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BACKGROUND: Cardiac hypertrophy is characterized by remodeling of the myocardium, which involves alterations in the ECM (extracellular matrix) and cardiomyocyte structure. These alterations critically contribute to impaired contractility and relaxation, ultimately leading to heart failure. Emerging evidence implicates that extracellular signaling molecules are critically involved in the pathogenesis of cardiac hypertrophy and remodeling. The immunophilin CyPA (cyclophilin A) has been identified as a potential culprit. In this study, we aimed to unravel the interplay between eCyPA (extracellular CyPA) and myocardial dysfunction and evaluate the therapeutic potential of inhibiting its extracellular accumulation to improve heart function. METHODS: Employing a multidisciplinary approach encompassing in silico, in vitro, in vivo, and ex vivo experiments we studied a mouse model of cardiac hypertrophy and human heart specimen to decipher the interaction of CyPA and the cardiac microenvironment in highly relevant pre-/clinical settings. Myocardial expression of CyPA (immunohistology) and the inflammatory transcriptome (NanoString) was analyzed in human cardiac tissue derived from patients with nonischemic, noninflammatory congestive heart failure (n=187). These analyses were paralleled by a mouse model of Ang (angiotensin) II-induced heart failure, which was assessed by functional (echocardiography), structural (immunohistology, atomic force microscopy), and biomolecular (Raman spectroscopy) analyses. The effect of inhibiting eCyPA in the cardiac microenvironment was evaluated using a newly developed neutralizing anti-eCyPA monoclonal antibody. RESULTS: We observed a significant accumulation of eCyPA in both human and murine-failing hearts. Importantly, higher eCyPA expression was associated with poor clinical outcomes in patients (P=0.043) and contractile dysfunction in mice (Pearson correlation coefficient, -0.73). Further, myocardial expression of eCyPA was critically associated with an increase in myocardial hypertrophy, inflammation, fibrosis, stiffness, and cardiac dysfunction in vivo. Antibody-based inhibition of eCyPA prevented (Ang II)-induced myocardial remodeling and dysfunction in mice. CONCLUSIONS: Our study provides strong evidence of the pathogenic role of eCyPA in remodeling, myocardial stiffening, and dysfunction in heart failure. The findings suggest that antibody-based inhibition of eCyPA may offer a novel therapeutic strategy for nonischemic heart failure. Further research is needed to evaluate the translational potential of these interventions in human patients with cardiac hypertrophy.
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BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.
Assuntos
Doença da Artéria Coronariana , MicroRNAs , Infarto do Miocárdio , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Camundongos , Animais , Plaquetas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Remodelação Ventricular , Traumatismo por Reperfusão Miocárdica/metabolismo , Isquemia Miocárdica/metabolismo , Infarto do Miocárdio/patologia , Doença da Artéria Coronariana/metabolismo , Inflamação/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND: Aortic stenosis (AS) is driven by progressive inflammatory and fibrocalcific processes regulated by circulating inflammatory and valve resident endothelial and interstitial cells. The impact of platelets, platelet-derived mediators, and platelet-monocyte interactions on the acceleration of local valvular inflammation and mineralization is presently unknown. METHODS: We prospectively enrolled 475 consecutive patients with severe symptomatic AS undergoing aortic valve replacement. Clinical workup included repetitive echocardiography, analysis of platelets, monocytes, chemokine profiling, aortic valve tissue samples for immunohistochemistry, and gene expression analysis. RESULTS: The patients were classified as fast-progressive AS by the median ∆Vmax of 0.45 m/s per year determined by echocardiography. Immunohistological aortic valve analysis revealed enhanced cellularity in fast-progressive AS (slow- versus fast-progressive AS; median [interquartile range], 247 [142.3-504] versus 717.5 [360.5-1234]; P<0.001) with less calcification (calcification area, mm2: 33.74 [27.82-41.86] versus 20.54 [13.52-33.41]; P<0.001). MIF (macrophage migration inhibitory factor)-associated gene expression was significantly enhanced in fast-progressive AS accompanied by significantly elevated MIF plasma levels (mean±SEM; 6877±379.1 versus 9959±749.1; P<0.001), increased platelet activation, and decreased intracellular MIF expression indicating enhanced MIF release upon platelet activation (CD62P, %: median [interquartile range], 16.8 [11.58-23.8] versus 20.55 [12.48-32.28], P=0.005; MIF, %: 4.85 [1.48-9.75] versus 2.3 [0.78-5.9], P<0.001). Regression analysis confirmed that MIF-associated biomarkers are strongly associated with an accelerated course of AS. CONCLUSIONS: Our findings suggest a key role for platelet-derived MIF and its interplay with circulating and valve resident monocytes/macrophages in local and systemic thromboinflammation during accelerated AS. MIF-based biomarkers predict an accelerated course of AS and represent a novel pharmacological target to attenuate progression of AS.
