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BACKGROUND AND AIMS: Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, KD: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. METHODS: Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. RESULTS: Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. CONCLUSIONS: EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk.
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BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a lifesaving therapy in patients with acute respiratory distress syndrome (ARDS). Hemostatic complications are frequently observed in patients on ECMO and limit the success of this therapy. Platelets are key mediators of hemostasis enabling activation, aggregation, and thrombus formation by coming in contact with exposed matrix proteins via their surface receptors such as glycoprotein (GP) VI or GPIb/V/IX. Recent research has elucidated a regulatory role of the GPV subunit. The cleaved soluble GPV (sGPV) ectodomain was identified to spatiotemporally control fibrin formation through complex formation with thrombin. OBJECTIVES: We aimed to decipher the impact of ECMO on platelet phenotype and function, including the role of GPV and plasmatic sGPV. METHODS: We recruited 36 patients with ARDS in the wake of COVID-19 pneumonia and performed a longitudinal comparison of platelet phenotype and function in non-ECMO (n = 23) vs ECMO (n = 13) compared with those of healthy controls. Patients were assessed at up to 3 time points (t1 = days 1-3; t2 = days 4-6; and t3 = days 7-14 after cannulation/study inclusion). RESULTS: Agonist-induced platelet activation was assessed by flow cytometry and revealed decreased GPIIb/IIIa activation and α-granule release in all ARDS patients. During ECMO treatment, agonist-induced δ-granule release continuously decreased, which was independently confirmed by electron microscopy and was associated with a prolonged in vitro bleeding time. GPV expression on the platelet surface markedly decreased in ECMO patients compared with that in non-ECMO patients. Plasma sGPV levels were increased in ECMO patients and were associated with poor outcome. CONCLUSION: Our data demonstrate an ECMO-intrinsic platelet δ-granule deficiency and hemostatic dysfunction beyond the underlying ARDS.
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Plaquetas , COVID-19 , Oxigenação por Membrana Extracorpórea , Síndrome do Desconforto Respiratório , Humanos , COVID-19/complicações , COVID-19/sangue , COVID-19/terapia , Síndrome do Desconforto Respiratório/terapia , Síndrome do Desconforto Respiratório/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Plaquetas/metabolismo , Adulto , Idoso , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Fenótipo , SARS-CoV-2 , Estudos de Casos e Controles , Complexo Glicoproteico GPIb-IX de PlaquetasRESUMO
BACKGROUND: How platelets interact with and influence the tumor microenvironment (TME) remains poorly characterized. METHODS: We compared the presence and participation of platelets in the TME of two tumors characterized by highly different TME, PyMT AT-3 mammary tumors and B16F1 melanoma. RESULTS: We show that whereas firmly adherent platelets continuously line tumor vessels of both AT-3 and B16F1 tumors, abundant extravascular stromal clusters of platelets from thrombopoietin-independent origin were present only in AT-3 mammary tumors. We further show that platelets influence the angiogenic and inflammatory profiles of AT-3 and B16F1 tumors, though with very different outcomes according to tumor type. Whereas thrombocytopenia increased bleeding in both tumor types, it further caused severe endothelial degeneration associated with massive vascular leakage, tumor swelling, and increased infiltration of cytotoxic cells, only in AT-3 tumors. CONCLUSIONS: These results indicate that while platelets are integral components of solid tumors, their localization and origin in the TME, as well as their impact on its shaping, are tumor type-dependent.
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Neoplasias Mamárias Animais , Microambiente Tumoral , Animais , HumanosRESUMO
ABSTRACT: Pulmonary defense mechanisms are critical for host integrity during pneumonia and sepsis. This defense is fundamentally dependent on the activation of neutrophils during the innate immune response. Recent work has shown that semaphorin 7A (Sema7A) holds significant impact on platelet function, yet its role on neutrophil function within the lung is not well understood. This study aimed to identify the role of Sema7A during pulmonary inflammation and sepsis. In patients with acute respiratory distress syndrome (ARDS), we were able to show a correlation between Sema7A and oxygenation levels. During subsequent workup, we found that Sema7A binds to the neutrophil PlexinC1 receptor, increasing integrins, and L-selectin on neutrophils. Sema7A prompted neutrophil chemotaxis in vitro and the formation of platelet-neutrophil complexes in vivo. We also observed altered adhesion and transmigration of neutrophils in Sema7A-/-animals in the lung during pulmonary inflammation. This effect resulted in increased number of neutrophils in the interstitial space of Sema7A-/- animals but reduced numbers of neutrophils in the alveolar space during pulmonary sepsis. This finding was associated with significantly worse outcome of Sema7A-/- animals in a model of pulmonary sepsis. Sema7A has an immunomodulatory effect in the lung, affecting pulmonary sepsis and ARDS. This effect influences the response of neutrophils to external aggression and might influence patient outcome. This trial was registered at www.ClinicalTrials.gov as #NCT02692118.
