RESUMO
INTRODUCTION: Patients with stroke are at a high risk of recurrence, and although they receive antiplatelet therapies such as clopidogrel for secondary prevention of non-cardioembolic stroke, the recurrence rate remains high. Three phase 3 trials (PRASTRO-I/II/III) were conducted to determine the efficacy of prasugrel in preventing recurrent stroke. Here, we performed an integrated analysis of these studies to confirm the generalizability of the PRASTRO-III findings and to supplement the small sample size of the study. METHODS: Patients from PRASTRO-I, PRASTRO-II, and PRASTRO-III with ischemic stroke (large-artery atherosclerosis or small-artery occlusion) and at least one of the following were included: hypertension, dyslipidemia, diabetes mellitus, chronic kidney disease, or ischemic stroke history. The primary efficacy endpoint was the composite incidence of ischemic stroke, myocardial infarction (MI), and death from other vascular causes in the intention-to-treat population. Bleeding events (life-threatening bleeding, major bleeding, and clinically relevant bleeding) were evaluated as the primary safety endpoint. Cumulative incidences and 95% confidence intervals (CIs) were calculated for the study outcomes using the Kaplan-Meier method. Hazard ratios (HRs) and 95% CIs were calculated using the Cox regression model. RESULTS: The data of 2,184, 274, and 230 patients from PRASTRO-I, PRASTRO-II, and PRASTRO-III, respectively, were analyzed (N = 2,688; prasugrel, N = 1,337; clopidogrel, N = 1,351). Stroke at enrollment was classified as large-artery atherosclerosis in 49.3% of patients and small-artery occlusion in 50.7% of patients. The primary efficacy endpoint composite incidence (prasugrel vs. clopidogrel) was 3.4% versus 4.3% (HR: 0.771, 95% CI: 0.522-1.138). The incidence of each component of the primary efficacy endpoint for prasugrel versus clopidogrel was 3.1% (n = 41) versus 4.1% (n = 55) for ischemic stroke, 0.3% (n = 4) versus 0.2% (n = 3) for MI, and no events of death from other vascular causes. For the primary safety endpoint, bleeding events were reported in 6.0% of patients in the prasugrel group versus 5.5% of patients in the clopidogrel group (HR: 1.074, 95% CI: 0.783-1.473). CONCLUSIONS: This integrated analysis supports the findings of PRASTRO-III. Prasugrel is a promising treatment that results in a numerical reduction in the composite incidence of ischemic stroke, MI, and death from other vascular causes in patients with ischemic stroke who are at a high risk of stroke recurrence. No major safety issues were observed for prasugrel.
Assuntos
Síndrome Coronariana Aguda , Aterosclerose , AVC Isquêmico , Infarto do Miocárdio , Acidente Vascular Cerebral , AVC Trombótico , Humanos , Síndrome Coronariana Aguda/induzido quimicamente , Síndrome Coronariana Aguda/complicações , Aterosclerose/induzido quimicamente , Aterosclerose/complicações , Clopidogrel/efeitos adversos , Hemorragia/induzido quimicamente , Hemorragia/complicações , AVC Isquêmico/complicações , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/prevenção & controle , Inibidores da Agregação Plaquetária/efeitos adversos , Cloridrato de Prasugrel/efeitos adversos , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/prevenção & controle , AVC Trombótico/induzido quimicamente , Resultado do TratamentoRESUMO
Kappa-carrageenan (KCG), which is used to induce thrombosis in laboratory animals for antithrombotic drug screening, can trigger platelet aggregation. However, the cell-surface receptor and related signaling pathways remain unclear. In this study, we investigated the molecular basis of KCG-induced platelet activation using light-transmittance aggregometry, flow cytometry, western blotting, and surface plasmon resonance assays using platelets from platelet receptor-deficient mice and recombinant proteins. KCG-induced tail thrombosis was also evaluated in mice lacking the platelet receptor. We found that KCG induces platelet aggregation with α-granule secretion, activated integrin αIIbß3, and phosphatidylserine exposure. As this aggregation was significantly inhibited by the Src family kinase inhibitor and spleen tyrosine kinase (Syk) inhibitor, a tyrosine kinase-dependent pathway is required. Platelets exposed to KCG exhibited intracellular tyrosine phosphorylation of Syk, linker activated T cells, and phospholipase C gamma 2. KCG-induced platelet aggregation was abolished in platelets from C-type lectin-like receptor-2 (CLEC-2)-deficient mice, but not in platelets pre-treated with glycoprotein VI-blocking antibody, JAQ1. Surface plasmon resonance assays showed a direct association between murine/human recombinant CLEC-2 and KCG. KCG-induced thrombosis and thrombocytopenia were significantly inhibited in CLEC-2-deficient mice. Our findings show that KCG induces platelet activation via CLEC-2.
