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Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.
Assuntos
Envelhecimento , Plaquetas , Diferenciação Celular , Células-Tronco Hematopoéticas , Trombose , Animais , Células-Tronco Hematopoéticas/metabolismo , Plaquetas/metabolismo , Trombose/patologia , Trombose/metabolismo , Camundongos , Humanos , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Células Progenitoras de Megacariócitos/metabolismo , MasculinoRESUMO
We report here that expression of the ribosomal protein, RPL22, is frequently reduced in human myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML); reduced RPL22 expression is associated with worse outcomes. Mice null for Rpl22 display characteristics of an MDS-like syndrome and develop leukemia at an accelerated rate. Rpl22-deficient mice also display enhanced hematopoietic stem cell (HSC) self-renewal and obstructed differentiation potential, which arises not from reduced protein synthesis but from increased expression of the Rpl22 target, ALOX12, an upstream regulator of fatty acid oxidation (FAO). The increased FAO mediated by Rpl22-deficiency also persists in leukemia cells and promotes their survival. Altogether, these findings reveal that Rpl22 insufficiency enhances the leukemia potential of HSC via non-canonical de-repression of its target, ALOX12, which enhances FAO, a process that may serve as a therapeutic vulnerability of Rpl22 low MDS and AML leukemia cells. Highlights: RPL22 insufficiency is observed in MDS/AML and is associated with reduced survivalRpl22-deficiency produces an MDS-like syndrome and facilitates leukemogenesisRpl22-deficiency does not impair global protein synthesis by HSCRpl22 controls leukemia cell survival by non-canonical regulation of lipid oxidation eTOC: Rpl22 controls the function and transformation potential of hematopoietic stem cells through effects on ALOX12 expression, a regulator of fatty acid oxidation.
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Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Trombose , Camundongos , Animais , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Ligantes , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Anticoagulantes/efeitos adversos , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Hemorragia/tratamento farmacológico , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêuticoRESUMO
Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.
Assuntos
Araquidonato 12-Lipoxigenase , Plaquetas , Humanos , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Plaquetas/metabolismo , Polimorfismo de Nucleotídeo Único , Deutério , Medição da Troca de Deutério , Lipossomos/metabolismo , Hidrogênio/metabolismoRESUMO
Coronavirus-associated coagulopathy (CAC) is a morbid and lethal sequela of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. CAC results from a perturbed balance between coagulation and fibrinolysis and occurs in conjunction with exaggerated activation of monocytes/macrophages (MO/Mφs), and the mechanisms that collectively govern this phenotype seen in CAC remain unclear. Here, using experimental models that use the murine betacoronavirus MHVA59, a well-established model of SARS-CoV-2 infection, we identify that the histone methyltransferase mixed lineage leukemia 1 (MLL1/KMT2A) is an important regulator of MO/Mφ expression of procoagulant and profibrinolytic factors such as tissue factor (F3; TF), urokinase (PLAU), and urokinase receptor (PLAUR) (herein, "coagulopathy-related factors") in noninfected and infected cells. We show that MLL1 concurrently promotes the expression of the proinflammatory cytokines while suppressing the expression of interferon alfa (IFN-α), a well-known inducer of TF and PLAUR. Using in vitro models, we identify MLL1-dependent NF-κB/RelA-mediated transcription of these coagulation-related factors and identify a context-dependent, MLL1-independent role for RelA in the expression of these factors in vivo. As functional correlates for these findings, we demonstrate that the inflammatory, procoagulant, and profibrinolytic phenotypes seen in vivo after coronavirus infection were MLL1-dependent despite blunted Ifna induction in MO/Mφs. Finally, in an analysis of SARS-CoV-2 positive human samples, we identify differential upregulation of MLL1 and coagulopathy-related factor expression and activity in CD14+ MO/Mφs relative to noninfected and healthy controls. We also observed elevated plasma PLAU and TF activity in COVID-positive samples. Collectively, these findings highlight an important role for MO/Mφ MLL1 in promoting CAC and inflammation.