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Antígenos CD , Neutrófilos , Pneumonia , Semaforinas , Sepse , Semaforinas/metabolismo , Sepse/imunologia , Sepse/metabolismo , Neutrófilos/metabolismo , Neutrófilos/imunologia , Humanos , Animais , Camundongos , Antígenos CD/metabolismo , Pneumonia/metabolismo , Pneumonia/imunologia , Proteínas Ligadas por GPI/metabolismo , Masculino , Modelos Animais de Doenças , Camundongos Knockout , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/metabolismo , FemininoRESUMO
The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.
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Plaquetas , Receptores de IgG , Humanos , Receptores de IgG/metabolismo , Plaquetas/química , Plaquetas/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais , Colágeno/metabolismoRESUMO
The Beige and Chediak-Higashi (BEACH) domain-containing, Neurobeachin-like 2 (NBEAL2) protein is a molecule with a molecular weight of 300 kDa. Inactivation of NBEAL2 by loss-of-function mutations in humans as well as deletion of the Nbeal2 gene in mice results in functional defects in cells of the innate immune system such as neutrophils, NK-cells, megakaryocytes, platelets and of mast cells (MCs). To investigate the detailed function of NBEAL2 in murine MCs we generated MCs from wild type (wt) and Nbeal2-/- mice, and deleted Nbeal2 by CRISPR/Cas9 technology in the murine mast cell line MC/9. We also predicted the structure of NBEAL2 to infer its function and to examine potential mechanisms for its association with interaction partners by using the deep learning-based method RoseTTAFold and the Pymol© software. The function of NBEAL2 was analysed by molecular and immunological techniques such as co-immunoprecipitation (co-IP) experiments, western blotting, enzyme-linked immunosorbent assay and flow cytometry. We identified RPS6 as an interaction partner of NBEAL2. Thereby, the NBEAL2/RPS6 complex formation is probably required to control the protein homeostasis of RPS6 in MCs. Consequently, inactivation of NBEAL2 leads to accumulation of strongly p90RSK-phosphorylated RPS6 molecules which results in the development of an abnormal MC phenotype characterised by prolonged growth factor-independent survival and in a pro-inflammatory MC-phenotype.
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Mastócitos , Proteína S6 Ribossômica , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Mastócitos/metabolismo , Neutrófilos/metabolismo , Proteína S6 Ribossômica/metabolismoRESUMO
BACKGROUND: Endoplasmic reticulum (ER) stress is a key feature of lipid-laden macrophages and contributes to the development of atherosclerotic plaques. Blood platelets are known to interact with macrophages and fine-tune effector functions such as inflammasome activation and phagocytosis. However, the effect of platelets on ER stress induction is unknown. OBJECTIVES: The objective of this study is to elucidate the potential of platelets in regulating ER stress in macrophages in vitro. METHODS: Bone marrow-derived macrophages and RAW 264.7 cells were incubated with isolated murine platelets, and ER stress and inflammation markers were determined by reverse transcription-quantitative polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. ER morphology was investigated by electron microscopy. Cell viability, lipid accumulation, and activation were measured by flow cytometry. To gain mechanistic insights, coincubation experiments were performed with platelet decoys/releasates as well as lipopolysaccharide, blocking antibodies, and TLR4 inhibitors. RESULTS: Coincubation of platelets and macrophages led to elevated levels of ER stress markers (BIP, IRE1α, CHOP, and XBP1 splicing) in murine and human macrophages, which led to a pronounced enlargement of the ER. Macrophage ER stress was accompanied by increased release of proinflammatory cytokines and intracellular lipid accumulation, but not cell death. Platelet decoys, but not platelet releasates or lysate from other cells, phenocopied the effect of platelets. Blocking TLR4 inhibited inflammatory activation of macrophages but did not affect ER stress induction by platelet coincubation. CONCLUSION: To our knowledge, this study is the first to demonstrate that platelets induce ER stress and unfolded protein response in macrophages by heat-sensitive membrane proteins, independent of inflammatory activation of macrophages.
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Plaquetas , Estresse do Retículo Endoplasmático , Macrófagos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases , Proteína 1 de Ligação a X-Box , Animais , Plaquetas/metabolismo , Macrófagos/metabolismo , Humanos , Camundongos , Células RAW 264.7 , Proteína 1 de Ligação a X-Box/metabolismo , Proteína 1 de Ligação a X-Box/genética , Receptor 4 Toll-Like/metabolismo , Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Citocinas/metabolismo , Chaperona BiP do Retículo Endoplasmático , Fator de Transcrição CHOP/metabolismo , Transdução de Sinais , Lipopolissacarídeos/farmacologia , Proteínas de Choque Térmico/metabolismo , Metabolismo dos Lipídeos , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Sobrevivência CelularRESUMO
AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.
