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1.
Arterioscler Thromb Vasc Biol ; 44(6): 1283-1301, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38572646

RESUMO

BACKGROUND: Glycoursodeoxycholic acid (GUDCA) has been acknowledged for its ability to regulate lipid homeostasis and provide benefits for various metabolic disorders. However, the impact of GUDCA on arterial thrombotic events remains unexplored. The objective of this study is to examine the effects of GUDCA on thrombogenesis and elucidate its underlying mechanisms. METHODS: Plasma samples from patients with arterial thrombotic events and diet-induced obese mice were collected to determine the GUDCA concentrations using mass spectrometry. Multiple in vivo murine thrombosis models and in vitro platelet functional assays were conducted to comprehensively evaluate the antithrombotic effects of GUDCA. Moreover, lipidomic analysis was performed to identify the alterations of intraplatelet lipid components following GUDCA treatment. RESULTS: Plasma GUDCA level was significantly decreased in patients with arterial thrombotic events and negatively correlated with thrombotic propensity in diet-induced obese mice. GUDCA exhibited prominent suppressing effects on platelet reactivity as evidenced by the attenuation of platelet activation, secretion, aggregation, spreading, and retraction (P<0.05). In vivo, GUDCA administration robustly alleviated thrombogenesis (P<0.05) without affecting hemostasis. Mechanistically, GUDCA inhibited DGK (diacylglycerol kinase) activity, leading to the downregulation of the phosphatidic acid-mediated signaling pathway. Conversely, phosphatidic acid supplementation was sufficient to abolish the antithrombotic effects of GUDCA. More importantly, long-term oral administration of GUDCA normalized the enhanced DGK activity, thereby remarkably alleviating the platelet hyperreactivity as well as the heightened thrombotic tendency in diet-induced obese mice (P<0.05). CONCLUSIONS: Our study implicated that GUDCA reduces platelet hyperreactivity and improves thrombotic propensity by inhibiting DGKs activity, which is a potentially effective prophylactic approach and promising therapeutic agent for arterial thrombotic events.


Assuntos
Plaquetas , Diacilglicerol Quinase , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Trombose , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/metabolismo , Trombose/prevenção & controle , Trombose/sangue , Trombose/enzimologia , Trombose/tratamento farmacológico , Humanos , Masculino , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/metabolismo , Camundongos , Ativação Plaquetária/efeitos dos fármacos , Feminino , Agregação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Pessoa de Meia-Idade , Fibrinolíticos/farmacologia , Estudos de Casos e Controles , Camundongos Obesos , Obesidade/tratamento farmacológico , Obesidade/enzimologia , Obesidade/sangue , Inibidores da Agregação Plaquetária/farmacologia
2.
J Thromb Haemost ; 22(7): 1806-1818, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38518897

RESUMO

There have been substantial advances in vascular protein disulfide isomerases (PDIs) in platelet function and thrombosis in recent years. There are 4 known prothrombotic thiol isomerases; PDI, endoplasmic reticulum protein (ERp)57, ERp72, and ERp46, and 1 antithrombotic PDI; transmembrane protein 1. A sixth PDI, ERp5, may exhibit either prothrombotic or antithrombotic properties in platelets. Studies on ERp46 in platelet function and thrombosis provide insight into the mechanisms by which these enzymes function. ERp46-catalyzed disulfide cleavage in the αIIbß3 platelet integrin occurs prior to PDI-catalyzed events to maximally support platelet aggregation. The transmembrane PDI transmembrane protein 1 counterbalances the effect of ERp46 by inhibiting activation of αIIbß3. Recent work on the prototypic PDI found that oxidized PDI supports platelet aggregation. The a' domain of PDI is constitutively oxidized, possibly by endoplasmic reticulum oxidoreductase-1α. However, the a domain is normally reduced but becomes oxidized under conditions of oxidative stress. In contrast to the role of oxidized PDI in platelet function, reduced PDI downregulates activation of the neutrophil integrin αMß2. Intracellular platelet PDI cooperates with Nox1 and contributes to thromboxane A2 production to support platelet function. Finally, αIIb and von Willebrand factor contain free thiols, which alter the functions of these proteins, although the extent to which the PDIs regulate these functions is unclear. We are beginning to understand the substrates and functions of vascular thiol isomerases and the redox network they form that supports hemostasis and thrombosis. Moreover, the disulfide bonds these enzymes target are being defined. The clinical implications of the knowledge gained are wide-ranging.