Assuntos
COVID-19 , Histona-Lisina N-Metiltransferase , Animais , Humanos , Camundongos , COVID-19/complicações , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Inflamação/metabolismo , Monócitos/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , SARS-CoV-2/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND: This study evaluated E-selectin inhibition with GMI-1271 (Uproleselan [GMI]) alone and in combination with the standard of care low-molecular-weight heparin (LMWH) to improve vein recanalization, decrease vein wall inflammation and protect against adverse bleeding in a primate model. We sought to examine this novel treatment of venous thrombosis. METHODS: Using a well-documented primate animal model, iliac vein thrombosis was induced by balloon occlusion of the iliac vein for 6 hours. Starting on day 2 after thrombosis, animals began treatment in two phases. In phase one, nontreated controls received no treatment (n = 5) vs animals treated with the E-selectin inhibitor GMI, 25 mg/kg, subcutaneous (SC), once daily (n = 4) for 21 days (previously published data). In phase two, animals were treated with GMI plus a combination of LMWH 1.5 mg/kg or 40 mg (GMI + LMWHc) SC once daily (n = 8) for 19 days; and animals treated with LMWH 1.5 mg/kg or 40 mg (LMWHc) SC once daily (n = 6) for 19 days. Animals were evaluated by magnetic resonance venography for vein recanalization and inflammation by gadolinium extravasation, duplex ultrasound, coagulation tests (thromboelastography, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen) and complete blood count at baseline, days 2, 7, 14, and 21 at euthanasia. Statistical analysis included using unpaired t test with Welch's correction for direct comparisons and one-way analysis of variance for comparison between the groups. RESULTS: Percent vein recanalization by magnetic resonance venography was highest in the GMI alone group followed by GMI + LMWHc, both significantly different from control. On ultrasound examination, animals treated with GMI alone had no decrease in open vein lumen by day 21, whereas decreases were observed in groups GMI + LMWHc (-26%), LMWHc (-27%), and controls (-80%). Vein wall inflammation decreased significantly in all treated groups. Intimal fibrosis and intimal thickness was best preserved in the GMI alone group. An analysis of total vein wall collagen revealed a trend in all treatment groups of decreasing vein wall collagen. No clinically significant bleeding events were noted in any group. The LMWH groups trended to have prolonged coagulation test values, whereas E-selectin inhibition with GMI did not cause clinically significant changes in coagulation measures. CONCLUSIONS: Treatment with E-selectin inhibition results in improved vein recanalization, a decrease in vein wall inflammation and vein wall intimal thickness and fibrosis, with no changes in markers of coagulation. E-selectin inhibition with GMI alone is superior to E-selectin inhibition combined with LMWH, LMWH alone, and no treatment in this deep vein thrombosis model of iliac vein thrombosis.
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Anticoagulantes/uso terapêutico , Selectina E/antagonistas & inibidores , Glicolipídeos/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Trombose Venosa/tratamento farmacológico , Animais , PapioRESUMO
BACKGROUND: Immune checkpoint inhibition is effective in several cancers. Expression of programmed death-ligand 1 (PD-L1) on circulating tumor or immune effector cells could provide insights into selection of patients for immune checkpoint inhibition. METHODS: Whole blood was collected at serial timepoints from metastatic breast cancer patients and healthy donors for circulating tumor cell (CTC) and platelet PD-L1 analysis with a phycoerythrin-labeled anti-human PD-L1 monoclonal antibody (Biolegend clone 29E.2A3) using the CellSearch® assay. CTC PD-L1 was considered positive if detected on at least 1% of the cells; platelet PD-L1 was considered positive if ≥100 platelets per CellSearch frame expressed PD-L1. RESULTS: A total of 207 specimens from 124 metastatic breast cancer patients were collected. 52/124 (42%) samples at timepoint-1 (at or close to time of progressive disease) had ≥5 CTC/7.5ml whole blood. Of those, 21 (40%) had positive CTC PD-L1. In addition, platelet PD-L1 expression was observed in 35/124 (28%) at timepoint-1. Platelet PD-L1 was not detected in more than 70 specimens from 12 healthy donors. Platelet PD-L1 was associated with ≥5 CTC/7.5ml whole blood (p = 0.0002), less likely in patients with higher red blood cell counts (OR = 0.72, p<0.001) and a history of smoking tobacco (OR = 0.76, p<0.001). Platelet PD-L1 staining was not associated with tumor marker status, recent procedures or treatments, platelet-affecting drugs, or CTC PD-L1 expression. CONCLUSION: PD-L1 expression was found in metastatic breast cancer patients on both CTC and platelets in an independent fashion. Inter-patient platelet PD-L1 expression was highly heterogeneous suggesting that it is a biological event associated with cancer in some but not all patients. Taken together, our data suggest that CTC and platelet PD-L1 expression could play a role in predicting which patients should receive immune checkpoint inhibition and as a pharmacodynamics biomarker during treatment.
