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2.
Clin Appl Thromb Hemost ; 26: 1076029620954913, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33030036

RESUMO

INTRODUCTION: Sulodexide represents a mixture of fast-moving heparin (FMH) and dermatan sulfate (DS) and has been used for the management of venous diseases such as DVT and related disorders. The purpose of this study is to compare sulodexide and its components with unfractionated heparin (UFH) to determine its suitability for the indications in which UFH is used. MATERIALS AND METHOD: Active pharmaceutical ingredients (API) versions of sulodexide, FMH and DS were obtained from Alfasigma. API versions of UFH were obtained from Medefil Inc. Normal human citrated plasma was obtained from blood bank of the Loyola University Medical Center. Each of the individual agents were supplemented in plasma at a graded concentration of 0.0-10 µg/mL. Clotting assays (PiCT, aPTT, PT and TT), anti-Xa and anti-IIa and thrombin generation studies were carried out. Results were compiled as mean ± SD of 3 individual determination. RESULT: In the clot based (PiCT, aPTT and TT), anti-Xa and IIa assays, both the UFH and FMH produced stronger activities in these assays followed by sulodexide. DS did not show any anticoagulant activity. In the thrombin generation assay, FMH and UFH produced comparable inhibition of thrombin generation as measured by various parameters. Sulodexide was slightly weaker in this assay, whereas DS produced relatively weaker effects. CONCLUSION: In comparison to sulodexide, both UFH and FMH exhibit comparable anticoagulant activity despite differences in their molecular weight. These results suggest that sulodexide can be developed as a parenteral anticoagulant for indications in which UFH is used.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Trombina/farmacologia , Anticoagulantes/administração & dosagem , Antitrombinas/administração & dosagem , Antitrombinas/farmacologia , Glicosaminoglicanos/administração & dosagem , Heparina/administração & dosagem , Heparina/farmacologia , Humanos , Itália , Sensibilidade e Especificidade , Trombina/administração & dosagem
3.
Food Chem ; 332: 127384, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615384

RESUMO

Dairy polar lipids (PL) seem to exhibit antiplatelet effects. However, it is not known what molecular species may be responsible. In this study, we confirmed using C30 reversed-phase (C30RP) ultra-high-performance liquid chromatography (UHPLC) coupled to high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) that fermentation of yoghurts from ovine milk using specific starter cultures altered the PL composition. These lipid alterations occurred concomitant with increased antithrombotic properties of the yoghurts PL fractions against platelet-activating factor (PAF) and thrombin-induced platelet aggregation. Specifically, elevation in phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylcholine (PC) and their molecular species were observed following yoghurt fermentation. Furthermore, PC(18:0/18:1), PE(18:1/18:2), SM(d18:0/22:0) and several other molecular species were significantly inversely correlated with the inhibition of PAF and thrombin. These molecular species were abundant in the most bioactive yoghurts fermented by S. thermophilus and L. acidophilus, which suggest that fermentation by these microorganisms increases the antithrombotic properties of ovine milk PL.


Assuntos
Lipídeos/análise , Leite/metabolismo , Inibidores da Agregação de Plaquetas/análise , Iogurte/análise , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Fermentação , Lipídeos/farmacologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Ovinos , Esfingomielinas/metabolismo , Espectrometria de Massas em Tandem , Trombina/farmacologia
4.
Arterioscler Thromb Vasc Biol ; 40(8): 1905-1917, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32580633

