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1.
Atherosclerosis ; 294: 17-24, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31945614

RESUMO

BACKGROUND AND AIMS: Circulating platelet microparticles (PMP) are the most abundant in bloodstream, are highly procoagulant and contribute to cross-talk with inflammatory cells. The aim of the present study was to investigate the interactions of PMP with platelets and explore the involvement of toll-like receptor 4 (TLR-4). METHODS: PMP were separated by ultracentrifugation of expired platelet concentrates and added to: i) washed platelets, to confirm uptake, by flow cytometry and confocal and transmission electron microscopy, ii) platelet rich plasma (PRP), to assess changes in platelet function due to uptake by aggregometry in response to ADP; and iii) whole blood, to evaluate heterotypic aggregate (HA) formation by flow cytometry. Moreover, whole blood previously enriched with platelets with internalized PMP was used to explore modifications in thromboelastometry parameters (ROTEM). The inhibitory action of anti-TLR-4 was investigated. RESULTS: Confocal and ultrastructural microscopy studies revealed PMP internalization by platelets. Flow cytometry showed PMP-platelet association (p < 0.01 vs controls, at different PMP dilutions). PMP, at 1/20 dilution, increased HA (p < 0.05 vs controls), the percentage of maximal platelet aggregation to ADP (p < 0.05 vs controls), and accelerated clotting and clot formation times (p < 0.05 vs controls). Incubation of platelets with anti-TLR-4 prior to exposure to PMP reduced PMP-platelet association (p < 0.05 vs absence of the antibody), prevented HA formation, reduced maximal platelet aggregation and normalized ROTEM parameters. CONCLUSIONS: Platelets exhibit internalization ability towards their own PMP, a process that potentiates their thrombogenicity and is partially mediated by the innate immunity receptor TLR-4.

2.
J Cell Mol Med ; 24(2): 1713-1723, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31782253

RESUMO

Endothelial dysfunction is an earlier contributor to the development of atherosclerosis in chronic kidney disease (CKD), in which the role of epigenetic triggers cannot be ruled out. Endothelial protective strategies, such as defibrotide (DF), may be useful in this scenario. We evaluated changes induced by CKD on endothelial cell proteome and explored the effect of DF and the mechanisms involved. Human umbilical cord vein endothelial cells were exposed to sera from healthy donors (n = 20) and patients with end-stage renal disease on haemodialysis (n = 20). Differential protein expression was investigated by using a proteomic approach, Western blot and immunofluorescence. HDAC1 and HDAC2 overexpression was detected. Increased HDAC1 expression occurred at both cytoplasm and nucleus. These effects were dose-dependently inhibited by DF. Both the HDACs inhibitor trichostatin A and DF prevented the up-regulation of the endothelial dysfunction markers induced by the uraemic milieu: intercellular adhesion molecule-1, surface Toll-like receptor-4, von Willebrand Factor and reactive oxygen species. Moreover, DF down-regulated HDACs expression through the PI3/AKT signalling pathway. HDACs appear as key modulators of the CKD-induced endothelial dysfunction as specific blockade by trichostatin A or by DF prevents endothelial dysfunction responses to the CKD insult. Moreover, DF exerts its endothelial protective effect by inhibiting HDAC up-regulation likely through PI3K/AKT.

3.
Clin J Am Soc Nephrol ; 14(12): 1719-1732, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694864

RESUMO

BACKGROUND AND OBJECTIVES: Atypical hemolytic uremic syndrome is a form of thrombotic microangiopathy caused by dysregulation of the alternative complement pathway. There is evidence showing complement activation in other thrombotic microangiopathies. The aim of this study was to evaluate complement activation in different thrombotic microangiopathies and to monitor treatment response. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Complement activation was assessed by exposing endothelial cells to sera or activated-patient plasma-citrated plasma mixed with a control sera pool (1:1)-to analyze C5b-9 deposits by immunofluorescence. Patients with atypical hemolytic uremic syndrome (n=34) at different stages of the disease, HELLP syndrome (a pregnancy complication characterized by hemolysis, elevated liver enzymes, and low platelet count) or severe preeclampsia (n=10), and malignant hypertension (n=5) were included. RESULTS: Acute phase atypical hemolytic uremic syndrome-activated plasma induced an increased C5b-9 deposition on endothelial cells. Standard and lower doses of eculizumab inhibited C5b-9 deposition in all patients with atypical hemolytic uremic syndrome, except in two who showed partial remission and clinical relapse. Significant fibrin formation was observed together with C5b-9 deposition. Results obtained using activated-plasma samples were more marked and reproducible than those obtained with sera. C5b-9 deposition was also increased with samples from patients with HELLP (all cases) and preeclampsia (90%) at disease onset. This increase was sustained in those with HELLP after 40 days, and levels normalized in patients with both HELLP and preeclampsia after 6-9 months. Complement activation in those with malignant hypertension was at control levels. CONCLUSIONS: The proposed methodology identifies complement overactivation in patients with atypical hemolytic uremic syndrome at acute phase and in other diseases such as HELLP syndrome and preeclampsia. Moreover, it is sensitive enough to individually assess the efficiency of the C5 inhibition treatment.