Thrombosis is a serious medical condition that occurs when blood clots form in the blood vessels and can lead to heart attacks or strokes. Animal models are important for evaluating the effectiveness of drugs in thrombosis treatment. Kappa-carrageenan (KCG) is a food thickener and a substance used to induce clot formation in laboratory animals. In this study, we investigated the molecular basis of KCG-induced platelet activation, which is an important step in thrombosis development. We found that KCG activates platelets via a receptor called C-type lectin-like receptor 2 (CLEC-2), leading to a prothrombotic state in mice. We also showed that KCG-induced tail thrombosis (CTT) is significantly inhibited in CLEC-2 deficient mice. Our findings suggest that CLEC-2-mediated platelet activation plays a key role in the pathogenesis of thrombosis and CLEC-2 May participate in innate immunity as a receptor for sulfate-polysaccharide.Abbreviation; CLEC-2: C-type lectin-like receptor 2; CRP: collagen-related peptide; CTT: KCGN-induced tail thrombosis; DIC: disseminated intravascular coagulation; EDTA: ethylenediaminetetraacetic acid; GPVI: glycoprotein VI; HRP: horseradish peroxidase; KCG: Κ-Carrageenan; LAT: linker activated T cells; LDS: lithium dodecyl sulfate; LTA: light-transmittance aggregometry; MFI: mean fluorescence intensity; PFA: paraformaldehyde; PLCγ2: phospholipase C gamma 2; PS: phosphatidylserine; Syk: spleen tyrosine kinase; Co-HP: Cobalt-hematoporphyrin.
Assuntos
Glicoproteínas de Membrana , Trombose , Animais , Humanos , Camundongos , Carragenina/efeitos adversos , Carragenina/metabolismo , Glicoproteínas de Membrana/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Cauda/metabolismo , Agregação Plaquetária , Plaquetas/metabolismo , Ativação Plaquetária , Quinase Syk/metabolismo , Fosforilação , Proteínas de Transporte/metabolismo , Trombose/metabolismoRESUMO
Predicting the clinical course and allocating limited medical resources appropriately is crucial during the COVID-19 pandemic. Platelets are involved in microthrombosis, a critical pathogenesis of COVID-19; however, the role of soluble CLEC-2 (sCLEC-2), a novel platelet activation marker, in predicting the prognosis of COVID-19 remains unexplored. We enrolled 108 patients with COVID-19, hospitalized between January 2021 and May 2022, to evaluate the clinical use of sCLEC-2 as a predictive marker. sCLEC-2 levels were measured in plasma sampled on admission, as well as interleukin-6, cell-free DNA, von Willebrand factor, and thrombomodulin. We retrospectively classified the patients into two groups - those who required oxygenation during hospitalization (oxygenated group) and those who did not (unoxygenated group) - and compared their clinical and laboratory characteristics. The correlation between sCLEC-2 and the other parameters was validated. The sCLEC-2 level was significantly higher in the oxygenated group (188.8 pg/mL vs. 296.1 pg/mL). Multivariate analysis identified high sCLEC-2 levels (odds ratio per 10 pg/mL:1.25) as an independent predictor of oxygen therapy requirement. sCLEC-2 was positively correlated with cell-free DNA, supporting the association between platelet activation and neutrophil extracellular traps. In conclusion, sCLEC-2 is a clinically valuable marker in predicting oxygen therapy requirements for patients with COVID-19.