RESUMO

OBJECTIVE: Remodeling of the extracellular matrix plays a vital role in cardiovascular diseases. Using a mouse model of postnatal ascending aortic aneurysms (termed Fbln4SMKO), we have reported that abnormal mechanosensing led to aneurysm formation in Fbln4SMKO with an upregulation of the mechanosensitive transcription factor, Egr1 (Early growth response 1). However, the role of Egr1 and its upstream regulator(s) in the initiation of aneurysm development and their relationship to an aneurysmal microenvironment are unknown. Approach and Results: To investigate the contribution of Egr1 in the aneurysm development, we deleted Egr1 in Fbln4SMKO mice and generated double knockout mice (DKO, Fbln4SMKO; Egr1-/-). Aneurysms were prevented in DKO mice (42.8%) and Fbln4SMKO; Egr1+/- mice (26%). Ingenuity Pathway Analysis identified PAR1 (protease-activated receptor 1) as a potential Egr1 upstream gene. Protein and transcript levels of PAR1 were highly increased in Fbln4SMKO aortas at postnatal day 1 before aneurysm formed, together with active thrombin and MMP (matrix metalloproteinase)-9, both of which serve as a PAR1 activator. Concordantly, protein levels of PAR1, Egr1, and thrombin were significantly increased in human thoracic aortic aneurysms. In vitro cyclic stretch assays (1.0 Hz, 20% strain, 8 hours) using mouse primary vascular smooth muscle cells induced marked expression of PAR1 and secretion of prothrombin in response to mechanical stretch. Thrombin was sufficient to induce Egr1 expression in a PAR1-dependent manner. CONCLUSIONS: We propose that thrombin, MMP-9, and mechanical stimuli in the Fbln4SMKO aorta activate PAR1, leading to the upregulation of Egr1 and initiation of ascending aortic aneurysms.


Assuntos
Aneurisma da Aorta Torácica/etiologia , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Receptor PAR-1/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas da Matriz Extracelular/deficiência , Feminino , Humanos , Masculino , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Pessoa de Meia-Idade , Receptor PAR-1/antagonistas & inibidores , Estresse Mecânico , Trombina/farmacologia
5.
PLoS One ; 15(5): e0231944, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365105

RESUMO

Intrauterine bleeding during pregnancy is a major risk factor for preterm birth. Thrombin, the most abundant coagulation factor in blood, is associated with uterine myometrial contraction. Here, we investigated the molecular mechanism and signaling of thrombin-induced myometrial contraction. First, histologic studies of placental abruption, as a representative intrauterine bleeding, revealed that thrombin was expressed within the infiltrating hemorrhage and that thrombin receptor (protease-activated receptor 1, PAR1) was highly expressed in myometrial cells surrounding the hemorrhage. Treatment of human myometrial cells with thrombin resulted in augmented contraction via PAR1. Thrombin-induced signaling to myosin was then mediated by activation of myosin light chain kinase- and Rho-induced phosphorylation of myosin light chain-2. In addition, thrombin increased prostaglandin-endoperoxidase synthase-2 (PTGS2 or COX2) mRNA and prostaglandin E2 and F2α synthesis in human myometrial cells. Thrombin significantly increased the mRNA level of interleukine-1ß, whereas it decreased the expressions of prostaglandin EP3 and F2α receptors. Progesterone partially blocked thrombin-induced myometrial contractions, which was accompanied by suppression of the thrombin-induced increase of PTGS2 and IL1B mRNA expressions as well as suppression of PAR1 expression. Collectively, thrombin induces myometrial contractions by two mechanisms, including direct activation of myosin and indirect increases in prostaglandin synthesis. The results suggest a therapeutic potential of progesterone for preterm labor complicated by intrauterine bleeding.


Assuntos
Miométrio/efeitos dos fármacos , Trombina/farmacologia , Contração Uterina/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/metabolismo , Feminino , Humanos , Contração Muscular/efeitos dos fármacos , Miométrio/fisiologia , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Gravidez , Progesterona/metabolismo , Progesterona/farmacologia , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Imagem com Lapso de Tempo , Contração Uterina/fisiologia
6.
Am J Physiol Regul Integr Comp Physiol ; 318(6): R1068-R1077, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32320636