Assuntos
Plaquetas , Oxirredução , Isomerases de Dissulfetos de Proteínas , Trombose , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Plaquetas/metabolismo , Plaquetas/enzimologia , Trombose/sangue , Trombose/enzimologia , Animais , Agregação Plaquetária , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Compostos de Sulfidrila/metabolismo , Glicoproteínas de Membrana/metabolismo
3.
Int J Mol Sci ; 23(1)2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35008919

RESUMO

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s-1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.


Assuntos
Resistência ao Cisalhamento , Quinase Syk/metabolismo , Trombose/enzimologia , Colágeno/metabolismo , Humanos , Fosforilação , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Anticorpos de Domínio Único/metabolismo , Quinase Syk/antagonistas & inibidores , Temperatura , Trombina/farmacologia
4.
J Am Soc Nephrol ; 32(11): 2834-2850, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34716244

RESUMO

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.


Assuntos
Indicã/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/sangue , Cinurenina/fisiologia , Terapia de Alvo Molecular , Complicações Pós-Operatórias/enzimologia , Insuficiência Renal Crônica/enzimologia , Trombose/enzimologia , Procedimentos Cirúrgicos Vasculares/efeitos adversos , Animais , Aorta , Lesões das Artérias Carótidas/complicações , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/prevenção & controle , Meios de Cultura/farmacologia , Indução Enzimática/efeitos dos fármacos , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/deficiência , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Insuficiência Renal Crônica/tratamento farmacológico , Tromboplastina/metabolismo , Trombose/sangue , Trombose/etiologia , Trombose/prevenção & controle , Triptofano/metabolismo , Uremia/sangue
5.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070931

RESUMO

It is now about 20 years since the first case of a gain-of-function mutation involving the as-yet-unknown actor in cholesterol homeostasis, proprotein convertase subtilisin/kexin type 9 (PCSK9), was described. It was soon clear that this protein would have been of huge scientific and clinical value as a therapeutic strategy for dyslipidemia and atherosclerosis-associated cardiovascular disease (CVD) management. Indeed, PCSK9 is a serine protease belonging to the proprotein convertase family, mainly produced by the liver, and essential for metabolism of LDL particles by inhibiting LDL receptor (LDLR) recirculation to the cell surface with the consequent upregulation of LDLR-dependent LDL-C levels. Beyond its effects on LDL metabolism, several studies revealed the existence of additional roles of PCSK9 in different stages of atherosclerosis, also for its ability to target other members of the LDLR family. PCSK9 from plasma and vascular cells can contribute to the development of atherosclerotic plaque and thrombosis by promoting platelet activation, leukocyte recruitment and clot formation, also through mechanisms not related to systemic lipid changes. These results further supported the value for the potential cardiovascular benefits of therapies based on PCSK9 inhibition. Actually, the passive immunization with anti-PCSK9 antibodies, evolocumab and alirocumab, is shown to be effective in dramatically reducing the LDL-C levels and attenuating CVD. While monoclonal antibodies sequester circulating PCSK9, inclisiran, a small interfering RNA, is a new drug that inhibits PCSK9 synthesis with the important advantage, compared with PCSK9 mAbs, to preserve its pharmacodynamic effects when administrated every 6 months. Here, we will focus on the major understandings related to PCSK9, from its discovery to its role in lipoprotein metabolism, involvement in atherothrombosis and a brief excursus on approved current therapies used to inhibit its action.