What is the context? During the COVID-19 epidemic with tremendous damage to healthcare systems worldwide, predicting the clinical course of patients and allocating limited medical resources appropriately is crucial.Platelets are involved in microthrombosis - a critical pathogenesis of COVID-19. The role of soluble CLEC-2 (sCLEC-2), a novel in vivo platelet activation marker, in predicting the prognosis of COVID-19 remains unexplored.What is new? sCLEC-2 is an independent predictive marker of oxygen therapy requirement in COVID-19.What is the impact? In most cases, patients requiring oxygen therapy must be hospitalized. The ability to predict such cases during the COVID-19 epidemic, when medical recourses are depleted, may contribute to the appropriate allocation of medical resources.
Assuntos
COVID-19 , Ácidos Nucleicos Livres , Humanos , COVID-19/terapia , Pandemias , Estudos Retrospectivos , Lectinas Tipo C , OxigênioRESUMO
BACKGROUND AND PURPOSE: High blood pressure increases bleeding risk during treatment with antithrombotic medication. The association between blood pressure levels and the risk of recurrent stroke during long-term secondary stroke prevention with thienopyridines (particularly prasugrel) has not been well studied. METHODS: This was a post hoc analysis of the randomized, double-blind, multicenter PRASTRO-I trial (Comparison of Prasugrel and Clopidogrel in Japanese Patients With Ischemic Stroke-I). Patients with noncardioembolic stroke were randomly assigned (1:1) to receive prasugrel 3.75 mg/day or clopidogrel 75 mg/day for 96 to 104 weeks. Risks of any ischemic or hemorrhagic stroke, combined ischemic events, and combined bleeding events were determined based on the mean level and visit-to-visit variability, including successive variation, of systolic blood pressure (SBP) throughout the observational period. These risks were also compared between quartiles of mean SBP level and successive variation of SBP. RESULTS: A total of 3747 patients (age 62.1±8.5 years, 797 women), with a median average SBP level during the observational period of 132.5 mm Hg, were studied. All the risks of any stroke (146 events; hazard ratio, 1.318 [95% CI, 1.094-1.583] per 10-mm Hg increase), ischemic stroke (133 events, 1.219 [1.010-1.466]), hemorrhagic stroke (13 events, 3.247 [1.660-6.296]), ischemic events (142 events, 1.219 [1.020-1.466]), and bleeding events (47 events, 1.629 [1.172-2.261]) correlated with increasing mean SBP overall. Similarly, an increased risk of these events correlated with increasing successive variation of SBP (hazard ratio, 3.078 [95% CI, 2.220-4.225] per 10-mm Hg increase; 3.051 [2.179-4.262]; 3.276 [1.172-9.092]; 2.865 [2.042-4.011]; 2.764 [1.524-5.016], respectively). Event rates did not differ between the clopidogrel and prasugrel groups within each quartile of SBP or successive variation of SBP. CONCLUSIONS: Both high mean SBP level and high visit-to-visit variability in SBP were significantly associated with the risk of recurrent stroke during long-term medication with either prasugrel or clopidogrel after stroke. Control of hypertension would be important regardless of the type of antiplatelet drugs. Registration: URL: https://www.clinicaltrials.jp; Unique identifier: JapicCTI-111582.
Assuntos
Clopidogrel/uso terapêutico , Hipertensão/complicações , AVC Isquêmico/complicações , AVC Isquêmico/tratamento farmacológico , Cloridrato de Prasugrel/uso terapêutico , Idoso , Pressão Sanguínea , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/uso terapêutico , Prevenção Secundária/métodos , Tromboembolia/prevenção & controleRESUMO
Cholestatic liver injury leads to liver dysfunction. The available evidence suggests that platelets can either promote or reduce liver injury and fibrosis. This study focused on the functions of the C-type lectin-like receptor 2 (CLEC-2), a new special platelet receptor that binds with podoplanin-activating platelets. The role of CLEC-2 and podoplanin in cholestatic liver injury was investigated. Mice were injected intraperitoneally with weekly doses of anti-CLEC-2 antibody (2A2B10) to achieve effective CLEC-2 inhibition in their platelets. Next, left and middle hepatic bile duct ligation (BDL) procedures were performed, and mice were euthanized 1 week later (2A2B10-BDL group). In addition, mice were prepared for control groups, and relevant histological and laboratory variables were compared among these groups. The inhibition of CLEC-2 resulted in increasing hepatocellular necrosis, hepatic inflammation, and liver fibrosis. In addition, podoplanin was strongly expressed in hepatic sinusoidal endothelial cells in BDL-treated mice. Moreover, in 2A2B10-BDL mice, total plasma bile acid levels were significantly increased. In summary, podoplanin is expressed on hepatic sinusoidal endothelial cells upon BDL. Platelets bind with podoplanin via CLEC-2 and become activated. As a result, the total bile acid pool is decreased. Therefore, the CLEC-2-podoplanin interaction promotes liver protection and inhibits liver fibrosis after cholestatic liver injury.