RESUMO

Severe trauma can produce a postinjury "metabolic self-destruction" characterized by catabolic metabolism and hyperglycemia. The severity of the hyperglycemia is highly correlated with posttrauma morbidity and mortality. Although no mechanism has been posited to connect severe trauma with a loss of autonomic control over metabolism, traumatic injury causes other failures of autonomic function, notably, gastric stasis and ulceration ("Cushing's ulcer"), which has been connected with the generation of thrombin. Our previous studies established that proteinase-activated receptors (PAR1; "thrombin receptors") located on astrocytes in the autonomically critical nucleus of the solitary tract (NST) can modulate gastric control circuit neurons to cause gastric stasis. Hindbrain astrocytes have also been implicated as important detectors of low glucose or glucose utilization. When activated, these astrocytes communicate with hindbrain catecholamine neurons that, in turn, trigger counterregulatory responses (CRR). There may be a convergence between the effects of thrombin to derange hindbrain gastrointestinal control and the hindbrain circuitry that initiates CRR to increase glycemia in reaction to critical hypoglycemia. Our results suggest that thrombin acts within the NST to increase glycemia through an astrocyte-dependent mechanism. Blockade of purinergic gliotransmission pathways interrupted the effect of thrombin to increase glycemia. Our studies also revealed that thrombin, acting in the NST, produced a rapid, dramatic, and potentially lethal suppression of respiratory rhythm that was also a function of purinergic gliotransmission. These results suggest that the critical connection between traumatic injury and a general collapse of autonomic regulation involves thrombin action on astrocytes.


Assuntos
Astrócitos/efeitos dos fármacos , Glicemia , Neurônios/efeitos dos fármacos , Rombencéfalo/efeitos dos fármacos , Trombina/farmacologia , Animais , Masculino , Nervo Frênico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Taxa Respiratória/efeitos dos fármacos , Núcleo Solitário/efeitos dos fármacos
7.
Biochem Pharmacol ; 177: 113975, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32298692

RESUMO

BACKGROUND AND PURPOSE: Rapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. EXPERIMENTAL APPROACH: The effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. KEY RESULTS: Rapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. CONCLUSIONS AND IMPLICATIONS: Rapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.


Assuntos
Plaquetas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Plaquetas/citologia , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Regulação da Expressão Gênica , Humanos , Ionomicina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/metabolismo , Morfolinas/farmacologia , Peptídeos/farmacologia , Fosfatidilserinas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Cultura Primária de Células , Pirazóis/farmacologia , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Tacrolimo/farmacologia , Trombina/metabolismo , Trombina/farmacologia
8.
Eur J Histochem ; 64(2)2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32236088

RESUMO

Schwann cells (SC) are characterized by a remarkable plasticity that enables them to promptly respond to nerve injury promoting axonal regeneration. In peripheral nerves after damage SC convert to a repair-promoting phenotype activating a sequence of supportive functions that drive myelin clearance, prevent neuronal death, and help axon growth and guidance. Regeneration of peripheral nerves after damage correlates inversely with thrombin levels. Thrombin is not only the key regulator of the coagulation cascade but also a protease with hormone-like activities that affects various cells of the central and peripheral nervous system mainly through the protease-activated receptor 1 (PAR1). Aim of the present study was to investigate if and how thrombin could affect the axon supportive functions of SC. In particular, our results show that the activation of PAR1 in rat SC cultures with low levels of thrombin or PAR1 agonist peptides induces the release of molecules, which favor neuronal survival and neurite elongation. Conversely, the stimulation of SC with high levels of thrombin or PAR1 agonist peptides drives an opposite effect inducing SC to release factors that inhibit the extension of neurites. Moreover, high levels of thrombin administered to sciatic nerve ex vivo explants induce a dramatic change in SC morphology causing disappearance of the Cajal bands, enlargement of the Schmidt-Lanterman incisures and calcium-mediated demyelination of the paranodes. Our results indicate thrombin as a novel modulator of SC plasticity potentially able to favor or inhibit SC pro-regenerative properties according to its level at the site of lesion.


Assuntos
Neurogênese/efeitos dos fármacos , Nós Neurofibrosos/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Trombina/farmacologia , Animais , Cálcio/metabolismo , Feminino , Masculino , Neuritos/efeitos dos fármacos , Células PC12 , Pirróis/farmacologia , Quinazolinas/farmacologia , Ratos , Ratos Wistar , Receptor PAR-1/metabolismo , Nervo Isquiático/efeitos dos fármacos , Tapsigargina/farmacologia
9.
PLoS One ; 15(4): e0230507, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255777

RESUMO

The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.