Assuntos
Aterosclerose/genética , LDL-Colesterol/metabolismo , Dislipidemias/genética , Placa Aterosclerótica/genética , Pró-Proteína Convertase 9/genética , Trombose/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Aterosclerose/tratamento farmacológico , Aterosclerose/enzimologia , Aterosclerose/patologia , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Plaquetas/patologia , LDL-Colesterol/antagonistas & inibidores , Dislipidemias/tratamento farmacológico , Dislipidemias/enzimologia , Dislipidemias/patologia , Fibrinolíticos/uso terapêutico , Regulação da Expressão Gênica , Humanos , Hipolipemiantes/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Inibidores de PCSK9 , Placa Aterosclerótica/tratamento farmacológico , Placa Aterosclerótica/enzimologia , Placa Aterosclerótica/patologia , Ativação Plaquetária/efeitos dos fármacos , Pró-Proteína Convertase 9/biossíntese , RNA Interferente Pequeno/uso terapêutico , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transdução de Sinais , Trombose/enzimologia , Trombose/patologia , Trombose/prevenção & controle
6.
Prep Biochem Biotechnol ; 51(2): 191-200, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32845203

RESUMO

Fibrinolytic enzymes have been considered promising for treatment and protection of healthy circulation due its ability to dissolve the fibrin in blood clots. Extractive fermentation is a not explored and efficient downstream process which segregates the desired product simultaneously in a fermentation process fast and economically. Extraction of fibrinolytic enzymes by Bacillus stearothermophilus DPUA 1729 employing conventional aqueous two-phase systems (ATPS) and extractive fermentation with ATPS was evaluated. The results of both systems were compared using a factorial design with PEG molar mass, PEG and salt concentrations as independent variables and extraction parameters as a response. In all conditions evaluated it was observed a similar partitioning of fibrinolytic enzymes through the phases, both in conventional ATPS and extractive fermentation. Salt concentration and interaction among PEG and salt concentration influenced in the partition coefficient. The fibrinolytic activity was determined by hydrolysis of fibrin in plate using the extract of one condition from extractive fermentation. The zone degradation presented a diameter of 7.03 ± 0.94 mm. In conclusion, there was no significant difference among the results obtained using conventional ATPS and extractive fermentation, however, the second one presents more advantages and can integrate production and extraction in one single step, reducing the costs.


Assuntos
Fermentação , Geobacillus stearothermophilus/metabolismo , Peptídeo Hidrolases/metabolismo , Trombose/enzimologia , Animais , Fibrinólise , Hidrólise , Testes de Sensibilidade Microbiana , Polietilenoglicóis , Ratos , Ratos Wistar , Software , Alimentos de Soja , Sulfatos , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tipo Uroquinase/química , Água
7.
J Ethnopharmacol ; 267: 113508, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33169693

RESUMO

Influence of quail egg on pathologies has increased research interests and series of investigations are currently being done on its influence against these pathologies. The influence of quail egg against 2-butoxyethanol induced hemolysis and disseminated thrombosis was investigated to determine the enzymatic regulations that ensue in the amelioration of deleterious hemolytic and disseminated thrombosis displayed in female Wistar rats. Quail egg was separated into three (3) components (extracts)-quail egg yolk water soluble (QYWS) and fat soluble (QYFS), and albumen extract (QA) and the inorganic and organic compositions were characterized. Depranocytotic assaults was achieved by 250 mg/kg of 2-Butoxyethanol administered for 4 days, the clinical observation revealed a dark purple-red discoloration on the distal tails of the rats and therapeutic applications followed with 1000 mg/kg BWT of QYWS, QYFS and QA, and 15 mg/kg BWT of hydroxyurea. Morphological evaluation, haematological estimations and biochemical evaluations of the influence on the activities of sphingosine kinase-1, RNase, red cell carbonic anhydrase, lactate dehydrogenase, glutathione peroxidase and caspase-3, vis a vis the concentrations of sphingosine-1 phosphate, selenium and zinc (plasma and urine). In vitro anti-inflammatory influence of quail egg components were investigated against hemolysis and key enzymes of inflammation-cycloxygenase, lipoxygenase and ß-glucuronidase. The in vitro anti-inflammatory effects of QYWS, QYFS and QA were concentration dependent from 200 to 800 µg/ml against hemolysis and the key enzymes of inflammation. The characterization of inorganic and organic bioactive composition of the yolk and albumen revealed the presence of folic acid, cobalamin, pyridine, riboflavin, ascorbic acid as well as vitamins D and E, selenium, zinc, iron and calcium. These had reflected in the attenuation of the induced hemolytic and disseminated thrombosis by regulations of enzymes linked to the infarction, apoptosis and oxidative stress characterized in sickle cell index.