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Signaling lipids are key players in cellular processes. Despite their importance, no method currently allows their comprehensive monitoring in one analytical run. Challenges include a wide dynamic range, isomeric and isobaric species, and unwanted interaction along the separation path. Herein, we present a sensitive and robust targeted liquid chromatography-mass spectrometry (LC-MS/MS) approach to overcome these challenges, covering a broad panel of 17 different signaling lipid classes. It involves a simple one-phase sample extraction and lipid analysis using bioinert reversed-phase liquid chromatography coupled to targeted mass spectrometry. The workflow shows excellent sensitivity and repeatability in different biological matrices, enabling the sensitive and robust monitoring of 388 lipids in a single run of only 20 min. To benchmark our workflow, we characterized the human plasma signaling lipidome, quantifying 307 endogenous molecular lipid species. Furthermore, we investigated the signaling lipidome during platelet activation, identifying numerous regulations along important lipid signaling pathways. This highlights the potential of the presented method to investigate signaling lipids in complex biological systems, enabling unprecedentedly comprehensive analysis and direct insight into signaling pathways.
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Lipídeos , Transdução de Sinais , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Humanos , Lipídeos/análise , Lipídeos/sangue , Cromatografia Líquida/métodos , Lipidômica/métodos , Ativação Plaquetária , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post-myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A-mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia-sera/immunoglobulin G-triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.
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Receptores CXCR/metabolismo , Trombose , Plaquetas/metabolismo , Humanos , Inflamação/metabolismo , Lipidômica , Lipídeos , Espectrometria de Massas em Tandem , Trombina/metabolismo , Tromboinflamação , Trombose/metabolismoRESUMO
[Figure: see text].
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Anexina A7/metabolismo , Plaquetas/metabolismo , Metabolismo dos Lipídeos , Trombose/metabolismo , Animais , Anexina A7/genética , Araquidonato 12-Lipoxigenase/metabolismo , Sinalização do Cálcio , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismoRESUMO
AIMS: P-selectin is an activatable adhesion molecule on platelets promoting platelet aggregation, and platelet-leukocyte complex (PLC) formation. Increased numbers of PLC are circulating in the blood of patients shortly after acute myocardial infarction and predict adverse outcomes. These correlations led to speculations about whether PLC may represent novel therapeutic targets. We therefore set out to elucidate the pathomechanistic relevance of PLC in myocardial ischemia and reperfusion injury. METHODS AND RESULTS: By generating P-selectin deficient bone marrow chimeric mice, the post-myocardial infarction surge in PLC numbers in blood was prevented. Yet, intravital microscopy, flow cytometry and immunohistochemical staining, echocardiography, and gene expression profiling showed unequivocally that leukocyte adhesion to the vessel wall, leukocyte infiltration, and myocardial damage post-infarction were not altered in response to the lack in PLC. CONCLUSION: We conclude that myocardial infarction associated sterile inflammation triggers PLC formation, reminiscent of conserved immunothrombotic responses, but without PLC influencing myocardial ischemia and reperfusion injury in return. Our experimental data do not support a therapeutic concept of selectively targeting PLC formation in myocardial infarction.
