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Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcµR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.
Assuntos
Plaquetas , COVID-19 , Ativação do Complemento , Imunoglobulina M , Trombose , Humanos , Trombose/imunologia , Animais , Imunoglobulina M/imunologia , Ativação do Complemento/imunologia , Camundongos , Plaquetas/imunologia , Plaquetas/metabolismo , COVID-19/imunologia , COVID-19/complicações , SARS-CoV-2/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Ativação Plaquetária/imunologia , Imunoglobulina G/imunologia , MasculinoRESUMO
Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.
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Aterosclerose , Placa Aterosclerótica , Humanos , Macrófagos/metabolismo , Aterosclerose/metabolismo , Placa Aterosclerótica/metabolismo , Quimiocinas/metabolismo , Inflamação/metabolismo , Necrose/metabolismoRESUMO
Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.
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Doenças Cardiovasculares , Infarto do Miocárdio , Trombose , Humanos , Megacariócitos , Trombopoese , Neutrófilos , Plaquetas/fisiologiaRESUMO
Platelets are key players in cardiovascular disease and platelet aggregation represents a central pharmacological target, particularly in secondary prevention. However, inhibition of adenosine diphosphate and thromboxane signaling has low efficacy in preventing venous thromboembolism, necessitating the inhibition of the plasmatic coagulation cascade in this disease. Anticoagulation carries a significantly higher risk of bleeding complications, highlighting the need of alternative therapeutic approaches. We hypothesized that procoagulant activation (PA) of platelets promotes venous thrombus formation and that targeting PA could alleviate venous thrombosis. Here, we found elevated levels of procoagulant platelets in the circulation of patients with deep vein thrombosis (DVT) and pulmonary embolism, and in mice developing DVT following inferior vena cava stenosis. Further, we detected procoagulant activation of recruited platelets within murine and human venous thrombi. Mice with platelet-specific deficiency in central pathways of procoagulant activation - cyclophilin D and transmembrane protein 16F - were more resistant towards low flow-induced venous thrombosis. Finally, we found that a clinically approved carbonic anhydrase inhibitor, methazolamide, reduced platelet procoagulant activity and alleviated murine thrombus formation without affecting trauma-associated hemostasis. These findings identify an essential role of platelet procoagulant function in venous thrombosis and delineate novel pharmacological strategies targeting platelets in preventing venous thromboembolism.
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The major weakness of most knock-in JAK2V617F mouse models is the presence of the JAK2 mutation in all rather than in a few hematopoietic stem cells (HSC), such as in human "early-stage" myeloproliferative neoplasms (MPN). Understanding the mechanisms of disease initiation is critical as underscored by the incidence of clonal hematopoiesis of indeterminate potential associated with JAK2V617F. Currently, such studies require competitive transplantation. Here, we report a mouse model obtained by crossing JAK2V617F/WT knock-in mice with PF4iCre transgenic mice. As expected, PF4iCre;JAK2V617F/WT mice developed an early thrombocytosis resulting from the expression of JAK2V617F in the megakaryocytes. However, these mice then developed a polycythemia vera-like phenotype at 10 weeks of age. Using mT/mG reporter mice, we demonstrated that Cre recombination was present in all hematopoietic compartments, including in a low number of HSC. The frequency of mutated cells increased along hematopoietic differentiation mimicking the clonal expansion observed in essential thrombocythemia and polycythemia vera patients. This model thus mimics the HSC compartment observed in early-stage MPN, with a small number of JAK2V617F HSC competing with a majority of JAK2WT HSC. PF4iCre;JAK2V617F/WT mice are a promising tool to investigate the mechanisms that regulate clonal dominance and progression to myelofibrosis.