Assuntos
Anemia Falciforme/prevenção & controle , Antidrepanocíticos/farmacologia , Extratos Celulares/farmacologia , Coturnix , Ovos , Enzimas/sangue , Eritrócitos/efeitos dos fármacos , Etilenoglicóis , Hemólise/efeitos dos fármacos , Trombose/prevenção & controle , Anemia Falciforme/sangue , Anemia Falciforme/induzido quimicamente , Anemia Falciforme/enzimologia , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Antidrepanocíticos/isolamento & purificação , Apoptose/efeitos dos fármacos , Extratos Celulares/isolamento & purificação , Modelos Animais de Doenças , Eritrócitos/enzimologia , Eritrócitos/patologia , Feminino , Fibrinolíticos/farmacologia , Mediadores da Inflamação/metabolismo , Estresse Oxidativo , Ratos Wistar , Trombose/sangue , Trombose/induzido quimicamente , Trombose/enzimologia
8.
Circ Res ; 128(3): 386-400, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33292062

RESUMO

RATIONALE: Current thrombolytic agents activate plasminogen to plasmin which triggers fibrinolysis to dissolve thrombi. Since plasmin is a nonspecific proteolytic enzyme, all of the current plasmin-dependent thrombolytics lead to serious hemorrhagic complications, demanding a new class of fibrinolytic enzymes independent from plasmin activation and undesirable side effects. We speculated that the mammalian version of bacterial heat-shock proteins could selectively degrade intravascular thrombi, a typical example of a highly aggregated protein mixture. OBJECTIVE: The objective of this study is to identify enzymes that can dissolve intravascular thrombi specifically without affecting fibrinogen and fibronectin so that the wound healing processes remain uninterrupted and tissues are not damaged. In this study, HtrA (high-temperature requirement A) proteins were tested for its specific proteolytic activity on intravascular thrombi independently from plasmin activation. METHODS AND RESULTS: HtrA1 and HtrA2/Omi proteins, collectively called as HtrAs, lysed ex vivo blood thrombi by degrading fibrin polymers. The thrombolysis by HtrAs was plasmin-independent and specific to vascular thrombi without causing the systemic activation of plasminogen and preventing nonspecific proteolysis of other proteins including fibrinogen and fibronectin. As expected, HtrAs did not disturb clotting and wound healing of excised wounds from mouse skin. It was further confirmed in a tail bleeding and a rebleeding assay that HtrAs allowed normal clotting and maintenance of clot stability in wounds, unlike other thrombolytics. Most importantly, HtrAs completely dissolved blood thrombi in tail thrombosis mice, and the intravenous injection of HtrAs to mice with pulmonary embolism completely dissolved intravascular thrombi and thus rescued thromboembolism. CONCLUSIONS: Here, we identified HtrA1 and HtrA2/Omi as plasmin-independent and highly specific thrombolytics that can dissolve intravascular thrombi specifically without bleeding risk. This work is the first report of a plasmin-independent thrombolytic pathway, providing HtrA1 and HtrA2/Omi as ideal therapeutic candidates for various thrombotic diseases without hemorrhagic complications.


Assuntos
Fibrina/metabolismo , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Serina Peptidase 1 de Requerimento de Alta Temperatura A/farmacologia , Serina Peptidase 2 de Requerimento de Alta Temperatura A/farmacologia , Embolia Pulmonar/tratamento farmacológico , Trombose/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Fibrinolíticos/toxicidade , Hemorragia/induzido quimicamente , Serina Peptidase 1 de Requerimento de Alta Temperatura A/toxicidade , Serina Peptidase 2 de Requerimento de Alta Temperatura A/toxicidade , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Embolia Pulmonar/sangue , Embolia Pulmonar/enzimologia , Proteínas Recombinantes/farmacologia , Trombose/sangue , Trombose/enzimologia , Cicatrização/efeitos dos fármacos
9.
Biochem J ; 477(22): 4327-4342, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33242335

RESUMO

Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, ß, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2ß), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.