4.
Front Immunol ; 10: 2339, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31649666

RESUMO

Angiogenesis and endothelial activation and dysfunction have been associated with acute graft-vs.-host disease (aGVHD), pointing to the endothelium as a potential target for pharmacological intervention. Defibrotide (DF) is a drug with an endothelium-protective effect that has been approved for the treatment of veno-occlusive disease/sinusoidal obstruction syndrome after allogeneic hematopoietic cell transplantation. Clinical data suggest that DF also reduces the incidence of aGVHD; however, the mechanisms of DF-mediated aGVHD regulation have not been examined. To investigate possible DF-mediated prophylactic and therapeutic mechanisms in aGVHD, we performed in vitro studies using endothelial cell (EC) lines. We found that DF significantly and dose-dependently suppressed EC proliferation and notably reduced their ability to form vascular tubes in Matrigel. To explore whether DF administered prophylactically or therapeutically has a significant effect on aGVHD endothelial dysfunction, ECs were exposed to media containing sera from patients with aGVHD (n = 22) in the absence or presence of DF and from patients that did not develop aGVHD (n = 13). ECs upregulated adhesion molecules (vascular cell adhesion molecule 1, intercellular adhesion molecule 1), the adherence junction protein VE-cadherin, von Willebrand factor (VWF), and Akt phosphorylation in response to aGVHD sera. These responses were suppressed upon treatment with DF. In summary, DF inhibits vascular angiogenesis and endothelial activation induced by sera from aGVHD patients. Our results support the view that DF has notable positive effects on endothelial biology during aGVHD.

6.
Transfusion ; 59(7): 2436-2445, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30946491

RESUMO

BACKGROUND: Comparative studies on the restoration of hemostasis with different reversal agents after dabigatran therapy have not been performed. We compared the efficacy and prothrombotic potential of the specific antidote idarucizumab with that of previously recommended non-specific procoagulant concentrates. STUDY DESIGN AND METHODS: We explored the in vitro effects of dabigatran (184 ng/mL) on fibrin and platelet-aggregate formation onto a damaged vessel under flow conditions (600 s-1 ). The reversal mechanisms and efficacy of idarucizumab (0.3-3 mg/mL) were compared with that of the non-specific procoagulant concentrates aPCC (25-75 U/Kg), PCC (70 U/Kg), or rFVIIa (120 µg/Kg). Generation of thrombin and prothrombin fragment (F1 + 2), and thromboelastometry parameters of clot formation were measured. RESULTS: Dabigatran caused pronounced reductions in fibrin (87%) and platelet interactions (36%) with damaged vessels (p < 0.01) and significantly impaired thrombin generation and thromboelastometric parameters (delayed dynamics and reduced firmness). Idarucizumab completely normalized rates of fibrin and platelet coverage to baseline values in flow studies; and reversed the alterations in thrombin generation, F1 + 2 and thromboelastometry parameters produced by dabigatran. In comparison, aPCC and PCC only partially compensated for the dabigatran-induced alterations in fibrin deposition, but were unable to fully restore them to baseline values. Reversal with aPCC or PCC improved the majority of alterations in coagulation-related tests, but tended to overcompensate thrombin generation kinetics and significantly increased F1 + 2 levels. CONCLUSION: Idarucizumab antagonizes alterations of direct and indirect biomarkers of hemostasis caused by dabigatran. In our studies, idarucizumab was clearly more efficacious than strategies with non-specific procoagulant concentrates and devoid of the excessive procoagulant tendency observed with the latter.