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Ciclofilina A , Trombose , Camundongos , Humanos , Animais , Ciclofilina A/genética , Ciclofilina A/metabolismo , Produtos Finais de Glicação Avançada , Ligantes , Inflamação , Basigina/metabolismo , Trombose/genéticaRESUMO
Pulmonary fibrosis is a chronic and progressive disease with limited therapeutic options. Nitric oxide (NO) is suggested to reduce the progression of pulmonary fibrosis via NO-sensitive guanylyl cyclase (NO-GC). The exact effects of NO-GC during pulmonary fibrosis are still elusive. Here, we used a NO-GC knockout mouse (GCKO) and examined fibrosis and inflammation after bleomycin treatment. Compared to wildtype (WT), GCKO mice showed an increased fibrotic reaction, as myofibroblast occurrence (p = 0.0007), collagen content (p = 0.0006), and mortality (p = 0.0009) were significantly increased. After fibrosis induction, lymphocyte accumulations were observed in the lungs of GCKO but not in WT littermates. In addition, the total number of immune cells, specifically lymphocytes (p = <0.0001) and neutrophils (p = 0.0047), were significantly higher in the bronchoalveolar lavage fluid (BALF) of GCKO animals compared to WT, indicating an increased inflammatory response in the absence of NO-GC. The pronounced fibrotic response in GCKO mice was paralleled by significantly increased levels of transforming growth factor ß (TGFß) in BALF (p = 0.0207), which correlated with the total number of immune cells. Taken together, our data show the effect of NO-GC deletion in the pathology of lung fibrosis and the effect on immune cells in BALF. In summary, our results show that NO-GC has anti-inflammatory and anti-fibrotic properties in the murine lung, very likely by attenuating TGFß-mediated effects.
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Fibrose Pulmonar , Camundongos , Animais , Guanilil Ciclase Solúvel/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Pulmão/patologia , Camundongos Knockout , Líquido da Lavagem Broncoalveolar , Fator de Crescimento Transformador beta/metabolismo , Anti-Inflamatórios/uso terapêutico , Bleomicina/farmacologia , Camundongos Endogâmicos C57BLRESUMO
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif-coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI-treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet-neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI-treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.
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Lesão Pulmonar Aguda , Pneumonia , Síndrome do Desconforto Respiratório , Animais , Camundongos , Lesão Pulmonar Aguda/patologia , Células Endoteliais/patologia , Inflamação/patologia , Lipopolissacarídeos/efeitos adversos , Pulmão/patologia , Infiltração de Neutrófilos , Neutrófilos/patologia , Pneumonia/patologia , Síndrome do Desconforto Respiratório/patologiaRESUMO
Hyperreactive platelets are commonly observed in diabetic patients indicating a potential link between glucose homeostasis and platelet reactivity. This raises the possibility that platelets may play a role in the regulation of metabolism. Pancreatic ß cells are the central regulators of systemic glucose homeostasis. Here, we show that factor(s) derived from ß cells stimulate platelet activity and platelets selectively localize to the vascular endothelium of pancreatic islets. Both depletion of platelets and ablation of major platelet adhesion or activation pathways consistently resulted in impaired glucose tolerance and decreased circulating insulin levels. Furthermore, we found platelet-derived lipid classes to promote insulin secretion and identified 20-Hydroxyeicosatetraenoic acid (20-HETE) as the main factor promoting ß cells function. Finally, we demonstrate that the levels of platelet-derived 20-HETE decline with age and that this parallels with reduced impact of platelets on ß cell function. Our findings identify an unexpected function of platelets in the regulation of insulin secretion and glucose metabolism, which promotes metabolic fitness in young individuals.
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Células Secretoras de Insulina , Humanos , Secreção de Insulina , Insulina/metabolismo , Plaquetas , Glucose/metabolismoRESUMO
Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms remain unclear. Outside the body, however, our ability to generate large number of functional platelets is poor. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates substantial platelet numbers, up to 3000 per megakaryocyte. Despite their large size, megakaryocytes are able repeatedly to passage through the lung vasculature, leading to enucleation and subsequent platelet generation intravascularly. Using ex vivo lung and an in vitro microfluidic chamber we determine how oxygenation, ventilation, healthy pulmonary endothelium and the microvascular structure support thrombopoiesis. We also show a critical role for the actin regulator Tropomyosin 4 in the final steps of platelet formation in lung vasculature. This work reveals the mechanisms of thrombopoiesis in lung vasculature and informs approaches to large-scale generation of platelets.