Assuntos
Modelos Animais de Doenças , Células-Tronco Hematopoéticas/patologia , Janus Quinase 2/genética , Megacariócitos/patologia , Mutação , Transtornos Mieloproliferativos/patologia , Policitemia Vera/patologia , Animais , Diferenciação Celular , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Transgênicos , Transtornos Mieloproliferativos/genética , Fenótipo , Policitemia Vera/genéticaRESUMO
Clinical observations implicate a role of eosinophils in cardiovascular diseases because markers of eosinophil activation are elevated in atherosclerosis and thrombosis. However, their contribution to atherosclerotic plaque formation and arterial thrombosis remains unclear. In these settings, we investigated how eosinophils are recruited and activated through an interplay with platelets. Here, we provide evidence for a central importance of eosinophil-platelet interactions in atherosclerosis and thrombosis. We show that eosinophils support atherosclerotic plaque formation involving enhanced von Willebrand factor exposure on endothelial cells and augmented platelet adhesion. During arterial thrombosis, eosinophils are quickly recruited in an integrin-dependent manner and engage in interactions with platelets leading to eosinophil activation as we show by intravital calcium imaging. These direct interactions induce the formation of eosinophil extracellular traps (EETs), which are present in human thrombi and constitute a substantial part of extracellular traps in murine thrombi. EETs are decorated with the granule protein major basic protein, which causes platelet activation by eosinophils. Consequently, targeting of EETs diminished thrombus formation in vivo, which identifies this approach as a novel antithrombotic concept. Finally, in our clinical analysis of coronary artery thrombi, we identified female patients with stent thrombosis as the population that might derive the greatest benefit from an eosinophil-inhibiting strategy. In summary, eosinophils contribute to atherosclerotic plaque formation and thrombosis through an interplay with platelets, resulting in mutual activation. Therefore, eosinophils are a promising new target in the prevention and therapy of atherosclerosis and thrombosis.
Assuntos
Aterosclerose/patologia , Plaquetas/patologia , Eosinófilos/patologia , Armadilhas Extracelulares/metabolismo , Trombose/patologia , Animais , Aterosclerose/metabolismo , Plaquetas/metabolismo , Eosinófilos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ativação Plaquetária/fisiologia , Trombose/metabolismoRESUMO
Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses.
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Células Endoteliais/metabolismo , Janus Quinase 2/biossíntese , Selectina-P/biossíntese , Trombose/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Janus Quinase 2/genética , Camundongos , Camundongos Transgênicos , Selectina-P/genética , Trombose/tratamento farmacológico , Trombose/genética , Trombose/patologiaRESUMO
OBJECTIVE: Cancer patients are at high risk of developing deep venous thrombosis (DVT) and venous thromboembolism, a leading cause of mortality in this population. However, it is largely unclear how malignant tumors drive the prothrombotic cascade culminating in DVT. APPROACH AND RESULTS: Here, we addressed the pathophysiology of malignant DVT compared with nonmalignant DVT and focused on the role of tumor microvesicles as potential targets to prevent cancer-associated DVT. We show that microvesicles released by pancreatic adenocarcinoma cells (pancreatic tumor-derived microvesicles [pcMV]) boost thrombus formation in a model of flow restriction of the mouse vena cava. This depends on the synergistic activation of coagulation by pcMV and host tissue factor. Unlike nonmalignant DVT, which is initiated and propagated by innate immune cells, thrombosis triggered by pcMV was largely independent of myeloid leukocytes or platelets. Instead, we identified externalization of the phospholipid phosphatidylethanolamine as a major mechanism controlling the prothrombotic activity of pcMV. Disrupting phosphatidylethanolamine-dependent activation of factor X suppressed pcMV-induced DVT without causing changes in hemostasis. CONCLUSIONS: Together, we show here that the pathophysiology of pcMV-associated experimental DVT differs markedly from innate immune cell-promoted nonmalignant DVT and is therefore amenable to distinct antithrombotic strategies. Targeting phosphatidylethanolamine on tumor microvesicles could be a new strategy for prevention of cancer-associated DVT without causing bleeding complications.
