , Infarto Cerebral/diagnóstico por imagem , Infarto Cerebral/genética , Circulação Cerebrovascular , Masculino , Camundongos , Selectina-P/biossíntese , Selectina-P/genética , Adesividade Plaquetária , Serpina E2/biossíntese , Serpina E2/genética , Acidente Vascular Cerebral/diagnóstico por imagem , Ultrassonografia DopplerRESUMO
OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbß3 (αIIbß3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbß3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbß3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbß3, and P-selectin.
Assuntos
Plaquetas , Doenças do Cão/sangue , Insuficiência Renal Crônica/veterinária , Animais , Doenças do Cão/fisiopatologia , Cães , Fibrinogênio/metabolismo , Citometria de Fluxo/veterinária , Selectina-P/biossíntese , Tempo de Tromboplastina Parcial , Ativação Plaquetária , Testes de Função Plaquetária/veterinária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Tempo de Protrombina/veterinária , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/fisiopatologia , Tromboelastografia/veterinária , Trombofilia/veterináriaRESUMO
Metastatic dissemination of cancer cells is one of the hallmarks of malignancy and accounts for approximately 90 % of human cancer deaths. Within the blood vasculature, tumor cells may aggregate with platelets to form clots, adhere to and spread onto endothelial cells, and finally extravasate to form metastatic colonies. We have previously shown that sphingolipids play a central role in the interaction of tumor cells with platelets; this interaction is a prerequisite for hematogenous tumor metastasis in at least some tumor models. Here we show that the interaction between melanoma cells and platelets results in rapid and transient activation and secretion of acid sphingomyelinase (Asm) in WT but not in P-selectin-deficient platelets. Stimulation of P-selectin resulted in activation of p38 MAPK, and inhibition of p38 MAPK in platelets prevented the secretion of Asm after interaction with tumor cells. Intravenous injection of melanoma cells into WT mice resulted in multiple lung metastases, while in P-selectin-deficient mice pulmonary tumor metastasis and trapping of tumor cells in the lung was significantly reduced. Pre-incubation of tumor cells with recombinant ASM restored trapping of B16F10 melanoma cells in the lung in P-selectin-deficient mice. These findings indicate a novel pathway in tumor metastasis, i.e., tumor cell mediated activation of P-selectin in platelets, followed by activation and secretion of Asm and in turn release of ceramide and tumor metastasis. The data suggest that p38 MAPK acts downstream from P-selectin and is necessary for the secretion of Asm.
Assuntos
Melanoma Experimental/genética , Selectina-P/genética , Esfingomielina Fosfodiesterase/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Plaquetas , Ceramidas/biossíntese , Humanos , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Camundongos , Metástase Neoplásica , Selectina-P/biossíntese , Agregação Plaquetária/genética , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismoRESUMO
We demonstrated similarities and differences in the effects of IFN-α and IFN-ß compared to IFN-γ on the production of factors deposited in the Weibel-Palade bodies in cultures of endothelial cells (intact and infected with herpes simplex virus 1). IFN-α and IFN-ß reduced the content of von Willebrand factor, endothelin-1, and soluble P-selectin and increased IL-8 concentration in the culture medium of human umbilical vein endothelial cells. IFN-γ reduced the content of all studied factors in the endothelial cell culture medium. Possible mechanisms of these effects are discussed.
Assuntos
Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interferons/fisiologia , Corpos de Weibel-Palade/metabolismo , Células Cultivadas , Endotelina-1/biossíntese , Herpes Simples/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Interleucina-8/biossíntese , Selectina-P/biossíntese , Fator de von Willebrand/biossínteseRESUMO
Statins have beneficial effects on cerebral circulation and brain parenchyma during ischemic stroke and reperfusion. The primary hypothesis of this randomized parallel trial was that treatment with 80âmg/day of atorvastatin administered early at admission after acute atherosclerotic ischemic stroke could reduce serum levels of markers of immune-inflammatory activation of the acute phase and that this immune-inflammatory modulation could have a possible effect on prognosis of ischemic stroke evaluated by some outcome indicators. We enrolled 42 patients with acute ischemic stroke classified as large arteries atherosclerosis stroke (LAAS) randomly assigned in a randomized parallel trial to the following groups: Group A, 22 patients treated with atorvastatin 80âmg (once-daily) from admission day until discharge; Group B, 20 patients not treated with atorvastatin 80âmg until discharge, and after discharge, treatment with atorvastatin has been started. At 72 hours and at 7 days after acute ischemic stroke, subjects of group A showed significantly lower plasma levels of tumor necrosis factor-α, interleukin (IL)-6, vascular cell adhesion molecule-1, whereas no significant difference with regard to plasma levels of IL-10, E-Selectin, and P-Selectin was observed between the 2 groups. At 72âhours and 7 days after admission, stroke patients treated with atorvastatin 80âmg in comparison with stroke subjects not treated with atorvastatin showed a significantly lower mean National Institutes of Health Stroke Scale and modified Rankin scores. Our findings provide the first evidence that atorvastatin acutely administered immediately after an atherosclerotic ischemic stroke exerts a lowering effect on immune-inflammatory activation of the acute phase of stroke and that its early use is associated to a better functional and prognostic profile.