7.
J Clin Psychopharmacol ; 39(3): 254-257, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925498

RESUMO

PURPOSE/BACKGROUND: According to available international clinical guides, tricyclic antidepressants are our first- or second-line treatment of choice for severe unipolar major depression. However, the therapeutic option after an unsuccessful response to a tricyclic antidepressant drug in unipolar major depression is still unclear. METHODS/PROCEDURES: This 10-week randomized open-label study assessed the effectiveness of add-on lithium (adjusted to plasma levels) compared with add-on citalopram (30 mg/d) in 104 severe unipolar major depressive patients after a 10-week unsuccessful imipramine (adjusted to plasma level). Efficacy analyses examined changes in the severity of depression symptoms from baseline visit to endpoint and the comparative remission rate between treatment subgroups. FINDINGS/RESULTS: The randomized sample consisted of 104 imipramine-resistant severe unipolar major depressed patients. Both, the percentage of remitters (40.4% vs 21.1%, P = 0.034) and the mean reduction of the Hamilton Depression Rating Scale score (58.8% vs 42.5%, P = 0.005) were significantly greater in the add-on citalopram subgroup at endpoint visit. IMPLICATIONS/CONCLUSIONS: Although we should be cautious about generalizing these results to patients with a less severe unipolar major episode, results from the present study suggest that add-on citalopram is a very effective treatment option in unipolar major depressive episodes after an unsuccessful imipramine regimen.


Assuntos
Citalopram/administração & dosagem , Transtorno Depressivo Maior/tratamento farmacológico , Imipramina/administração & dosagem , Compostos de Lítio/administração & dosagem , Adulto , Idoso , Antidepressivos/administração & dosagem , Antidepressivos Tricíclicos/administração & dosagem , Transtorno Depressivo Maior/fisiopatologia , Quimioterapia Combinada , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Resultado do Tratamento
8.
J Clin Psychopharmacol ; 39(1): 63-66, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30516574

RESUMO

PURPOSE/BACKGROUND: Newer-generation antidepressants used in monotherapy or in combination with other newer-generation antidepressants or other psychotropic drugs are usually preferred as first- or second-step treatment options in resistant depression. According to our clinical experience, tricyclic antidepressants still are one of our preferred first choices in treatment-resistant moderate to severe unipolar major depressive episodes. METHODS: This 10-week open-design randomized study assessed the effectiveness of switching to imipramine (adjusted to plasma levels) compared with add-on mirtazapine (30 mg/d) for treatment of moderate to severe unipolar major depressive episodes after a 10-week unsuccessful venlafaxine regimen (225-300 mg/d). Efficacy analyses examined the change in depressive symptoms severity from baseline visit to endpoint and the comparative remission rate between treatment subgroups. FINDINGS/RESULTS: The randomized sample consisted of 112 venlafaxine-resistant moderate to severe unipolar major depressed patients. Both the percentage of remitters (71.43% vs 39.28%) and the mean reduction of the Hamilton Depression Rating Scale score (76.94% vs 50.72%) were significantly larger in the imipramine subgroup. IMPLICATIONS/CONCLUSIONS: Even though we should be cautious about generalizing these results to patients with a less severe unipolar major episodes, our study suggest that switching to imipramine is a very effective treatment option in unipolar major depressive episodes after an unsuccessful venlafaxine regimen.


Assuntos
Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Resistente a Tratamento/tratamento farmacológico , Imipramina/uso terapêutico , Mirtazapina/uso terapêutico , Cloridrato de Venlafaxina/uso terapêutico , Adolescente , Adulto , Idoso , Antidepressivos/uso terapêutico , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto Jovem
9.
Cell Physiol Biochem ; 51(3): 1287-1300, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30481789