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Antígeno B7-H1/metabolismo , Plaquetas/metabolismo , Neoplasias da Mama/metabolismo , Células Neoplásicas Circulantes/metabolismo , Regulação para Cima , Antígeno B7-H1/genética , Neoplasias da Mama/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Metástase NeoplásicaRESUMO
Gene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents.
Assuntos
Carbazóis/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Trombose/prevenção & controle , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Músculos Abdominais/irrigação sanguínea , Trifosfato de Adenosina/metabolismo , Animais , Arteríolas , Carbazóis/administração & dosagem , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Lasers , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Selectina-P/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
Uncontrolled bleeding associated with trauma and surgery is the leading cause of preventable death. Batroxobin, a snake venom-derived thrombin-like serine protease, has been shown to clot fibrinogen by cleaving fibrinopeptide A in a manner distinctly different from thrombin, even in the presence of heparin. The biochemical properties of batroxobin and its effect on coagulation have been well characterized in vitro. However, the efficacy of batroxobin on hemostatic clot formation in vivo is not well studied due to the lack of reliable in vivo hemostasis models. Here, we studied the efficacy of batroxobin and slounase, a batroxobin containing activated factor X, on hemostatic clot composition and bleeding using intravital microcopy laser ablation hemostasis models in micro and macro vessels and liver puncture hemostasis models in normal and heparin-induced hypocoagulant mice. We found that prophylactic treatment in wild-type mice with batroxobin, slounase and activated factor X significantly enhanced platelet-rich fibrin clot formation following vascular injury. In heparin-treated mice, batroxobin treatment resulted in detectable fibrin formation and a modest increase in hemostatic clot size, while activated factor X had no effect. In contrast, slounase treatment significantly enhanced both platelet recruitment and fibrin formation, forming a stable clot and shortening bleeding time and blood loss in wild-type and heparin-treated hypocoagulant mice. Our data demonstrate that, while batroxobin enhances fibrin formation, slounase was able to enhance hemostasis in normal mice and restore hemostasis in hypocoagulant conditions via the enhancement of fibrin formation and platelet activation, indicating that slounase is more effective in controlling hemorrhage.
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Batroxobina/uso terapêutico , Testes de Coagulação Sanguínea/métodos , Coagulação Sanguínea/efeitos dos fármacos , Hemorragia/tratamento farmacológico , Hemostáticos/uso terapêutico , Animais , Batroxobina/farmacologia , Hemostáticos/farmacologia , Humanos , Masculino , CamundongosRESUMO
INTRODUCTION: Despite the great promise for therapies using antisense oligonucleotides (ASOs), their adverse effects, which include pro-inflammatory effects and thrombocytopenia, have limited their use. Previously, these effects have been linked to the phosphorothioate (PS) backbone necessary to prevent rapid ASO degradation in plasma. The main aim of this study was to assess the impact of the nucleic acid portion of an ASO-type drug on platelets and determine if it may contribute to thrombosis or thrombocytopenia. METHODS: Platelets were isolated from healthy donors and men with advanced prostate cancer. Effects of antisense oligonucleotides (ASO), oligonucleotides, gDNA, and microRNA on platelet activation and aggregation were evaluated. A mouse model of lung thrombosis was used to confirm the effects of PS-modified oligonucleotides in vivo. RESULTS: Platelet exposure to gDNA, miRNA, and oligonucleotides longer than 16-mer at a concentration above 8 mM resulted in the formation of hypersensitive platelets, characterized by an increased sensitivity to low-dose thrombin (0.1 nM) and increase in p-Selectin expression (6-8 fold greater than control; p < 0.001). The observed nucleic acid (NA) effects on platelets were toll-like receptor (TLR) -7 subfamily dependent. Injection of a p-Selectin inhibitor significantly (p = 0.02) reduced the formation of oligonucleotide-associated pulmonary microthrombosis in vivo. CONCLUSION: Our results suggest that platelet exposure to nucleic acids independent of the presence of a PS modification leads to a generation of hypersensitive platelets and requires TLR-7 subfamily receptors. ASO studies conducted in cancer patients may benefit from testing the ASO effects on platelets ex vivo before initiation of patient treatment.