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Infarto do Miocárdio , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Traumatismo por Reperfusão , Camundongos , Animais , Selectina-P/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Leucócitos , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão/metabolismo , Isquemia Miocárdica/metabolismoRESUMO
Platelets express the transmembrane chemokine SR-PSOX/CXCL16, proteolytic cleavage of which generates the sCXCL16 soluble-(s) chemokine. The sCXCL16 engages CXCR6 on platelets to synergistically propagate degranulation, aggregation and thrombotic response. Currently, we have investigated the pro-thrombotic and prognostic association of platelet CXCL16−CXCR6 axis in CAD-(n = 240; CCS n = 62; ACS n = 178) patients. Platelet surface-associated-CXCL16 and CXCR6 surface expression ascertained by flow cytometry correlated significantly with platelet activation markers (CD62P denoting degranulation and PAC-1 binding denoting α2bß3-integrin activation). Higher platelet CXCL16 surface association (1st quartile vs. 2nd−4th quartiles) corresponded to significantly elevated collagen-induced platelet aggregation assessed by whole blood impedance aggregometry. Platelet-CXCL16 and CXCR6 expression did not alter with dyslipidemia, triglyceride, total cholesterol, or LDL levels, but higher (>median) plasma HDL levels corresponded with decreased platelet-CXCL16 and CXCR6. Although platelet-CXCL16 and CXCR6 expression did not change significantly with or correlate with troponin I levels, they corresponded with higher Creatine Kinase-(CK) activity and progressively deteriorating left ventricular ejection fraction (LVEF) at admission. Elevated-(4th quartile) platelet-CXCL16 (p = 0.023) and CXCR6 (p = 0.030) measured at admission were significantly associated with a worse prognosis. However, after Cox-PH regression analysis, only platelet-CXCL16 was ascertained as an independent predictor for all-cause of mortality. Therefore, the platelet CXCL16−CXCR6 axis may influence thrombotic propensity and prognosis in CAD patients.
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Plaquetas , Quimiocinas CXC , Doença da Artéria Coronariana , Plaquetas/metabolismo , Quimiocina CXCL16 , Quimiocinas CXC/metabolismo , Colesterol , Creatina Quinase , Humanos , Integrinas , Receptores CXCR6/metabolismo , Receptores Depuradores , Receptores Virais , Volume Sistólico , Triglicerídeos , Troponina I , Função Ventricular EsquerdaRESUMO
Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography-mass spectrometry-based workflow that separates phosphoinositide isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells, and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 and 625 fmol, respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple less-abundant phosphoinositides.
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Fosfatidilinositóis , Animais , Cromatografia Líquida , Espectrometria de Massas , Ratos , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
During thrombopoiesis, megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Phosphoinositide-dependent protein kinase-1 (PDK1) is required for Ca2+-dependent platelet activation, but its role in MK development and regulation of platelet production remained elusive. The present study explored the role of PDK1 in the regulation of MK maturation and polarization during thrombopoiesis using a MK/platelet-specific knockout approach. Pdk1-deficient mice (Pdk1-/-) developed a significant macrothrombocytopenia as compared with wild-type mice (Pdk1fl/fl). Pdk1 deficiency further dramatically increased the number of MKs without sinusoidal contact within the BM hematopoietic compartment, resulting in a pronounced MK hyperplasia and a significantly increased extramedullary thrombopoiesis. Cultured Pdk1-/- BM-MKs showed impaired spreading on collagen, associated with an altered actin cytoskeleton structure with less filamentous actin (F-actin) and diminished podosome formation, whereas the tubulin cytoskeleton remained unaffected. This phenotype was associated with abrogated phosphorylation of p21-activated kinase (PAK) as well as its substrates LIM domain kinase and cofilin, supporting the hypothesis that the defective F-actin assembly results from increased cofilin activity in Pdk1-deficient MKs. Pdk1-/- BM-MKs developed increased ploidy and exhibited an abnormal ultrastructure with disrupted demarcation membrane system (DMS). Strikingly, Pdk1-/- BM-MKs displayed a pronounced defect in DMS polarization and produced significantly less proplatelets, indicating that PDK1 is critically required for proplatelet formation. In human MKs, genetic PDK1 knockdown resulted in increased maturity but reduced platelet-like particles formation. The present observations reveal a pivotal role of PDK1 in the regulation of MK cytoskeletal dynamics and polarization, proplatelet formation, and thrombopoiesis.