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Plaquetas , Microfluídica , Camundongos , Animais , Megacariócitos , Trombopoese , PulmãoRESUMO
Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.
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Trombose , Animais , Camundongos , Plaquetas , Proteínas de Transporte/farmacologia , Trombina/farmacologiaRESUMO
BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.
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Proteínas de Transporte de Cátions , Homeostase , Infarto da Artéria Cerebral Média , AVC Isquêmico , Trombose , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Cálcio/metabolismo , Cátions/metabolismo , AVC Isquêmico/genética , AVC Isquêmico/complicações , AVC Isquêmico/metabolismo , Magnésio/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombose/genética , Trombose/metabolismo , Canal de Cátion TRPC6/metabolismo , Proteínas de Transporte de Cátions/deficiênciaRESUMO
The platelet specific integrin αIIbß3 mediates platelet adhesion, aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbß3 is expressed on the membrane surface and in intracellular compartments. Upon activation, the number of surface-expressed αIIbß3 is increased by the translocation of internal granule pools to the plasma membrane. The WASH complex is the major endosomal actin polymerization-promoting complex and has been implicated in the generation of actin networks involved in endocytic trafficking of integrins in other cell types. The role of the WASH complex and its subunit Strumpellin in platelet function is still unknown. Here, we report that Strumpellin-deficient murine platelets display an approximately 20% reduction in integrin αIIbß3 surface expression. While exposure of the internal αIIbß3 pool after platelet activation was unaffected, the uptake of the αIIbß3 ligand fibrinogen was delayed. The number of platelet α-granules was slightly but significantly increased in Strumpellin-deficient platelets. Quantitative proteome analysis of isolated αIIbß3-positive vesicular structures revealed an enrichment of protein markers, which are associated with the endoplasmic reticulum, Golgi complex and early endosomes in Strumpellin-deficient platelets. These results point to a so far unidentified role of the WASH complex subunit Strumpellin in integrin αIIbß3 trafficking in murine platelets.
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Integrina alfa2 , Integrina beta3 , Peptídeos e Proteínas de Sinalização Intracelular , Animais , Camundongos , Plaquetas/metabolismo , Integrina alfa2/metabolismo , Integrina beta3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Megacariócitos/metabolismo , Camundongos KnockoutRESUMO
The activation of platelets and coagulation at vascular injury sites is crucial for haemostasis but can promote thrombosis and inflammation in vascular pathologies. Here, we delineate an unexpected spatio-temporal control mechanism of thrombin activity that is platelet orchestrated and locally limits excessive fibrin formation after initial haemostatic platelet deposition. During platelet activation, the abundant platelet glycoprotein (GP) V is cleaved by thrombin. We demonstrate with genetic and pharmacological approaches that thrombin-mediated shedding of GPV does not primarily regulate platelet activation in thrombus formation, but rather has a distinct function after platelet deposition and specifically limits thrombin-dependent generation of fibrin, a crucial mediator of vascular thrombo-inflammation. Genetic or pharmacologic defects in haemostatic platelet function are unexpectedly attenuated by specific blockade of GPV shedding, indicating that the spatio-temporal control of thrombin-dependent fibrin generation also represents a potential therapeutic target to improve haemostasis.
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CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.
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Lectinas Tipo C , Anticorpos de Domínio Único , Animais , Humanos , Camundongos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Anticorpos de Domínio Único/farmacologia , Quinase Syk/metabolismoRESUMO
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
In tumor therapy anti-angiogenic approaches have the potential to increase the efficacy of a wide variety of subsequently or co-administered agents, possibly by improving or normalizing the defective tumor vasculature. Successful implementation of the concept of vascular normalization under anti-angiogenic therapy, however, mandates a detailed understanding of key characteristics and a respective scoring metric that defines an improved vasculature and thus a successful attempt. Here, we show that beyond commonly used parameters such as vessel patency and maturation, anti-angiogenic approaches largely benefit if the complex vascular network with its vessel interconnections is both qualitatively and quantitatively assessed. To gain such deeper insight the organization of vascular networks, we introduce a multi-parametric evaluation of high-resolution angiographic images based on light-sheet fluorescence microscopy images of tumors. We first could pinpoint key correlations between vessel length, straightness and diameter to describe the regular, functional and organized structure observed under physiological conditions. We found that vascular networks from experimental tumors diverted from those in healthy organs, demonstrating the dysfunctionality of the tumor vasculature not only on the level of the individual vessel but also in terms of inadequate organization into larger structures. These parameters proofed effective in scoring the degree of disorganization in different tumor entities, and more importantly in grading a potential reversal under treatment with therapeutic agents. The presented vascular network analysis will support vascular normalization assessment and future optimization of anti-angiogenic therapy.