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Adenocarcinoma/complicações , Coagulação Sanguínea , Micropartículas Derivadas de Células/metabolismo , Neoplasias Pancreáticas/complicações , Veia Cava Inferior/metabolismo , Trombose Venosa/etiologia , Adenocarcinoma/sangue , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Bacteriocinas/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/efeitos dos fármacos , Micropartículas Derivadas de Células/patologia , Modelos Animais de Doenças , Desenho de Fármacos , Fator Xa/metabolismo , Fibrinolíticos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Peptídeos/farmacologia , Fosfatidiletanolaminas/antagonistas & inibidores , Fosfatidiletanolaminas/sangue , Transdução de Sinais , Tromboplastina/metabolismo , Veia Cava Inferior/efeitos dos fármacos , Veia Cava Inferior/patologia , Trombose Venosa/sangue , Trombose Venosa/patologia , Trombose Venosa/prevenção & controleRESUMO
Persistent or chronic immune thrombocytopenias (P/C-ITP) are acquired blood disorders lasting more than 3 months or 1 year, respectively. The pathogenesis of these disorders is thought to be immunological. We hypothesized that some patients with P/C-ITP might have an intrinsic megakaryopoiesis defect. We identified a group of P/C-ITP patients with acquired isolated mild thrombocytopenia (30-100 × 10(9) /l), undetectable anti-platelet antibodies, negative autoimmune investigations and no need for treatment. We examined in vitro megakaryocyte differentiation and compared these patients' results with those of acute-ITP patients and healthy controls. No difference in proliferation, ploidy or expression of surface markers was found. In contrast, P/C-ITP patients had significantly fewer proplatelet-forming megakaryocytes. This novel observation demonstrated that some patients diagnosed with P/C-ITP have an intrinsic megakaryopoiesis defect independent of the bone-marrow environment. Further investigations are needed to dissect mechanisms underlying this impaired proplatelet formation in these patients.
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Autoanticorpos/imunologia , Plaquetas/imunologia , Diferenciação Celular/imunologia , Megacariócitos/imunologia , Mielopoese/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Adulto , Idoso , Plaquetas/patologia , Doença Crônica , Feminino , Humanos , Masculino , Megacariócitos/patologia , Pessoa de Meia-Idade , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/patologiaRESUMO
BACKGROUND: Neutrophils participate in the pathogenesis of thrombosis through the formation of neutrophil extracellular traps (NETs). Thrombosis is the main cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Recent studies have shown an increase in NET formation (NETosis) both in patients with JAK2V617F neutrophils and in mouse models, and reported the participation of NETosis in the pathophysiology of thrombosis in mice. OBJECTIVES: This study investigated whether JAK2V617F neutrophils are sufficient to promote thrombosis or whether their cooperation with other blood cell types is necessary. METHODS: NETosis was studied in PF4iCre;Jak2V617F/WT mice expressing JAK2V617F in all hematopoietic lineages, as occurs in MPNs, and in MRP8Cre;Jak2V617F/WT mice in which JAK2V617F is expressed only in leukocytes. RESULTS: In PF4iCre;Jak2V617F/WT mice, an increase in NETosis and spontaneous lung thrombosis abrogated by DNAse administration were observed. The absence of spontaneous NETosis or lung thrombosis in MRP8Cre;Jak2V617F/WT mice suggested that mutated neutrophils alone are not sufficient to induce thrombosis. Ex vivo experiments demonstrated that JAK2V617F-mutated platelets trigger NETosis by JAK2V617F-mutated neutrophils. Aspirin treatment in PF4iCre;Jak2V617F/WT mice reduced NETosis and reduced lung thrombosis. In cytoreductive-therapy-free patients with MPN treated with aspirin, plasma NET marker concentrations were lower than that in patients with MPN not treated with aspirin. CONCLUSION: Our study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis; rather, platelets cooperate with neutrophils to promote NETosis in vivo. A new role for aspirin in thrombosis prevention in MPNs was also identified.
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Armadilhas Extracelulares , Transtornos Mieloproliferativos , Neoplasias , Trombose , Trombose Venosa , Humanos , Camundongos , Animais , Neutrófilos/metabolismo , Armadilhas Extracelulares/metabolismo , Neoplasias/metabolismo , Transtornos Mieloproliferativos/genética , Janus Quinase 2/genética , Trombose Venosa/metabolismo , AspirinaRESUMO
BACKGROUND: Deep vein thrombosis (DVT) is a common condition associated with significant mortality due to pulmonary embolism. Despite advanced prevention and anticoagulation therapy, the incidence of venous thromboembolism remains unchanged. Individuals with elevated hematocrit and/or excessively high erythropoietin (EPO) serum levels are particularly susceptible to DVT formation. We investigated the influence of short-term EPO administration compared to chronic EPO overproduction on DVT development. Additionally, we examined the role of the spleen in this context and assessed its impact on thrombus composition. METHODS: We induced ligation of the caudal vena cava (VCC) in EPO-overproducing Tg(EPO) mice as well as wildtype mice treated with EPO for two weeks, both with and without splenectomy. The effect on platelet circulation time was evaluated through FACS analysis, and thrombus composition was analyzed using immunohistology. RESULTS: We present evidence for an elevated thrombogenic phenotype resulting from chronic EPO overproduction, achieved by combining an EPO-overexpressing mouse model with experimental DVT induction. This increased thrombotic state is largely independent of traditional contributors to DVT, such as neutrophils and platelets. Notably, the pronounced prothrombotic effect of red blood cells (RBCs) only manifests during chronic EPO overproduction and is not influenced by splenic RBC clearance, as demonstrated by splenectomy. In contrast, short-term EPO treatment does not induce thrombogenesis in mice. Consequently, our findings support the existence of a differential thrombogenic effect between chronic enhanced erythropoiesis and exogenous EPO administration. CONCLUSION: Chronic EPO overproduction significantly increases the risk of DVT, while short-term EPO treatment does not. These findings underscore the importance of considering EPO-related factors in DVT risk assessment and potential therapeutic strategies.