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Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Megacariócitos/metabolismo , Trombopoese/fisiologia , Animais , Plaquetas/citologia , Humanos , Megacariócitos/citologia , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Gremlin-1 is a cystine knot protein and is expressed in organs developing fibrosis. Transient ischaemia leads to myocardial fibrosis, a major determinant of impaired myocardial function. MATERIALS AND METHODS: Expression of Gremlin-1 was investigated in infarcted myocardium by real-time PCR, Western blot analysis, histological and immunohistochemistry staining. We further elaborated the colocalization of Gremlin-1 and TGF-ß proteins by confocal microscopy and co-immunoprecipitation experiments. The interaction between Gremlin-1 and TGF-ß was analysed by photon correlation spectroscopy. Gremlin-1 modulation of the TGF-ß-dependent collagen I synthesis in fibroblasts was investigated using ELISA and immunohistochemistry experiments. The effect of prolonged administration of recombinant Gremlin-1 on myocardial function following ischaemia/reperfusion was accessed by echocardiography and immunohistochemistry. RESULTS: Gremlin-1 is expressed in myocardial tissue and infiltrating cells after transient myocardial ischaemia (P < .05). Gremlin-1 colocalizes with the pro-fibrotic cytokine transforming growth factor-ß (TGF-ß) expressed in fibroblasts and inflammatory cell infiltrates (P < .05). Gremlin-1 reduces TGF-ß-induced collagen production of myocardial fibroblasts by approximately 20% (P < .05). We found that Gremlin-1 binds with high affinity to TGF-ß (KD = 54 nmol/L) as evidenced by photon correlation spectroscopy and co-immunoprecipitation. intravenous administration of m Gremlin-1-Fc, but not of equivalent amount of Fc control, significantly reduced infarct size by approximately 20%. In the m Gremlin-1-Fc group, infarct area was reduced by up to 30% in comparison with mice treated with Fc control (I/LV: 4.8 ± 1.2% vs 6.0 ± 1.2% P < .05; I/AaR: 15.2 ± 1.5% vs 21.1 ± 5%, P < .05). CONCLUSIONS: The present data disclose Gremlin-1 as an antagonist of TGF-ß and presume a role for Gremlin-1/TGF-ß interaction in myocardial remodelling following myocardial ischaemia.
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Fibroblastos/metabolismo , Coração/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Infarto do Miocárdio/genética , Traumatismo por Reperfusão Miocárdica/genética , Miocárdio/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Colágeno Tipo I/metabolismo , Ecocardiografia , Células Endoteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose , Coração/diagnóstico por imagem , Coração/efeitos dos fármacos , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Microscopia Confocal , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Proteínas Recombinantes , Fator de Crescimento Transformador beta/efeitos dos fármacos , Remodelação Ventricular/genéticaRESUMO
Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.
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Antígenos CD/metabolismo , Apirase/metabolismo , Fibrinolíticos/farmacologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Citometria de Fluxo , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Modelos Biológicos , Nanopartículas , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/químicaRESUMO
Platelets play a significant role in atherothrombosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of LDL metabolism and interacts with platelet function. The effect of PCSK9 in platelet function is poorly understood. The authors of this article sought to characterize platelets as a major source of PCSK9 and PCSK9's role in atherothrombosis. In a large cohort of patients with coronary artery disease (CAD), platelet count, platelet reactivity, and platelet-derived PCSK9 release were analyzed. The role of platelet PCSK9 on platelet and monocyte function was investigated in vitro. Platelet count and hyper-reactivity correlated with plasma LDL in CAD. The circulating platelets express on their surface and release substantial amounts of PCSK9. Release of PCSK9 augmented platelet-dependent thrombosis, monocyte migration, and differentiation into macrophages/foam cells. Platelets and PCSK9 accumulated in tissue derived from atherosclerotic carotid arteries in areas of macrophages. PCSK9 inhibition reduced platelet activation and platelet-dependent thrombo-inflammation. The authors identified platelets as a source of PCSK9 in CAD, which may have an impact on LDL metabolism. Furthermore, platelet-derived PCSK9 contributes to atherothrombosis, and inhibition of PCSK9 attenuates thrombo-inflammation, which may contribute to the reported beneficial clinical effects.
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Aterosclerose/metabolismo , Plaquetas/fisiologia , Doença da Artéria Coronariana/metabolismo , Lipoproteínas LDL/metabolismo , Pró-Proteína Convertase 9/fisiologia , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas/estatística & dados numéricos , Trombose/metabolismoRESUMO
Thrombocyte formation from megakaryocyte and their progenitor cells is tightly regulated by thrombopoietin (TPO) and its receptor c-MPL, thereby maintaining physiological functionality and numbers of circulating platelets. In patients, dysfunction of this regulation could cause thrombocytopenia or myeloproliferative syndromes. Since regulation of this pathway is still not completely understood, we investigated the role of the ubiquitin ligase c-Cbl which was previously shown to negatively regulated c-MPL signalling. We developed a new conditional mouse model using c-Cblfl/fl Pf4Cre mice and demonstrated that platelet-specific knockout of c-Cbl led to severe microthrombocytosis and impaired uptake of TPO and c-MPL receptor internalization. Furthermore, we characterized a constitutive STAT5 activation c-Cbl KO platelets. This study identified c-Cbl as a potential player in causing megakaryocytic and thrombocytic disorders.