Assuntos
Plaquetas/metabolismo , Células da Medula Óssea/citologia , Animais , Antígenos CD34/metabolismo , Plaquetas/citologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Suínos , Trombina/farmacologia
10.
Curr Pharm Des ; 26(18): 2109-2115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32250213

RESUMO

Discovery and selection of the potential targets are some of the important issues in pharmacology. Even when all the reactions and the proteins in a biological network are known, how does one choose the optimal target? Here, we review and discuss the application of the computational methods to address this problem using the blood coagulation cascade as an example. The problem of correct antithrombotic targeting is critical for this system because, although several anticoagulants are currently available, all of them are associated with bleeding risks. The advantages and the drawbacks of different sensitivity analysis strategies are considered, focusing on the approaches that emphasize: 1) the functional modularity and the multi-tasking nature of this biological network; and 2) the need to normalize hemostasis during the anticoagulation therapy rather than completely suppress it. To illustrate this effect, we show the possibility of the differential regulation of lag time and endogenous thrombin potential in the thrombin generation. These methods allow to identify the elements in the blood coagulation cascade that may serve as the targets for the differential regulation of this system.


Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea , Biologia de Sistemas , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Trombina/farmacologia
11.
Curr Med Sci ; 40(1): 55-62, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32166665

RESUMO

The present study aimed to explore the molecular mechanisms underlying the increase of nicotinamide adenine dinucleotide phosphate:quinine oxidoreductase 1 (NQO1) and γ-glutamylcysteine synthetase (γ-GCS) in brain tissues after intracerebral hemorrhage (ICH). The microglial cells obtained from newborn rats were cultured and then randomly divided into the normal control group (NC group), model control group (MC group), rosiglitazone (RSG) intervention group (RSG group), retinoic-acid intervention group (RSG+RA group), and sulforaphane group (RSG+SF group). The expression levels of NQO1, γ-GCS, and nuclear factor E2-related factor 2 (Nrf2) were measured by real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. The results showed that the levels of NQO1, γ-GCS and Nrf2 were significantly increased in the MC group and the RSG group as compared with those in the NC group (P<0.01). They were found to be markedly decreased in the RSG+RA group and increased in the RSG+SF group when compared with those in the MC group or the RSG group (P<0.01). The RSG+SF group displayed the highest levels of NQO1, γ-GCS, and Nrf2 among the five groups. In conclusion, a medium dose of RSG increased the anti-oxidative ability of thrombin-activated microglia by increasing the expression of NQO1 and γ-GCS. The molecular mechanisms underlying the increase of NQO1 and γ-GCS in thrombin-activated microglia may be associated with the activation of Nrf2.


Assuntos
Hemorragia Cerebral/genética , Glutamato-Cisteína Ligase/genética , Microglia/citologia , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/genética , PPAR gama/genética , Trombina/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/metabolismo , Modelos Animais de Doenças , Feminino , Glutamato-Cisteína Ligase/metabolismo , Isotiocianatos/administração & dosagem , Isotiocianatos/farmacologia , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , PPAR gama/metabolismo , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Rosiglitazona/administração & dosagem , Rosiglitazona/farmacologia , Tretinoína/administração & dosagem , Tretinoína/farmacologia
12.
Arterioscler Thromb Vasc Biol ; 40(5): 1256-1274, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32160773