Assuntos
Plaquetas/enzimologia , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Trombose/enzimologia , Trombose/terapia , Animais , Plaquetas/patologia , Humanos , Isoenzimas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Trombose/patologia
10.
Arterioscler Thromb Vasc Biol ; 40(10): e262-e272, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32814440

RESUMO

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.


Assuntos
Coagulação Sanguínea , Plaquetas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Janus Quinase 2/sangue , Megacariócitos/enzimologia , Ativação Plaquetária , Mielofibrose Primária/enzimologia , Trombose/enzimologia , Animais , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/genética , Modelos Animais de Doenças , Janus Quinase 2/genética , Camundongos Transgênicos , Mutação , Agregação Plaquetária , Mielofibrose Primária/sangue , Mielofibrose Primária/genética , Trombopoese , Trombose/sangue , Trombose/genética
11.
Arterioscler Thromb Vasc Biol ; 40(10): 2391-2403, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787521

RESUMO

OBJECTIVE: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. CONCLUSIONS: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.


Assuntos
Plaquetas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Moléculas de Adesão Celular Neuronais/sangue , Proteínas da Matriz Extracelular/sangue , Proteínas do Tecido Nervoso/sangue , Neuropeptídeos/sangue , Fosfolipase C gama/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Serina Endopeptidases/sangue , Trombose/enzimologia , Proteínas rac1 de Ligação ao GTP/sangue , Proteína rhoA de Ligação ao GTP/sangue , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Coagulação Sanguínea , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/etiologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Retração do Coágulo , Citoesqueleto/enzimologia , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Camundongos da Linhagem 129 , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Ativação Plaquetária , Proteína Reelina , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Trombose/sangue , Trombose/etiologia
12.
Open Heart ; 7(1)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32532805

RESUMO

The high rate of thrombotic complications associated with COVID-19 seems likely to reflect viral infection of vascular endothelial cells, which express the ACE2 protein that enables SARS-CoV-2 to invade cells. Various proinflammatory stimuli can promote thrombosis by inducing luminal endothelial expression of tissue factor (TF), which interacts with circulating coagulation factor VII to trigger extrinsic coagulation. The signalling mechanism whereby these stimuli evoke TF expression entails activation of NADPH oxidase, upstream from activation of the NF-kappaB transcription factor that drives the induced transcription of the TF gene. When single-stranded RNA viruses are taken up into cellular endosomes, they stimulate endosomal formation and activation of NADPH oxidase complexes via RNA-responsive toll-like receptor 7. It is therefore proposed that SARS-CoV-2 infection of endothelial cells evokes the expression of TF which is contingent on endosomal NADPH oxidase activation. If this hypothesis is correct, hydroxychloroquine, spirulina (more specifically, its chromophore phycocyanobilin) and high-dose glycine may have practical potential for mitigating the elevated thrombotic risk associated with COVID-19.


Assuntos
Betacoronavirus/patogenicidade , Coagulação Sanguínea , Infecções por Coronavirus/virologia , Endossomos/virologia , Células Endoteliais/virologia , NADPH Oxidases/metabolismo , Pneumonia Viral/virologia , Tromboplastina/metabolismo , Trombose/virologia , Animais , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Coagulação Sanguínea/efeitos dos fármacos , COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/enzimologia , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática , Fibrinolíticos/uso terapêutico , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , SARS-CoV-2 , Transdução de Sinais , Trombose/sangue , Trombose/enzimologia , Trombose/prevenção & controle , Tratamento Farmacológico da COVID-19
13.
J Vasc Surg ; 72(3): 1025-1034, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32067878

RESUMO

OBJECTIVE: The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. METHODS: Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. RESULTS: A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. CONCLUSIONS: These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.