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Endocitose , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores de Trombopoetina/metabolismo , Animais , Integrases/metabolismo , Linfocitose , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico , Transdução de Sinais , Trombocitose , Trombopoese , Trombopoetina/metabolismoRESUMO
Platelet integrity and function critically depend on lipid composition. However, the lipid inventory in platelets was hitherto not quantified. Here, we examined the lipidome of murine platelets using lipid-category tailored protocols on a quantitative lipidomics platform. We could show that the platelet lipidome comprises almost 400 lipid species and covers a concentration range of 7 orders of magnitude. A systematic comparison of the lipidomics network in resting and activated murine platelets, validated in human platelets, revealed that <20% of the platelet lipidome is changed upon activation, involving mainly lipids containing arachidonic acid. Sphingomyelin phosphodiesterase-1 (Smpd1) deficiency resulted in a very specific modulation of the platelet lipidome with an order of magnitude upregulation of lysosphingomyelin (SPC), and subsequent modification of platelet activation and thrombus formation. In conclusion, this first comprehensive quantitative lipidomic analysis of platelets sheds light on novel mechanisms important for platelet function, and has therefore the potential to open novel diagnostic and therapeutic opportunities.
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Plaquetas/metabolismo , Lipídeos/análise , Fosforilcolina/análogos & derivados , Esfingomielina Fosfodiesterase/fisiologia , Esfingosina/análogos & derivados , Trombose/fisiopatologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilcolina/metabolismo , Ativação Plaquetária , Esfingosina/metabolismo , Trombose/metabolismoRESUMO
Permanent pacemaker implantation (PPI) is a widely recognized complication associated with TAVI (incidence up to 20%). Smaller registries have identified several variables associated with PPI. The objective was to validate patient- and transcatheter aortic valve implantation (TAVI)-related procedural variables associated with PPI. We performed a retrospective analysis of patients from six European centers undergoing TAVI with the Edwards SAPIEN 3 prosthesis. Baseline variables and pre-procedural ECG characteristics and CT-scans were taken into account. Data for 1745 patients were collected; 191 (10.9%) required PPI after TAVI. The baseline variables pulmonary hypertension (OR 1.64; 95% CI 1.01-2.59), QRS duration > 117 ms (OR 2.58; 95% CI 1.73-3.84), right bundle branch block (RBBB; OR 5.14; 95% CI 3.39-7.72), left anterior hemi block (OR 1.92; 95% CI 1.19-3.02) and first-degree atrioventricular block (AVB, OR 1.63; 95%CI 1.05-2.46) were significantly associated with PPI. RBBB (OR 8.11; 95% CI 3.19-21.86) and first-degree AVB (OR 2.39; 95% CI 1.18-4.66) remained significantly associated in a multivariate analysis. Procedure-related variables included access site (TF; OR 1.97; 95% CI 1.07-4.05), implanted valve size (29 mm; OR 1.88; 95% CI 1.35-2.59), mean TAVI valve implantation depth below the annulus > 30% (OR 3.75; 95% CI 2.01-6.98). Patients receiving PPI had longer ICU stays and later discharges. Acute kidney injury stage 2/3 was more common in patients with PPI until discharge (15.2 vs. 3.1%; p = 0.007), but was not statistically significant thereafter. Further differences in outcomes at 30 days did not reach significance. The data will aid pre- and post-procedural patient management and prevent adverse long-term outcomes.Clinical Trial: NCT03497611.
Assuntos
Estenose da Valva Aórtica/cirurgia , Valva Aórtica/cirurgia , Valvuloplastia com Balão/efeitos adversos , Estimulação Cardíaca Artificial , Bloqueio Cardíaco/terapia , Frequência Cardíaca , Próteses Valvulares Cardíacas , Marca-Passo Artificial , Substituição da Valva Aórtica Transcateter/instrumentação , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/fisiopatologia , Europa (Continente) , Feminino , Bloqueio Cardíaco/diagnóstico , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/fisiopatologia , Humanos , Masculino , Sistema de Registros , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Substituição da Valva Aórtica Transcateter/efeitos adversos , Resultado do TratamentoRESUMO
BACKGROUND: Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant. METHODS: We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry. RESULTS: We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca2+ influx. Using C3aR-/- mice or C3-/- mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR-/- mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR-/- mice with C3aR+/+ platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation. CONCLUSIONS: Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.
Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Imunidade Inata , Infarto do Miocárdio/sangue , Receptores de Complemento/sangue , Acidente Vascular Cerebral/sangue , Trombose/sangue , Animais , Plaquetas/imunologia , Sinalização do Cálcio , Ativação do Complemento , Complemento C3/genética , Complemento C3/imunologia , Complemento C3/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/imunologia , Ativação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Receptores de Complemento/deficiência , Receptores de Complemento/genética , Receptores de Complemento/imunologia , Acidente Vascular Cerebral/imunologia , Trombose/imunologiaRESUMO
Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.
Assuntos
Caseína Quinase II/fisiologia , Fragmentos de Peptídeos/fisiologia , Ativação Plaquetária , Trombopoese , Trombose/patologia , Animais , Plaquetas , Sinalização do Cálcio , Caseína Quinase II/deficiência , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/ultraestrutura , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/deficiência , Trombose/etiologia , Trombose/metabolismoRESUMO
Platelet aggregation, dense granule secretion and thrombus formation are dependent on sphingolipids like ceramide and sphingosine as well as sphingosine-1 phosphate. Sphingosine/ceramide metabolism involves ceramide synthases and ceramidases. However, the role of ceramide synthase and ceramidase in the regulation of platelet function remained ill-defined. The present study determined transmission light aggregometry, employed luciferase based ATP release measurements and studied in vitro thrombus formation under high arterial shear rates in order to define the impact of pharmacological inhibition of serine palmitoyltransferase, ceramide synthase and ceramidase on platelet function. As a result, inhibition of ceramidase significantly blunted collagen related peptide (CRP) induced glyocoprotein VI (GPVI)-dependent platelet aggregation, ATP release and thrombus formation on a collagen-coated surface under shear rates of 1700-sec. Defective platelet aggregation after ceramidase inhibition could partially be overcome by exogenous sphingosine treatment reflecting a pivotal role of ceramidase-derived sphingosine in platelet function. Inhibition of serine palmitoyltransferase and ceramide synthase did not significantly modify GPVI-dependent platelet activation. In conclusion, the present study unraveled ceramidase as a crucial player in sphingosine-induced platelet activation following GPVI-dependent signaling.
Assuntos
Plaquetas/enzimologia , Ceramidases/metabolismo , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombose/enzimologia , Trombose/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , CamundongosRESUMO
AIMS: Hyperlipidaemia enhances susceptibility to thrombosis, while platelet oxidixed LDL (oxLDL) binding in acute coronary syndrome (ACS) correlates with activation status. This study explores the platelet lipidome in symptomatic coronary artery disease (CAD) patients and the functional consequences of the chemokine CXCL12 and its receptors CXCR-4/-7 on lipid uptake in platelets. METHODS AND RESULTS: Platelet-oxLDL detected by flow cytometry was enhanced (P = 0.04) in CAD patients, moderately correlated with platelet CXCR7 surface expression (ρ = 0.39; P < 0.001), while inversely with CXCR4 (ρ = 0.35; P < 0.001). Platelet-oxLDL was elevated (P = 0.01) in ACS patients with angiographic evidence of intracoronary thrombi. Ex vivo analysis of intracoronary thrombi sections revealed oxLDL deposition in platelet-enriched areas verified by immunofluorescence confocal microscopy. LDL-oxLDL uptake enhanced reactive oxygen species, mitochondrial superoxide generation, intraplatelet LDL to oxLDL conversion, and lipid peroxidation, counteracted by SOD2-mimetic MnTMPyP. Lipidomic analysis revealed enhanced intraplatelet-oxidized phospholipids, cholesteryl esters, sphingomyelin, ceramides, di- and triacylglycerols, acylcarnitines in CAD patients compared with age-matched controls as ascertained by liquid chromatography hyphenated to high-resolution mass spectrometry. LDL-oxLDL induced degranulation, αIIbß3-integrin activation, apoptosis, thrombin generation estimated by calibrated automated thrombinoscopy, and shape change verified by live imaging using scanning ion conductance microscopy. Further, LDL-oxLDL enhanced thrombus formation ex vivo and in vivo in mice (ferric chloride-induced carotid artery injury). LDL-oxLDL enhanced platelet CXCL12 release, differentially regulated CXCR4-CXCR7 surface exposure, while CXCL12 prompted LDL-oxLDL uptake and synergistically augmented the LDL-oxLDL-induced pro-oxidative, thrombogenic impact on platelet function. CONCLUSION: An altered platelet lipidome might be associated with thrombotic disposition in CAD, a mechanism potentially regulated by CXCL12-CXCR4-CXCR7 axis.