RESUMO

OBJECTIVE: In view of our previous observations on differential expression of LMCD1 (LIM and cysteine-rich domains 1) in human versus rodents, we asked the question whether LMCD1 plays a species-specific role in the development of vascular lesions. Approach and Results: A combination of genetic, molecular, cellular, and disease models were used to test species-specific role of LMCD1 in the pathogenesis of vascular lesions. Here, we report species-specific regulation of LMCD1 expression in mediating vascular smooth muscle cell proliferation and migration during vascular wall remodeling in humans versus mice. Thrombin induced LMCD1 expression in human aortic smooth muscle cells but not mouse aortic smooth muscle cells via activation of Par1 (protease-activated receptor 1)-Gαq/11 (Gα protein q/11)-PLCß3 (phospholipase Cß3)-NFATc1 (nuclear factor of activated T cells 1) signaling. Furthermore, although LMCD1 mediates thrombin-induced proliferation and migration of both human aortic smooth muscle cells and mouse aortic smooth muscle cells via influencing E2F1 (E2F transcription factor 1)-mediated CDC6 (cell division cycle 6) expression and NFATc1-mediated IL (interleukin)-33 expression, respectively, in humans, it acts as an activator, and in mice, it acts as a repressor of these transcriptional factors. Interestingly, LMCD1 repressor activity was nullified by N-myristoyltransferase 2-mediated myristoylation in mouse. Besides, we found increased expression of LMCD1 in human stenotic arteries as compared to nonstenotic arteries. On the other hand, LMCD1 expression was decreased in neointimal lesions of mouse injured arteries as compared to noninjured arteries. CONCLUSIONS: Together, these observations reveal that LMCD1 acts as an activator and repressor of E2F1 and NFATc1 in humans and mice, respectively, in the induction of CDC6 and IL-33 expression during development of vascular lesions. Based on these findings, LMCD could be a potential target for drug development against restenosis and atherosclerosis in humans.


Assuntos
Proteínas Correpressoras/metabolismo , Fator de Transcrição E2F1/metabolismo , Interleucina-33/metabolismo , Proteínas com Domínio LIM/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Remodelação Vascular , Lesões do Sistema Vascular/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteínas Correpressoras/genética , Modelos Animais de Doenças , Fator de Transcrição E2F1/genética , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-33/genética , Proteínas com Domínio LIM/genética , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Ácido Mirístico/metabolismo , Fatores de Transcrição NFATC/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Especificidade da Espécie , Trombina/farmacologia , Remodelação Vascular/efeitos dos fármacos , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologia
13.
Int J Mol Sci ; 21(1)2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31948107

RESUMO

Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin ß2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin ß2 translocation are specifically or predominantly dependent on coronin 1.


Assuntos
Plaquetas/metabolismo , Cadeias beta de Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Colágeno/farmacologia , AMP Cíclico/metabolismo , Epoprostenol/farmacologia , Integrina alfa2/genética , Integrina alfa2/metabolismo , Cadeias beta de Integrinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Ligação Proteica , Transporte Proteico , Trombina/farmacologia
14.
Am J Pathol ; 190(2): 388-399, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31955792

RESUMO

Preterm premature rupture of membranes (PPROM) and thrombin generation by decidual cell-expressed tissue factor often accompany abruptions. Underlying mechanisms remain unclear. We hypothesized that thrombin-induced colony-stimulating factor-2 (CSF-2) in decidual cells triggers paracrine signaling via its receptor (CSF2R) in trophoblasts, promoting fetal membrane weakening and abruption-associated PPROM. Decidua basalis sections from term (n = 10), idiopathic preterm birth (PTB; n = 8), and abruption-complicated pregnancies (n = 8) were immunostained for CSF-2. Real-time quantitative PCR measured CSF2 and CSF2R mRNA levels. Term decidual cell (TDC) monolayers were treated with 10-8 mol/L estradiol ± 10-7 mol/L medroxyprogesterone acetate (MPA) ± 1 IU/mL thrombin pretreatment for 4 hours, washed, and then incubated in control medium with estradiol ± MPA. TDC-derived conditioned media supernatant effects on fetal membrane weakening were analyzed. Immunostaining localized CSF-2 primarily to decidual cell cytoplasm and cytotrophoblast cell membranes. CSF-2 immunoreactivity was higher in abruption-complicated or idiopathic PTB specimens versus normal term specimens (P < 0.001). CSF2 mRNA was higher in TDCs versus cytotrophoblasts (P < 0.05), whereas CSF2R mRNA was 1.3 × 104-fold higher in cytotrophoblasts versus TDCs (P < 0.001). Thrombin enhanced CSF-2 secretion in TDC cultures fourfold (P < 0.05); MPA reduced this effect. Thrombin-pretreated TDC-derived conditioned media supernatant weakened fetal membranes (P < 0.05), which MPA inhibited. TDC-derived CSF-2, acting via trophoblast-expressed CSFR2, contributes to thrombin-induced fetal membrane weakening, eliciting abruption-related PPROM and PTB.