Assuntos
Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Elastase de Leucócito/análise , Imageamento por Ressonância Magnética , Metaloproteinase 9 da Matriz/análise , Trombose/diagnóstico por imagem , Idoso , Aorta Abdominal/enzimologia , Aorta Abdominal/cirurgia , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/cirurgia , Colágeno Tipo III/análise , Estudos Transversais , Elastina/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Proteoglicanas/análise , Proteólise , Trombose/enzimologia , Trombose/cirurgia
14.
Arterioscler Thromb Vasc Biol ; 40(3): e65-e77, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31893947

RESUMO

OBJECTIVE: In patients with diabetes mellitus, increased platelet reactivity predicts cardiac events. Limited evidence suggests that DPP-4 (dipeptidyl peptidase 4) influences platelets via GLP-1 (glucagon-like peptide 1)-dependent effects. Because DPP-4 inhibitors are frequently used in diabetes mellitus to improve the GLP-1-regulated glucose metabolism, we characterized the role of DPP-4 inhibition and of native intact versus DPP-4-cleaved GLP-1 on flow-dependent thrombus formation in mouse and human blood. Approach and Results: An ex vivo whole blood microfluidics model was applied to approach in vivo thrombosis and study collagen-dependent platelet adhesion, activation, and thrombus formation under shear-flow conditions by multiparameter analyses. In mice, in vivo inhibition or genetic deficiency of DPP-4 (Dpp4-/-), but not of GLP-1-receptors (Glp1r-/-), suppressed flow-dependent platelet aggregation. In human blood, GLP-1(7-36), but not DPP-4-cleaved GLP-1(9-36), reduced thrombus volume by 32% and impaired whole blood thrombus formation at both low/venous and high/arterial wall-shear rates. These effects were enforced upon ADP costimulation and occurred independently of plasma factors and leukocytes. Human platelets did not contain detectable levels of GLP-1-receptor transcripts. Also, GLP-1(7-36) did not inhibit collagen-induced aggregation under conditions of stirring or stasis of platelets, pointing to a marked flow-dependent role. CONCLUSIONS: Native, intact GLP-1 is a natural suppressor of thrombus growth under physiological flow conditions, with DPP-4 inhibition and increased intact GLP-1 suppressing platelet aggregation under flow without a main relevance of GLP-1-receptor on platelets.


Assuntos
Plaquetas/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Fibrinolíticos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Linagliptina/farmacologia , Fosfato de Sitagliptina/farmacologia , Trombose/prevenção & controle , Animais , Plaquetas/metabolismo , Dipeptidil Peptidase 4/genética , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Transdução de Sinais , Trombose/enzimologia , Trombose/genética
15.
Platelets ; 31(2): 167-173, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30973035

RESUMO

Stent thrombosis remains an infrequent but significant complication following percutaneous coronary intervention. Preclinical models to rapidly screen and validate therapeutic compounds for efficacy are lacking. Herein, we describe a reproducible, high throughput and cost-effective method to evaluate candidate therapeutics and devices for either treatment or propensity to develop stent thrombosis in an in vitro bench-top model. Increasing degree of stent malapposition (0.00 mm, 0.10 mm, 0.25 mm and 0.50 mm) was associated with increasing thrombosis and luminal area occlusion (4.1 ± 0.5%, 6.3 ± 0.5%, 19.7 ± 4.5%, and 92.6 ± 7.4%, p < 0.0001, respectively). Differences in stent design in the form of bare-metal, drug-eluting, and bioresorbable vascular scaffolds demonstrated differences in stent thrombus burden (14.7 ± 3.8% vs. 20.5 ± 3.1% vs. 86.8 ± 5.3%, p < 0.01, respectively). Finally, thrombus burden was significantly reduced when healthy blood samples were incubated with Heparin, ASA/Ticagrelor (DAPT), and Heparin+DAPT compared to control (DMSO) at 4.1 ± 0.6%, 6.9 ± 1.7%, 4.5 ± 1.2%, and 12.1 ± 1.8%, respectively (p < 0.01). The reported model produces high throughput reproducible thrombosis results across a spectrum of antithrombotic agents, stent design, and degrees of apposition. Importantly, performance recapitulates clinical observations of antiplatelet/antithrombotic regimens as well as device and deployment characteristics. Accordingly, this model may serve as a screening tool for candidate therapies in preclinical evaluation.