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Ácidos Nucleicos , Preparações Farmacêuticas , Animais , Plaquetas , Humanos , Camundongos , Oligonucleotídeos Antissenso , Oligonucleotídeos FosforotioatosRESUMO
OBJECTIVE: There is an inter-relationship between thrombosis and inflammation. Previously, we have shown the importance of P-selectin in thrombogenesis and thrombus resolution in many preclinical animal models. The role of E-selectin has been explored in rodent models and in a small pilot study of clinical calf vein deep venous thrombosis. The purpose of this study was to determine the role of E-selectin in thrombosis in a primate model of proximal iliac vein thrombosis, a model close to the human condition. METHODS: Iliac vein thrombosis was induced with a well-characterized primate model. Through a transplant incision, the hypogastric vein and iliac vein branches were ligated. Thrombus was induced by balloon occlusion of the proximal and distal iliac vein for 6 hours. The balloons were then deflated, and the primates recovered. Starting on postocclusion day 2, animals were treated with the E-selectin inhibitor GMI-1271, 25 mg/kg subcutaneously, once daily until day 21 (n = 4). Nontreated control animals received no treatment (n = 5). All animals were evaluated by magnetic resonance venography (MRV); evaluation of vessel area by ultrasound, protein analysis, hematology (complete blood count), and coagulation tests (bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, and thromboelastography) were performed at baseline, day 2, day 7, day 14, and day 21 with euthanasia. In addition, platelet function and CD44 expression on leukocytes were determined. RESULTS: E-selectin inhibition by GMI-1271 significantly increased vein recanalization by MRV vs control animals on day 14 (P < .05) and day 21 (P < .0001). GMI-1271 significantly decreased vein wall inflammation by MRV with gadolinium vein wall enhancement vs control also on day 14 (P < .0001) and day 21 (P < .0001). The thromboelastographic measure of clot strength (maximum amplitude) showed significant decreases in animals treated with GMI-1271 vs controls at day 2 (P < .05) and day 7 (P < .05). Animals treated with GMI-1271 had significant vessel area increase by day 21 vs controls (P < .05) by ultrasound. Vein wall intimal thickening (P < .001) and intimal fibrosis (P < .05) scores were significantly decreased in GMI-1271-treated animals vs controls. Importantly, no significant differences in hematology or coagulation test results were noted between all groups, suggesting that E-selectin inhibition carries no bleeding potential. GMI-1271 did not affect platelet function or aggregation or CD44 expression on leukocytes. In addition, no episodes of bleeding were noted in either group. CONCLUSIONS: This study suggests that E-selectin modulates venous thrombus progression and that its inhibition will increase thrombus recanalization and decrease vein wall inflammation, without affecting coagulation. The use of an E-selectin inhibitor such as GMI-1271 could potentially change how we treat deep venous thrombosis.
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Anti-Inflamatórios/farmacologia , Selectina E/antagonistas & inibidores , Fibrinolíticos/farmacologia , Glicolipídeos/farmacologia , Veia Ilíaca/efeitos dos fármacos , Trombose Venosa/tratamento farmacológico , Animais , Modelos Animais de Doenças , Selectina E/metabolismo , Veia Ilíaca/diagnóstico por imagem , Veia Ilíaca/metabolismo , Papio , Transdução de Sinais , Trombose Venosa/diagnóstico por imagem , Trombose Venosa/metabolismoRESUMO
Deep vein thrombosis (DVT), caused by alterations in venous homeostasis is the third most common cause of cardiovascular mortality; however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39-deficiency conferred a >2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This was orchestrated by increased phosphorylation of the p65 subunit of NFκB, activation of the NLRP3 inflammasome, and interleukin-1ß (IL-1ß) release in CD39-deficient mice. Substantiating these findings, an IL-1ß-neutralizing antibody attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1ß is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways, and is a critical vascular checkpoint in venous thrombosis.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Trombose Venosa/metabolismo , Animais , Antígenos CD/genética , Apirase/genética , Modelos Animais de Doenças , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/metabolismo , Humanos , Inflamassomos/genética , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/genética , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Trombose Venosa/genética , Trombose Venosa/patologiaRESUMO
Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.