RESUMO

BACKGROUND/AIMS: Accelerated atherosclerosis in chronic kidney disease (CKD) is preceded by endothelial dysfunction (ED), which exhibits a proinflammatory and prothrombotic phenotype and enhanced oxidative stress. In this study, the effect of several compounds with anti-inflammatory and/or antioxidant properties on uremia-induced endothelial dysfunction has been evaluated in an in vitro model. METHODS: Endothelial cells (ECs) were exposed to sera from uremic patients in the absence and presence of the flavonoids apigenin, genistein and quercetin, the antioxidant enzyme mimetics (AEM) ebselen (glutathione peroxidase mimetic), EUK-134 and EUK-118 (both superoxide dismutase mimetics), and the pharmacological drug N-acetylcysteine (NAC). We explored changes in the expression of adhesion receptors on the cell surface, by immunofluorescence, the production of radical oxygen species (ROS), by fluorescence detection, and the activation of signaling proteins related to inflammation, by both a phosphospecific antibody cell-based ELISA and immunoblotting techniques. RESULTS: Uremic media induced a significantly increased expression of ICAM-1, overproduction of radical oxygen species (ROS) and activation of p38 mitogen activated protein kinase (p38MAPK) and Nuclear Factor kB (NFkB) in ECs. Quercetin, the AEM and NAC showed a significant inhibitory effect on both ICAM-1 expression and ROS generation (p<0.05). All the compounds reduced p38MAPK activation, but only the AEM, especially ebselen, and NAC, both potentiating the glutathione peroxidase pathway, also inhibited NFkB activation. These two compounds were capable of increasing endothelial glutathione levels, especially in response to uremia. CONCLUSION: Our results indicate that the potentiation of the antioxidant pathways can be an effective strategy to improve endothelial dysfunction in uremia and a potential target to reduce the cardiovascular risk in this population.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Idoso , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Bone Marrow Transplant ; 53(12): 1541-1547, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29706650

RESUMO

Autologous stem cell transplantation (PBSCT) is standard for young patients in MM and its TRM has decreased after the 2000s. Bortezomib and immunomodulatory agents (IMiDs) in MM have improved the outcome. However, they seem to boost pro-inflammatory stage increasing the incidence of engraftment syndrome (ES). Favorable factors in PBSCT such as G-CSF could increase inflammatory stage during transplant. Corticosteroids have shown an excellent response of ES and some authors propose them as prophylaxis for ES. The aim was to analyze the impact of G-CSF avoidance and corticosteroids' prophylaxis in 170 patients diagnosed of MM treated with bortezomib/IMiDs that underwent PBSCT. We established three groups: Group-I [(G-CSF_administration), 60 patients (35%)], group-II [(nonG-CSF), 60 patients (35%)] and group-III [(nonG-CSF plus corticosteroid's prophylaxis), 50 patients (30%)]. A decreased ES incidence among groups was observed: 62, 42, and 22% (P < 0.0001). The incidence of symptoms mimicking a capillary leak syndrome associated with ES dropped: 43, 32, and 0% (P = 0.03). The G-CSF avoidance and corticosteroids had impact over admission 24, 21, and 20 days (P = 0.001). The most important variables related to ES were HCT-CI >2 (p < 0.0001; HR 8.5) and risk groups (p < 0.0001; HR 7.2). Hence, G-CSF avoidance and corticosteroid's prophylaxis decrease morbidity in patients undergoing PBSCT with MM treated with bortezomib/IMiDs.


Assuntos
Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Quimioterapia de Indução/métodos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/terapia , Condicionamento Pré-Transplante/efeitos adversos , Transplante Autólogo/efeitos adversos , Adulto , Idoso , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Condicionamento Pré-Transplante/métodos , Transplante Autólogo/métodos
11.
Clin Endocrinol (Oxf) ; 88(3): 415-424, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29154455