Assuntos
Trombose Coronária/etiologia , Stents/efeitos adversos , Fenômenos Fisiológicos Sanguíneos/efeitos dos fármacos , Trombose Coronária/complicações , Trombose Coronária/diagnóstico por imagem , Trombose Coronária/enzimologia , Stents Farmacológicos/efeitos adversos , Enzimas/sangue , Humanos , Técnicas In Vitro , Modelos Biológicos , Intervenção Coronária Percutânea/efeitos adversos , Inibidores da Agregação Plaquetária/uso terapêutico , Trombose/sangue , Trombose/complicações , Trombose/enzimologia , Tomografia de Coerência Óptica
16.
Mol Neurobiol ; 57(1): 226-238, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31493239

RESUMO

In ischemic stroke, vascular occlusion rapidly induces tissue infarct. Over the ensuing hours, damage spreads to adjacent tissue and forms transition zone (penumbra), which is potentially salvageable. Epigenetic regulation of chromatin structure controls gene expression and protein synthesis. We studied the expression of histone deacetylases HDAC1 and HDAC2 in the penumbra at 4 or 24 h after photothrombotic stroke (PTS) in the rat brain cortex. PTS increased the expression of HDAC1 and HDAC2 in penumbra and caused the redistribution of HDAC1 but not HDAC2 from the neuronal nuclei to cytoplasm. In astrocytes, HDAC1 expression and localization did not change. In neurons, HDAC2 localized exclusively in nuclei, but in astrocytes, it was also observed in processes. PTS induced neuronal apoptosis in the penumbra. TUNEL-stained apoptotic neurons co-localized with HDAC2 but not HDAC1. These data suggest that HDAC2 may represent the potential target for anti-stroke therapy and its selective inhibition may be a promising strategy for the protection of the penumbra tissue after ischemic stroke.


Assuntos
Apoptose , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Luz , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/enzimologia , Trombose/complicações , Trombose/enzimologia , Animais , Astrócitos/metabolismo , Biomarcadores/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Ratos Wistar
17.
Handb Exp Pharmacol ; 259: 79-88, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31541323

RESUMO

Functions for phospholipase D1 and D2 (PLD1 and PLD2), the canonical isoforms of the PLD superfamily in mammals, have been explored using cell biological and animal disease models for two decades. PLD1 and PLD2, which are activated as a consequence of extracellular signaling events and generate the second messenger signaling lipid phosphatidic acid (PA), have been reported to play roles in settings ranging from platelet activation to the response to cardiac ischemia, viral infection, neurodegenerative disease, and cancer. Of these, the most tractable as therapeutic targets may be thrombotic disease and cancer, as will be discussed here in the context of ongoing efforts to develop small molecule PLD inhibitors.


Assuntos
Inibidores Enzimáticos/farmacologia , Neoplasias/enzimologia , Fosfolipase D/antagonistas & inibidores , Trombose/enzimologia , Animais , Humanos , Isoenzimas/antagonistas & inibidores , Transdução de Sinais
18.
Cytotherapy ; 21(4): 444-459, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30904331

RESUMO

BACKGROUND: Endothelial progenitor cells (EPCs) are circulating progenitor cells that can play an essential role in vascular remodelling. In this work, we compared the role of two EPCs cultivated with different mediums in the resolution of the arterial thrombus induced by FeCl3 lesion and in vessel re-endothelization in the mouse carotid artery. METHODS: Mice mononuclear cells were differentiated into EPCs using Dulbecco's Modified Eagle's Medium (DMEM) and vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and IGF (Insulin Growth Factor) called EPCs--M1) or with EGM2(endothelial growth medium) (media supplemented with growth factors from Lonza called (EPCs-M2) for 30days and characterized using flow cytometry. The animals received three EPC injections post-lesion, and we analyzed thrombosis time, vessel re-endothelization, metalloproteinases activities, eNOS (endothelial Nitric oxide synthase) presence and SDF-1(Stromal Derived Factor- 1) levels in circulation. RESULTS: EPC-M1 presented a more immature progenitor profile than EPC-M2 cells. The injection of EPC-M1 prolonged the thrombosis time, and the treatment with the different EPCs increased eNOS expression and MMP2 (Metalloproteinase 2) activity and decreased SDF-1 in plasma. Only EPC-M1 treatment increased both MMP2 and MMP9 and reduced thrombus after 7days. Also, both EPCs decreased platelet aggregation in vitro. CONCLUSIONS: EPCs-M1 were more efficient in all of the analyzed assays. EPCsM2 may be a more mature EPC, proliferating less and promoting a less significant matrix remodelling. EPCs can promote vascular remodelling by inhibiting thrombosis and stimulating vascular wall remodelling and the treatment with a more immature progenitor may be more efficient in this process.