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Proteínas Reguladoras de Apoptose/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Repressoras/fisiologia , Tromboplastina/biossíntese , Trombose/prevenção & controle , Animais , Antitrombina III/análise , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Transplante de Medula Óssea , Células Cultivadas , Cloretos/toxicidade , Imunoprecipitação da Cromatina , Regulação para Baixo , Feminino , Compostos Férricos/toxicidade , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeo Hidrolases/análise , Agregação Plaquetária , Interferência de RNA , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Tromboplastina/genética , Trombose/induzido quimicamente , Transcrição GênicaRESUMO
The NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 develop severe thrombocytopenia with lethality resulting from neonatal hemorrhage. Recent data in mammals reveal potential differences in embryonic and adult thrombopoiesis. Multiple studies in zebrafish have revealed mechanistic insights into hematopoiesis, although thrombopoiesis has been less studied. Rather than platelets, zebrafish possess thrombocytes, which are nucleated cells with similar functional properties. Using transcription activator-like effector nucleases to generate mutations in nfe2, we show that unlike mammals, zebrafish survive to adulthood in the absence of Nfe2. Despite developing severe thrombocytopenia, homozygous mutants do not display overt hemorrhage or reduced survival. Surprisingly, quantification of circulating thrombocytes in mutant 6-day-old larvae revealed no significant differences from wild-type siblings. Both wild-type and nfe2 null larvae formed thrombocyte-rich clots in response to endothelial injury. In addition, ex vivo thrombocytic colony formation was intact in nfe2 mutants, and adult kidney marrow displayed expansion of hematopoietic progenitors. These data suggest that loss of Nfe2 results in a late block in adult thrombopoiesis, with secondary expansion of precursors: features consistent with mammals. Overall, our data suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development and potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. Long-term homozygous mutant survival will facilitate in-depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production.
Assuntos
Plaquetas/metabolismo , Fator de Transcrição NF-E2/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Plaquetas/citologia , Códon de Terminação , Fibrinogênio/metabolismo , Mutação da Fase de Leitura , Edição de Genes , Humanos , Larva/metabolismo , Fator de Transcrição NF-E2/química , Fator de Transcrição NF-E2/genética , Alinhamento de Sequência , Trombopoese , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Platelets are essential for clotting in the blood and maintenance of normal hemostasis. Under pathologic conditions such as atherosclerosis, vascular injury often results in hyperactive platelet activation, resulting in occlusive thrombus formation, myocardial infarction, and stroke. Recent work in the field has elucidated a number of platelet functions unique from that of maintaining hemostasis, including regulation of tumor growth and metastasis, inflammation, infection, and immune response. Traditional therapeutic targets for inhibiting platelet activation have primarily been limited to cyclooxygenase-1, integrin αIIbß3, and the P2Y12 receptor. Recently identified signaling pathways regulating platelet function have made it possible to develop novel approaches for pharmacological intervention in the blood to limit platelet reactivity. In this review, we cover the newly discovered roles for platelets as well as their role in hemostasis and thrombosis. These new roles for platelets lend importance to the development of new therapies targeted to the platelet. Additionally, we highlight the promising receptor and enzymatic targets that may further decrease platelet activation and help to address the myriad of pathologic conditions now known to involve platelets without significant effects on hemostasis.
Assuntos
Plaquetas/fisiologia , Trombose/tratamento farmacológico , Animais , Plaquetas/efeitos dos fármacos , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Humanos , Terapia de Alvo Molecular , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Transdução de Sinais , Trombose/metabolismo , Trombose/fisiopatologiaRESUMO
OBJECTIVE: Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased Gq signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention. APPROACH AND RESULTS: Membranes expressing human PAR4-120 variants were reconstituted with either Gq or G13 to determine the kinetics of G protein activation. The kinetics of Gq and G13 activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y12 receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy. CONCLUSIONS: Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both Gq and G13 activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y12 receptor.
Assuntos
Aspirina/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Clopidogrel/uso terapêutico , Inibidores de Ciclo-Oxigenase/uso terapêutico , Resistência a Medicamentos , Inibidores da Agregação Plaquetária/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Receptores de Trombina/sangue , Negro ou Afro-Americano/genética , Coagulação Sanguínea/genética , Plaquetas/metabolismo , Ciclo-Oxigenase 1/sangue , Resistência a Medicamentos/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/sangue , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/sangue , Genótipo , Humanos , Cinética , Variantes Farmacogenômicos , Fenótipo , Agregação Plaquetária/genética , Polimorfismo de Nucleotídeo Único , Receptores Purinérgicos P2Y12/sangue , Receptores Purinérgicos P2Y12/efeitos dos fármacos , Receptores de Trombina/genética , Transdução de Sinais/efeitos dos fármacos , População Branca/genética , Proteína rhoA de Ligação ao GTP/sangueRESUMO
Platelets play an important role in the vessel. Following their formation from megakaryocytes, platelets exist in circulation for 5-7 days and primarily function as regulators of hemostasis and thrombosis. Following vascular insult or injury, platelets become activated in the blood resulting in adhesion to the exposed extracellular matrix underlying the endothelium, formation of a platelet plug, and finally formation and consolidation of a thrombus consisting of both a core and shell. In pathological conditions, platelets are essential for formation of occlusive thrombus formation and as a result are the primary target for prevention of arterial thrombus formation. In addition to regulation of hemostasis in the vessel, platelets have also been shown to play an important role in innate immunity as well as regulation of tumor growth and extravasations in the vessel. These primary functions of the platelet represent its normal function and versatility in circulation.