Assuntos
Descolamento Prematuro da Placenta/fisiopatologia , Decídua/patologia , Membranas Extraembrionárias/patologia , Ruptura Prematura de Membranas Fetais/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Nascimento Prematuro/etiologia , Trombina/farmacologia , Decídua/efeitos dos fármacos , Decídua/metabolismo , Membranas Extraembrionárias/metabolismo , Feminino , Ruptura Prematura de Membranas Fetais/etiologia , Ruptura Prematura de Membranas Fetais/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Gravidez , Nascimento Prematuro/metabolismo , Nascimento Prematuro/patologia , Transdução de Sinais , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Trofoblastos/patologia
15.
J Pharmacol Sci ; 142(3): 116-123, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31924407

RESUMO

Neutrophils constitute the major population of infiltrating leukocytes after stroke including intracerebral hemorrhage (ICH), and these cells may exhibit pro-inflammatory and anti-inflammatory phenotypes depending on the external stimuli. Here we constructed an experimental system to evaluate how the properties of neutrophils were influenced by the injured brain tissues. HL60 cells differentiated into neutrophils were added to the culture medium of neonatal rat cortico-striatal slices maintained at liquid-air interface. Thrombin was applied to the cultures to mimic the pathogenic events associated with ICH. HL60 cells responded to thrombin by increasing mRNA expression of pro-inflammatory IL-1ß and anti-inflammatory IL-10 with a different time course. Co-presence of cortico-striatal slice cultures significantly enhanced IL-1ß mRNA expression, whereas attenuated IL-10 mRNA expression, in HL60 cells. Toll-like receptor 4 (TLR4) agonist lipopolysaccharide synergistically enhanced IL-1ß mRNA expression with thrombin, and TLR4 inhibitor TAK-242 abolished thrombin-induced IL-1ß mRNA expression in the presence of slice cultures. On the other hand, thrombin-induced cell death in cortico-striatal cultures was attenuated by the presence of HL60 cells. This experimental system may provide a unique platform to elucidate complex cell-to-tissue interactions during ICH pathogenesis.


Assuntos
Comunicação Celular , Neutrófilos , Trombina/farmacologia , Alarminas , Animais , Células Cultivadas , Citocinas , Células HL-60 , Humanos , Inflamação , Ratos Wistar , Trombina/fisiologia
16.
Platelets ; 31(3): 360-364, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31161848

RESUMO

Dabigatran, a direct oral thrombin inhibitor, has two therapeutic effects: anticoagulation; and antiplatelet activity. In the clinical field, evaluation of the effect of dabigatran on thrombin-induced platelet aggregation is difficult because of fibrin clot formation and platelet aggregation. The aim of this study was to establish a new platelet aggregation method and to investigate the effects of dabigatran on thrombin-induced platelet aggregation. Platelet aggregation with thrombin was performed with automated light transmission aggregometry (CS2400; Sysmex, Kobe, Japan) in 40 healthy subjects. Thrombin-induced platelet aggregation was performed using thrombin and platelet-rich plasma (PRP), and thrombin-induced fibrin polymerization was inhibited by adding the peptide Gly-Pro-Arg-Pro (GPRP). The effect of dabigatran was then evaluated using the above method. Thrombin at < 0.2 U/mL did not induce platelet aggregation in most normal subjects. Median maximum aggregation percent (MA%) (25th-75th percentile) with 0.5 and 1.0 U/mL of thrombin was 87.0% (79.3-90.8%), and 90.2% (86.5-92.2%), respectively. The anti-platelet effects of dabigatran were then evaluated with these concentrations of thrombin. Dabigatran (final concentration, 2.5-1000 nM) inhibited platelet aggregation by 0.2-1.0 U/mL of thrombin in a concentration-dependent manner in vitro. Dabigatran showed potent inhibitory effects against platelet aggregation induced by 0.5 and 1.0 U/mL thrombin with half maximal inhibitory concentrations of 10.5 and 40.4 nM, respectively. A standard for thrombin-induced platelet aggregation was developed using the CS2400 in healthy subjects, and dabigatran was confirmed to inhibit thrombin-induced platelet aggregation in vitro with PRP.