RESUMO

OBJECTIVE: Sustained evidence from observational studies indicates that after remission of Cushing syndrome (CS) a cardiovascular risk phenotype persists. Here, we performed a translational study in active CS and CS in remission (RCS) to evaluate the subclinical cardiometabolic burden and to explore the direct pro-inflammatory and prothrombotic potential of their sera on the endothelium in an in vitro translational atherothrombotic cell model. PATIENTS: Cross sectional study. The groups were (n = 9/group): I. RCS; II. Active CS (ACS) and III. Controls (CTR), all matched for age, body mass index, sex, without other hormonal deficits. DESIGN: We evaluated in vivo: cardiometabolic profile; endothelial markers (sVCAM-1, NO); endothelial dysfunction (FMD); intima-media thickness and body composition (DEXA). In vitro endothelial cells (EC) were exposed to sera taken from the different subjects to evaluate inflammatory EC response (tisVCAM) and thrombogenicity of the generated extracellular matrix (ECM): von Willebrand factor (VWF) and platelet reactivity. RESULTS: Three of the 9 RCS subjects were on glucocorticoid replacement therapy (GC-RT). Patients on GC-RT had a shorter period of time in stable remission. In vivo analysis ACS showed typically metabolic features, while cardiometabolic markers reached statistical significance for RCS only for Hs-CRP (P < .01). In vitro:EC exposed to ACS and RCS sera displayed increased tisVCAM-1 (P < .01 for ACS and P < .05 for RCS vs CTR), VWF (P < .01 for ACS and P < .05 for RCS vs CTR) and platelet adhesion on ECM (P < .01 for ACC and P < .05 for RCS vs CTR). No statistically significant differences were observed between GC-RT RSC and RCS without GC-RT. CONCLUSIONS: The sera of premenopausal women with CS in remission, without atherothrombotic disease, contain circulatory endothelial deleterious factors with a direct thrombogenic and pro-inflammatory endothelial effect that could increase cardiovascular risk.


Assuntos
Doenças Cardiovasculares/etiologia , Síndrome de Cushing/sangue , Endotélio/lesões , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos Transversais , Síndrome de Cushing/complicações , Síndrome de Cushing/patologia , Endotélio/patologia , Feminino , Humanos , Inflamação/etiologia , Masculino , Pessoa de Meia-Idade , Pós-Menopausa , Indução de Remissão , Trombose/etiologia , Pesquisa Médica Translacional , Adulto Jovem
12.
Thromb Haemost ; 117(12): 2312-2321, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29212119

RESUMO

Background Serotonin reuptake inhibitors (SSRIs) may impair platelet function. Thrombin is a strong platelet agonist causing irreversible aggregation, release of granules' contents, cytoskeletal rearrangement and activation of signalling pathways. We investigated the effects of the SSRI escitalopram (SCIT) on thrombin-induced platelet response. Methods Isolated platelets were exposed to SCIT and activated with thrombin. We evaluated (1) platelet response by aggregometry and flow cytometry; (2) modifications in cytoskeleton proteins and signalling pathways by electrophoresis and Western blot; and (3) ultrastructural changes in platelets by electron microscopy. Results SCIT inhibited platelet response to thrombin, measured as platelet aggregation and expression of activation markers CD62-P and CD63 from platelet granules. Platelet aggregation decreased in a dose-dependent manner, reaching statistical significance with SCIT ≥32 µg/mL (65.4 ± 6.8% vs. 77.7 ± 2.5% for controls; p < 0.05). Expression of activation markers was statistically reduced with SCIT ≥20 µg/mL (p < 0.05). SCIT impaired the polymerization of the actin cytoskeleton and association of contractile proteins during activation with thrombin (p < 0.05 with SCIT ≥50 µg/mL). Resting platelets incubated with SCIT became most spherical, with increased platelet roundness (p < 0.01, SCIT 50 µg/mL vs. control). SCIT interfered with signalling pathways modulated by thrombin (RhoA, PKC, Erk1/2 and PI3K/AKT). Conclusions Our data indicate that SCIT inhibits thrombin-induced platelet response and interferes with cytoskeletal assembly and related signalling pathways, thus resulting in compromised release of granules' contents, reduced platelet activation and aggregation. These mechanisms may explain the antithrombotic benefits observed in patients treated with this SSRI, and could become new therapeutic targets for future antithrombotic strategies.


Assuntos
Citoesqueleto de Actina/metabolismo , Plaquetas/fisiologia , Citalopram/farmacologia , Inibidores de Captação de Serotonina/farmacologia , Plaquetas/efeitos dos fármacos , Células Cultivadas , Voluntários Saudáveis , Humanos , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo
13.
Adv Healthc Mater ; 6(19)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28714577