Assuntos
Plaquetas/fisiologia , Animais , Plaquetas/patologia , Hemostasia/fisiologia , Humanos , Trombose/sangue , Trombose/patologiaRESUMO
OBJECTIVE: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis. APPROACH AND RESULTS: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets. CONCLUSIONS: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.
Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacologia , Araquidonato 12-Lipoxigenase/sangue , Plaquetas/efeitos dos fármacos , Cromograninas/sangue , Fibrinolíticos/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/sangue , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Trombose/prevenção & controle , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Araquidonato 12-Lipoxigenase/deficiência , Araquidonato 12-Lipoxigenase/genética , Plaquetas/metabolismo , Moléculas de Adesão Celular/sangue , AMP Cíclico/sangue , Proteínas Quinases Dependentes de AMP Cíclico/sangue , Modelos Animais de Doenças , Fibrinolíticos/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/sangue , Oxirredução , Fosfoproteínas/sangue , Fosforilação , Agregação Plaquetária/efeitos dos fármacos , Complexo Shelterina , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação a Telômeros/sangue , Trombose/sangue , Trombose/enzimologia , Trombose/genética , Fatores de TempoRESUMO
OBJECTIVE: Black individuals are at an increased risk of myocardial infarction and stroke, 2 vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke, and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however, has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to antiplatelet therapies. APPROACH AND RESULTS: Washed human platelets from black volunteers were hyperaggregable in response to PAR4-mediated platelet stimulation compared with whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP (2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate), suggesting that the racial difference is independent of secondary feedback. Furthermore, stimulation of platelets from black donors with PAR4-activating peptide showed a potentiated level of activation through the Gq pathway compared with platelets from white donors. Differences in signaling included increased Ca(2+) mobilization, Rap1 (Ras-related protein 1) activation, and integrin αIIbß3 activation with no observed difference in platelet protein expression between the groups tested. CONCLUSIONS: Our study is the first to demonstrate that the Gq pathway is differentially regulated by race after PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of cyclooxygenase and P2Y12 receptor dual inhibition, suggesting that current antiplatelet therapy may provide less protection to blacks than whites.
Assuntos
População Negra , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/sangue , Ativação Plaquetária/fisiologia , Receptores de Trombina/sangue , População Branca , Adulto , Sinalização do Cálcio , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Humanos , Masculino , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Prostaglandina-Endoperóxido Sintases/sangue , Proteína Quinase C/sangue , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/sangue , Complexo Shelterina , Transdução de Sinais , Proteínas de Ligação a Telômeros/sangueRESUMO
Blood microRNA (miRNA) levels have been associated with and shown to participate in disease pathophysiology. However, the hematopoietic cell of origin of blood miRNAs and the individual blood cell miRNA profiles are poorly understood. We report the miRNA content of highly purified normal hematopoietic cells from the same individuals. Although T-cells, B-cells and granulocytes had the highest miRNA content per cell, erythrocytes contributed more cellular miRNA to the blood, followed by granulocytes and platelets. miRNA profiling revealed different patterns and different expression levels of miRNA specific for each lineage. miR-30c-5p was determined to be an appropriate reference normalizer for cross-cell qRT-PCR comparisons. miRNA profiling of 5 hematopoietic cell lines revealed differential expression of miR-125a-5p. We demonstrated endogenous levels of miR-125a-5p regulate reporter gene expression in Meg-01 and Jurkat cells by (1) constructs containing binding sites for miR-125a-5p or (2) over-expressing or inhibiting miR-125a-5p. This quantitative analysis of the miRNA profiles of peripheral blood cells identifies the circulating hematopoietic cellular miRNAs, supports the use of miRNA profiles for distinguishing different hematopoietic lineages and suggests that endogenously expressed miRNAs can be exploited to regulate transgene expression in a cell-specific manner.