Assuntos
Antitrombinas/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Dabigatrana/farmacologia , Agregação Plaquetária , Testes de Função Plaquetária , Trombina/metabolismo , Adulto , Biomarcadores , Coagulação Sanguínea/efeitos dos fármacos , Feminino , Humanos , Masculino , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Testes de Função Plaquetária/métodos , Trombina/farmacologia , Adulto Jovem
17.
Ir J Med Sci ; 189(1): 133-137, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31165346

RESUMO

BACKGROUND: Clinically evident arterial thrombosis is rare following thrombin injection therapy for femoral pseudoaneurysm. However, it is unclear to what extent injected thrombin may pass to the ipsilateral lower limb arteries. AIMS: To assess if technetium 99m injected at the time of thrombin injection for femoral artery pseudoaneurysm therapy passes into the adjacent lower limb arteries. METHODS: This was a prospective trial with institutional review board approval. Four consecutive patients with common femoral pseudoaneurysms and failed manual compression were enrolled. Under real-time colour flow doppler ultrasound, a mixture of 1000 IU thrombin and approximately 200 MBq technetium 99m was injected in 0.1-mL doses into the pseudoaneurysm until thrombosis occurred. Gamma camera imaging of the syringe before injection, the injected groin after thrombosis and the syringe after injection were performed. Analysis of the gamma camera information was performed to determine the amount of technetium 99m deposited in the arterial tree. RESULTS: All the procedures were technically successful. A mean of 33% (range 3-50%; SD 21) of the administered technetium 99m dose was deposited in the arterial circulation during pseudoaneurysm therapy. No clinically evident arterial thrombosis was identified. CONCLUSION: Technetium 99m is routinely deposited in the arterial circulation following injection of a mixture of thrombin and technetium for therapy of common femoral artery pseudoaneurysms. This suggests that arterial passage of thrombin is more common than clinically evident.


Assuntos
Falso Aneurisma/tratamento farmacológico , Terapia Combinada/métodos , Embolia/tratamento farmacológico , Artéria Femoral/anormalidades , Cintilografia/métodos , Tecnécio/uso terapêutico , Trombina/uso terapêutico , Ultrassonografia de Intervenção/métodos , Idoso , Feminino , Humanos , Masculino , Estudos Prospectivos , Tecnécio/farmacologia , Trombina/farmacologia
18.
Gen Thorac Cardiovasc Surg ; 68(1): 18-23, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31177484

RESUMO

OBJECTIVES: Fibrin glue is used to reinforce anastomosis in aortic surgery. There has not yet been a consensus on how it should be applied optimally. This study aimed to define the optimal condition of applying fibrin glue. METHODS: In experiment 1, we determined the optimal condition for spraying fibrin glue using an expanded polytetrafluoroethylene graft within a needle hole. The length and area of the fibrin cap within the hole were measured. In experiment 2, methods for applying fibrinogen were assessed by comparing brushing and spraying. In experiment 3, swine aorta segments sutured with a Dacron graft were divided into the following three groups: nothing was applied; fibrinogen was sprayed and rubbed using brush. The aorta was clamped and blood was infused from an occlusion catheter inserted into the graft. The pressure at the first appearance of blood leak was recorded. RESULTS: In experiment 1, among the four groups divided by the pressure and distance of spraying, the fibrin cap area in the group with 0.075 MPa and 2-cm spray distance was significantly larger than that in the group with 0.15 MPa and 2 cm (P < 0.01). In experiment 2, the fibrin cap area in the brushing group was significantly larger than that in the spraying group (P < 0.05). In experiment 3, the capacity to resist endoluminal pressure was higher in the brushing and combined spraying group compared with that in the sequential combined spraying group (P < 0.01). CONCLUSIONS: The brush and spray methods showed excellent hemostatic outcomes.