Assuntos
Células Progenitoras Endoteliais/transplante , Trombose/terapia , Animais , Artérias/patologia , Diferenciação Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Embolização Terapêutica , Células Progenitoras Endoteliais/metabolismo , Gelatinases/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Agregação Plaquetária , Trombose/enzimologia , Trombose/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Pediatr Blood Cancer ; 66(6): e27719, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30900794

RESUMO

Asparaginase therapy induces a transient antithrombin III (ATIII) deficiency, which contributes to the risk of asparaginase-induced thrombosis. At Cincinnati Children's Hospital Medical Center, management of asparaginase-induced thrombosis includes ATIII supplementation during therapeutic anticoagulation with enoxaparin. Due to the expense associated with ATIII, a capped dosing approach for ATIII was evaluated in this population. Peak ATIII levels were obtained following capped doses to evaluate response. In this pilot evaluation, 11 patients received a total of 138 capped doses for a total cost savings of $803 782. This pilot evaluation represents the first reported analysis of capped ATIII dosing in oncology patients.


Assuntos
Deficiência de Antitrombina III/tratamento farmacológico , Deficiência de Antitrombina III/economia , Antitrombina III/economia , Asparaginase/efeitos adversos , Análise Custo-Benefício , Enoxaparina/economia , Trombose/tratamento farmacológico , Adolescente , Adulto , Anticoagulantes/administração & dosagem , Anticoagulantes/economia , Antitrombina III/administração & dosagem , Antitrombina III/metabolismo , Deficiência de Antitrombina III/induzido quimicamente , Criança , Quimioterapia Combinada , Enoxaparina/administração & dosagem , Feminino , Seguimentos , Humanos , Masculino , Projetos Piloto , Prognóstico , Estudos Retrospectivos , Trombose/enzimologia , Trombose/patologia , Adulto Jovem
20.
Scand Cardiovasc J ; 53(1): 1-8, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30741027

RESUMO

OBJECTIVES: Nitric oxide (NO) represents the most powerful endogenous molecule with vasodilator action mainly produced by endothelial nitric oxide synthase (eNOS) enzyme. Polymorphisms and epigenetic-sensitive mechanisms can modulate the expression of eNOS gene, leading to the endothelial dysfunction. This review updates on the mechanistic role of NO in the regulation of platelet activation, as well as the impact of eNOS genetic and epigenetic modifications on arterial thrombosis onset. DESIGN: A systematic search was addressed to examination of PubMed databases with the following terms: nitric oxide; arterial thrombosis; endothelial dysfunction; DNA variations; epigenetic modifications; personalized therapy; network medicine. RESULTS: G894T, -786T/C, and 4b/4a variable number tandem repeat (VNTR), are the main classes of polymorphisms harbored in eNOS gene associated to increased arterial thrombosis risk. DNA methylation, histone/non-histone modifications, and microRNA (miRNAs) can modulate eNOS gene expression. Investigators largely focused on the role of miRNAs in modulating NO production in arterial thrombosis development. In detail, miR-195, and miR-582 are inversely correlated both to eNOS and NO levels, thus suggesting novel biomarkers. CONCLUSION: We are far from incorporating omics pathogenic data from bench to arterial thrombosis bedside. Network medicine is an emerging paradigm that ideally overcomes the current shortcomings of the reductionist approach. Despite several clinical limitations, the network-based analysis of the interactome might reveal the key nodes underlying the perturbations of the arterial thrombosis, thus advancing personalized therapy.


Assuntos
Arteriopatias Oclusivas/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Trombose/enzimologia , Animais , Arteriopatias Oclusivas/sangue , Arteriopatias Oclusivas/genética , Arteriopatias Oclusivas/fisiopatologia , Epigênese Genética , Predisposição Genética para Doença , Humanos , Repetições Minissatélites , Óxido Nítrico Sintase Tipo III/genética , Fenótipo , Ativação Plaquetária , Polimorfismo Genético , Fatores de Risco , Trombose/sangue , Trombose/genética , Trombose/fisiopatologia , Vasodilatação
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