Assuntos
Atorvastatina/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Inflamação/tratamento farmacológico , Acidente Vascular Cerebral/tratamento farmacológico , Doença Aguda , Idoso , Atorvastatina/farmacologia , Biomarcadores , Isquemia Encefálica/etiologia , Isquemia Encefálica/fisiopatologia , Selectina E/biossíntese , Feminino , Humanos , Inflamação/fisiopatologia , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-10/biossíntese , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Arteriosclerose Intracraniana/complicações , Masculino , Pessoa de Meia-Idade , Selectina-P/biossíntese , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/fisiopatologia , Fator de Necrose Tumoral alfa/biossíntese , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
We explored the synergistic effect of serine combined with several selenocompounds or used alone on the expression of selenoprotein P (SelP) and glutathione peroxidase (GPx) in this study. We first compared the SelP and GPx expression difference between HepG2 and Hela cells treated with serine and finally chose HepG2 as experimental cell. In the serine-used-alone experiment, three kinds of selenium nutritional models (low-, adequate-, and high-selenium) were established and serine was 10 times gradient diluted (0.01 to 100 µmol/L). In the combined experiment, the selenocompound doses were set as 0.01, 0.1, and 1 µmol Se/L and serine was set according to its molar ratio with the selenocompounds. We found that SelP and GPx concentrations in the low-, adequate-, and high-selenium models increased following with serine dose. When the concentration of sodium selenite and SeMet was 1 µmol Se/L while MeSeCys was 0.1 and 1 µmol Se/L, SelP concentrations for serine combined with selenocompounds groups were significantly higher than that of selenocompounds used alone. When the concentration of sodium selenite was 0.1 µmol Se/L, SeMet was 0.1 and 1 µmol Se/L while MeSeCys was 0.01 and 1 µmol Se/L, GPx concentrations for serine combined with selenocompounds groups were significantly higher than that of selenocompounds used alone. Our preliminary result indicated the beneficial effect of serine on the expression of SelP and GPx, which suggested that it might be a candidate for combined selenium supplement.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/biossíntese , Proteínas de Neoplasias/biossíntese , Selectina-P/biossíntese , Serina/farmacologia , Selenito de Sódio/farmacologia , Sinergismo Farmacológico , Células Hep G2 , Humanos , Serina/agonistas , Selenito de Sódio/agonistasRESUMO
Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.
Assuntos
Eletricidade , Fator de Crescimento Epidérmico/metabolismo , Plasma Rico em Plaquetas/citologia , Animais , Anexina A5/biossíntese , Anexina A5/genética , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Estimulação Elétrica , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Citometria de Fluxo , Expressão Gênica , Humanos , Octoxinol/farmacologia , Selectina-P/biossíntese , Selectina-P/genética , Ativação Plaquetária/efeitos dos fármacos , Plasma Rico em Plaquetas/metabolismo , Receptores de Trombina/química , Trombina/farmacologiaRESUMO
BACKGROUND AIMS: Dermatan sulfate (DS), an anticoagulant and antithrombotic glycosaminoglycan, also has anti-inflammatory activity. In this study, we investigated the effect of DS treatment in the presence or absence of bone marrow mononuclear cells (MNCs) or endothelial progenitor cells (EPCs) in the vascular response to carotid artery lesion in C57BL6 mice. METHODS: Thrombus formation, the expression of adhesion molecules and factors involved in vascular remodeling, inflammation or vascular tone were analyzed by histologic examination, Western blotting and enzyme-linked immunoassay 1 and 3 days after vascular injury. RESULTS: DS injections prevented thrombus formation and decreased P-selectin expression after 3 days of the injury. DS treatment also increased plasma SDF-1 levels but failed to rescue endothelial nitric oxide synthase (eNOS) expression, which is responsible for vascular tone. Treatment with MNCs alone failed to prevent thrombus formation 1 day after injury and increased intercellular adhesion molecule-1 expression, likely because of the inflammatory nature of these cells. Treatment with EPCs with DS was the most efficient among all therapies studied. Dual administration of EPCs and DS promoted an increase in the expression of adhesion molecules and, at the same time, induced a higher expression of eNOS at the injury site. Furthermore, it stimulated an elevated number of EPCs to migrate and adhere to the vascular wall. DISCUSSION: Simultaneous treatment with EPCs and DS increased the expression of adhesion molecules, prevented thrombosis, rescued the expression of eNOS and increased migration of EPCs to the site of injury, thereby affecting thrombus remodeling and inflammation and can be involved in vessel hemostasis.
Assuntos
Lesões das Artérias Carótidas/terapia , Dermatan Sulfato/uso terapêutico , Células Progenitoras Endoteliais/transplante , Fibrinolíticos/uso terapêutico , Trombose/prevenção & controle , Remodelação Vascular/fisiologia , Animais , Anti-Inflamatórios/farmacologia , Células da Medula Óssea/citologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/cirurgia , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Quimiocina CXCL12/biossíntese , Terapia Combinada , Molécula 1 de Adesão Intercelular/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/biossíntese , Selectina-P/biossínteseRESUMO
The aim of this study was to investigate the effect of leukocyte filtration on the P-selectin (CD62P) surface expression of apheresis platelets during the retention period. Ten bags of apheresis platelets stored for 1 day (0-24 h) and 10 bags of apheresis platelets stored for 2 days (24-48 h) were used for leukocyte filtration (experimental group). Ten bags of apheresis platelets with the corresponding retention periods but without filtration were used as a negative control (control group). Thereafter, 100 µL of platelet suspensions from apheresis platelets with or without leukocyte filtration were sampled before and after leukocyte filtration for the detection of CD62P surface expression by flow cytometry. No statistical difference in the CD62P surface expression of apheresis platelets was observed before and after leukocyte filtration (P > 0.05), neither did the CD62P surface expression exhibit any change among the different retention periods. Leukocyte filtration does not affect the CD62P surface expression of apheresis platelets stored for up to 2 days, which indicates that leukocyte filtration does not damage the activation of apheresis platelets within the retention period.