Assuntos
Aorta Torácica/cirurgia , Adesivo Tecidual de Fibrina/farmacologia , Hemostáticos/farmacologia , Administração Tópica , Animais , Prótese Vascular , Cateterismo , Adesivo Tecidual de Fibrina/administração & dosagem , Fibrinogênio/administração & dosagem , Fibrinogênio/farmacologia , Hemostáticos/administração & dosagem , Polietilenotereftalatos , Politetrafluoretileno , Suínos , Trombina/administração & dosagem , Trombina/farmacologia
19.
Anal Chim Acta ; 1094: 57-69, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31761048

RESUMO

In this study, a combined targeted/untargeted UHPLC-ESI-QTOF-MS/MS method for the targeted quantitative analysis of the primary platelet lipid mediators thromboxane B2 (TXB2), 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) and its oxidation product 12-keto-5Z,8E,10E-heptadecatrienoic acid (KHT) was developed, which allowed simultaneous untargeted profiling for the detection of other lipid biomarkers such as other oxylipins and fatty acids (FAs) in platelet releasates. A general procedure for the synthesis of keto-analogs from hydroxylated polyunsaturated FAs (PUFAs) using Dess-Martin periodinane oxidation reagent was proposed for the preparation of KHT standard. MS detection was performed in data independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) in the range of 50-500 Da with variable window sizes. The LC-MS/MS assay was validated for the targeted analytes and applied for analysis of supernatants derived from resting platelets and from platelets treated with thrombin. The targeted analytes KHT, HHT and TXB2 were found at highly elevated levels in the activated platelet releasates. On average, 13 ±â€¯7, 15 ±â€¯9, and 0.6 ±â€¯0.2 attomols per platelet were released upon thrombin-activation. Furthermore, the simultaneous untargeted profiling (n = 8 in each group) revealed that these oxylipins are released with a pool of other (significantly upregulated) oxidized (12-HETE, 12-HEPE) and non-oxidized PUFAs. All these compounds can be considered additional biomarkers of platelet activation complementing the primary platelet activation marker thromboxane B2. The other lipids may support platelet activation or trigger other biological actions with some potential implications in thromboinflammation.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos/sangue , Ativação Plaquetária/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Trombina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/síntese química , Humanos , Limite de Detecção , Tromboxano B2/sangue
20.
Int J Mol Sci ; 20(24)2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31817594

RESUMO

Kindlins are important proteins for integrin signaling and regulation of the cytoskeleton, but we know little about their precise function and regulation in platelets during acute ischemic events. In this work, we investigated kindlin-3 protein levels in platelets isolated from patients with ST-elevation myocardial infarction (STEMI) compared to patients with non-ischemic chest pain. Platelets from twelve patients with STEMI and twelve patients with non-ischemic chest pain were isolated and analyzed for kindlin-3 protein levels and intracellular localization by immunoblotting and two-dimensional gel electrophoresis. Platelet proteome analysis by two-dimensional gel electrophoresis and protein sequencing identified kindlin-3 as a protein that is cleaved in platelets from patients with myocardial infarction. Kindlin-3 full-length protein was significantly decreased in patients with STEMI compared to patients with non-ischemic chest pain (1.0 ± 0.2 versus 0.28 ± 0.2, p < 0.05) by immunoblotting. Kindlin-3 showed a differential distribution and was primarily cleaved in the cytosolic and membrane compartment of platelets in myocardial infarction. Platelet activation with thrombin alone did not affect kindlin-3 protein levels. The present study demonstrates that kindlin-3 protein levels become significantly reduced in platelets of patients with myocardial infarction compared to controls. The results suggest that kindlin-3 cleavage in platelets is associated with the ischemic event of myocardial infarction.


Assuntos
Plaquetas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Infarto do Miocárdio com Supradesnível do Segmento ST/metabolismo , Idoso , Plaquetas/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Feminino , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Trombina/farmacologia
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