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Venous thromboembolism (VTE) comprising deep venous thrombosis and pulmonary embolism is a major cause of morbidity and mortality. Short-term immobility-related conditions are a major risk factor for the development of VTE. Paradoxically, long-term immobilized free-ranging hibernating brown bears and paralyzed spinal cord injury (SCI) patients are protected from VTE. We aimed to identify mechanisms of immobility-associated VTE protection in a cross-species approach. Mass spectrometry-based proteomics revealed an antithrombotic signature in platelets of hibernating brown bears with heat shock protein 47 (HSP47) as the most substantially reduced protein. HSP47 down-regulation or ablation attenuated immune cell activation and neutrophil extracellular trap formation, contributing to thromboprotection in bears, SCI patients, and mice. This cross-species conserved platelet signature may give rise to antithrombotic therapeutics and prognostic markers beyond immobility-associated VTE.
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Plaquetas , Proteínas de Choque Térmico HSP47 , Hipocinesia , Traumatismos da Medula Espinal , Ursidae , Tromboembolia Venosa , Animais , Humanos , Camundongos , Fibrinolíticos/uso terapêutico , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/etnologia , Embolia Pulmonar/metabolismo , Fatores de Risco , Traumatismos da Medula Espinal/complicações , Ursidae/metabolismo , Tromboembolia Venosa/etiologia , Tromboembolia Venosa/metabolismo , Hipocinesia/complicações , Proteínas de Choque Térmico HSP47/metabolismo , Plaquetas/metabolismoRESUMO
Essentials To reliably study the respective roles of blood and endothelial cells in hemostasis, mouse models with a strong and specific endothelial expression of the Cre recombinase are needed. Using mT/mG reporter mice and conditional JAK2V617F/WT mice, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Comparison of recombination efficiency and specificity towards blood lineage reveals major differences between endothelial transgenic mice. Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2;JAK2V617F/WT transgenic mice. SUMMARY: Background The vessel wall, and particularly blood endothelial cells (BECs), are intensively studied to better understand hemostasis and target thrombosis. To understand the specific role of BECs, it is important to have mouse models that allow specific and homogeneous expression of genes of interest in all BEC beds without concomitant expression in blood cells. Inducible Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 transgenic mice are widely used for BEC targeting. However, issues remain in terms of recombination efficiency and specificity regarding hematopoietic cells. Objectives To determine which mouse model to choose when strong expression of a transgene is required in adult BECs from various organs, without concomitant expression in hematopoietic cells. Methods Using mT/mG reporter mice to measure recombination efficiency and conditional JAK2V617F/WT mice to assess specificity regarding hematopoietic cells, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Results Adult Cdh5(PAC)-CreERT2 mice are endothelial specific but require a dose of 10 mg of tamoxifen to allow constant Cre expression. Pdgfb-iCreERT2 mice injected with 5 mg of tamoxifen are appropriate for most endothelial research fields except liver studies, as hepatic sinusoid ECs are not recombined. Surprisingly, 2 months after induction of Cre-mediated recombination, all Pdgfb-iCreERT2;JAK2V617F/WT mice developed a myeloproliferative neoplasm that is related to the presence of JAK2V617F in hematopoietic cells, showing for the first time that Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2 transgenic mice. Conclusion This study provides useful guidelines for choosing the best mouse line to study the role of BECs in hemostasis and thrombosis.