RESUMO

The main drawbacks of cardiovascular bare-metal stents (BMS) are in-stent restenosis and stent thrombosis as a result of an incomplete endothelialization after stent implantation. Nano- and microscale modification of implant surfaces is a strategy to recover the functionality of the artery by stimulating and guiding molecular and biological processes at the implant/tissue interface. In this study, cobalt-chromium (CoCr) alloy surfaces are modified via direct laser interference patterning (DLIP) in order to create linear patterning onto CoCr surfaces with different periodicities (≈3, 10, 20, and 32 µm) and depths (≈20 and 800 nm). Changes in surface topography, chemistry, and wettability are thoroughly characterized before and after modification. Human umbilical vein endothelial cells' adhesion and spreading are similar for all patterned and plain CoCr surfaces. Moreover, high-depth series induce cell elongation, alignment, and migration along the patterned lines. Platelet adhesion and aggregation decrease in all patterned surfaces compared to CoCr control, which is associated with changes in wettability and oxide layer characteristics. Cellular studies provide evidence of the potential of DLIP topographies to foster endothelialization without enhancement of platelet adhesion, which will be of high importance when designing new BMS in the future.


Assuntos
Plaquetas/citologia , Plaquetas/fisiologia , Prótese Vascular , Ligas de Cromo/efeitos da radiação , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Stents , Adesão Celular/fisiologia , Células Cultivadas , Ligas de Cromo/química , Análise de Falha de Equipamento , Humanos , Lasers , Teste de Materiais , Desenho de Prótese , Doses de Radiação , Propriedades de Superfície/efeitos da radiação
14.
Blood ; 129(19): 2599-2600, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28495925

Assuntos
Plaquetas , Indicã , Humanos , Óxidos
15.
PLoS One ; 12(2): e0171486, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28192448

RESUMO

INTRODUCTION: Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions. METHODS: We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets. RESULTS: In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated. CONCLUSIONS: Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis.


Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Fibrina/metabolismo , Pirazóis/farmacologia , Piridonas/farmacologia , Trombose/metabolismo , Animais , Plaquetas/metabolismo , Relação Dose-Resposta a Droga , Inibidores do Fator Xa/farmacologia , Feminino , Hemorreologia , Hemostasia/efeitos dos fármacos , Humanos , Cinética , Ativação Plaquetária/efeitos dos fármacos , Coelhos , Tromboelastografia , Trombina/metabolismo
16.
J Biomed Mater Res A ; 105(4): 973-983, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28000367

RESUMO

Immobilization of bioactive peptide sequences on CoCr surfaces is an effective route to improve endothelialization, which is of great interest for cardiovascular stents. In this work, we explored the effect of physical and covalent immoblization of RGDS, YIGSR and their equimolar combination peptides on endothelial cells (EC) and smooth muscle cell (SMC) adhesion and on thrombogenicity. We extensively investigated using RT-qPCR, the expression by ECs cultured on functionalised CoCr surfaces of different genes. Genes relevant for adhesion (ICAM-1 and VCAM-1), vascularization (VEGFA, VEGFR-1 and VEGFR-2) and anti-thrombogenicity (tPA and eNOS) were over-expressed in the ECs grown to covalently functionalized CoCr surfaces compared to physisorbed and control surfaces. Pro-thrombogenic genes expression (PAI-1 and vWF) decreased over time. Cell co-cultures of ECs/SMCs found that functionalization increased the amount of adhered ECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. SMCs adhered less compared to ECs in all surfaces. All studied peptides showed a lower platelet cell adhesion compared to TCPS. Covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represented prevailing strategy to enhance the early stages of ECs adhesion and proliferation, while preventing SMCs and platelet adhesion. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 973-983, 2017.


Assuntos
Ligas de Cromo/química , Materiais Revestidos Biocompatíveis/química , Oclusão de Enxerto Vascular/prevenção & controle , Células Endoteliais da Veia Umbilical Humana/metabolismo , Oligopeptídeos/química , Trombose/prevenção & controle , Plaquetas/metabolismo , Oclusão de Enxerto Vascular/metabolismo , Humanos , Trombose/metabolismo
17.
Blood ; 127(13): 1719-27, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-26755708

RESUMO

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Polidesoxirribonucleotídeos/farmacocinética , Anti-Inflamatórios/farmacocinética , Antioxidantes/farmacocinética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Inflamação/patologia , Inflamação/prevenção & controle , Óxido Nítrico Sintase Tipo III/metabolismo , Pinocitose/efeitos dos fármacos , Temperatura Ambiente , Fatores de Tempo
18.
J Cell Biochem ; 117(2): 448-57, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26221761

RESUMO

Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.


Assuntos
Plaquetas/fisiologia , Antígenos CD36/metabolismo , Micropartículas Derivadas de Células/metabolismo , Citoesqueleto/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Tromboplastina/metabolismo , Células Cultivadas , Ativação Enzimática , Humanos , Integrina beta3/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Transporte Proteico , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo
19.
Transfusion ; 56(3): 580-6, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26450525

RESUMO

BACKGROUND: Cryopreserved platelet (CPP) concentrates exhibit a variety of morphologic and functional alterations that may affect the action of CPP with accelerated platelet (PLT) response and clotting. The objective of this study was to compare the in vitro hemostatic effect of CPP with fresh whole blood (WB) and standard 5-day PLT concentrates (PCs). STUDY DESIGN AND METHODS: WB collected from eight healthy donors was used to prepare fresh WB, PLT-depleted WB (TPN), and PLT-restored TPN using CPP (TPN-CPP) or PC (TPN-PC). Clot properties were evaluated with thromboelastometry (ROTEM); adhesion and aggregate formation under high shear (Impact-R); and PLT adhesion, aggregate formation, fibrin formation, and prothrombin activation under medium shear in a perfusion system. RESULTS: TPN-CPP had faster clot initiation (ROTEM clot time--TPN-CPP 115 sec, WB 194 sec, TPN-PC 161 sec), and CPP contributes to a strong clot with PLT involvement (maximum clot firmness--TPN-CPP 32 mm, WB 62 mm, TPN-PC 59 mm). The Impact-R PLT-covered area with TPN-CPP was less than those of WB and PCs, but aggregate size was the same as WB. PLT coverage in perfusion studies was observed with TPN-CPP, although generally less than both WB and PC. Fibrin was deposited with CPP-restored samples, but did not exceed the level of WB. CONCLUSION: CPPs present a phenotype supporting a moderate increase in the rate of clot formation, form stable PLT clots, and do not present a hypercoagulable phenotype during in vitro functional tests.


Assuntos
Plaquetas/citologia , Preservação de Sangue/efeitos adversos , Criopreservação , Coagulação Sanguínea/fisiologia , Testes de Coagulação Sanguínea , Hemostasia/fisiologia , Hemostáticos , Humanos , Leucócitos/citologia , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/fisiologia
20.
Transfus Med Rev ; 29(4): 242-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26364029

RESUMO

We evaluated the hemostatic alterations in blood from healthy individuals treated for 5 days with direct oral anticoagulants (DOACs) rivaroxaban (20 mg/d) or dabigatran (150 mg/12 h) in a single-blind clinical trial with crossover assignment (NCT01478282). We assessed the potential of prothrombin complex concentrates, activated prothrombin complex concentrates, or recombinant activated factor VII, when added ex vivo, to reverse the alterations caused by these DOACs. Blood was drawn at maximum plasma concentration after the last dose of each DOAC, and modifications in coagulation biomarkers were evaluated using a series of tests performed under steady conditions including routine coagulation, thrombin generation, and thromboelastometry assays. Additional studies in standardized flow devices were applied to evaluate alterations on platelet deposition and fibrin formation on damaged vascular surfaces exposed to flowing blood. Both DOACs caused important modifications of all coagulation biomarkers and significantly reduced fibrin formation in flow studies. Alterations in biomarkers observed in steady laboratory tests were normalized and occasionally overcompensated by procoagulant strategies. In contrast, reductions in fibrin formation observed in studies with flowing blood were improved, although never completely restored to baseline levels. Effects of dabigatran in flow studies appeared more resistant to reversal strategies than those of rivaroxaban. Inconsistencies between results of coagulation studies in steady or flowing assays not only raise concerns about the adequacy of the earlier tests to predict the restoration of the coagulopathy induced by DOACs but also suggest limitations of nonspecific procoagulant strategies to control severe coagulopathy in patients inadvertently overexposed these agents.


Assuntos
Anticoagulantes/efeitos adversos , Fatores de Coagulação Sanguínea/uso terapêutico , Dabigatrana/efeitos adversos , Fibrina/metabolismo , Rivaroxabana/efeitos adversos , Adulto , Coagulação Sanguínea/efeitos dos fármacos , Feminino , Voluntários Saudáveis , Hemostasia/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Método Simples-Cego , Falha de Tratamento , Adulto Jovem
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