Assuntos
Plaquetas/metabolismo , Colestase/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Colestase/patologia , Células Endoteliais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação Plaquetária/fisiologiaRESUMO
The third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE-/- mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE-/- mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE-/- mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.
Assuntos
Doenças Cardiovasculares/induzido quimicamente , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/toxicidade , Piridazinas/toxicidade , Microangiopatias Trombóticas/complicações , Animais , Aorta/metabolismo , Endotélio/metabolismo , Humanos , Isquemia/induzido quimicamente , Camundongos , Camundongos Knockout , Adesividade Plaquetária/efeitos dos fármacos , Inibidores de Proteínas Quinases/toxicidade , Disfunção Ventricular/induzido quimicamente , Fator de von Willebrand/efeitos dos fármacos , Fator de von Willebrand/metabolismoRESUMO
Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-ß depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-ß signaling, thus regulating normal lung development.
Assuntos
Plaquetas/metabolismo , Diferenciação Celular/fisiologia , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Alvéolos Pulmonares/embriologia , Transdução de Sinais/fisiologia , Animais , Plaquetas/citologia , Células Endoteliais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Lectinas Tipo C/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Alvéolos Pulmonares/citologia , Mucosa Respiratória/citologia , Mucosa Respiratória/embriologiaRESUMO
Prasugrel, a novel P2Y12 receptor antagonist, has been shown to be more effective than clopidogrel for preventing cardiovascular events in patients with acute coronary syndromes undergoing percutaneous coronary intervention. We investigated the dose-response antiplatelet effects of prasugrel compared with clopidogrel in Japanese patients with non-cardioembolic stroke. The influence of cytochrome P450 (CYP) polymorphisms on the antiplatelet effects of both drugs was also compared. In this multicenter randomized active-control comparative study, patients were randomized to receive prasugrel 2.5 mg, 5 mg, or 7.5 mg (double blind) or clopidogrel 75 mg (open label) once daily for 14 days. The primary endpoint was inhibition of platelet aggregation (IPA) in response to adenosine diphosphate 20 µM within 8 h of study drug administration on day 14. Of the 66 patients randomized, data from 63 (prasugrel 2.5 mg, 5 mg, and 7.5 mg groups, n = 14, 16, and 18, respectively; clopidogrel group, n = 15) were used in the pharmacodynamic assessment. IPA (arithmetic mean ± SD) after prasugrel administration increased dose-dependently (33 ± 9%, 44 ± 11%, and 53 ± 14%, at 2.5 mg, 5 mg, and 7.5 mg, respectively) and was higher in these groups than after clopidogrel (23 ± 16%). In a subgroup of CYP2C19 intermediate metabolizers, IPA was higher in the prasugrel 5 mg and 7.5 mg groups than in the clopidogrel group. No death or serious adverse events were reported. Prasugrel was well tolerated at doses up to 7.5 mg/day and had antiplatelet effects higher than those of clopidogrel 75 mg/day. CYP2C19 polymorphisms may have reduced clopidogrel-induced IPA.
Assuntos
Citocromo P-450 CYP2C19/genética , Inibidores da Agregação Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Polimorfismo Genético , Cloridrato de Prasugrel , Acidente Vascular Cerebral , Adulto , Idoso , Clopidogrel/administração & dosagem , Clopidogrel/farmacocinética , Citocromo P-450 CYP2C19/administração & dosagem , Citocromo P-450 CYP2C19/farmacocinética , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cloridrato de Prasugrel/administração & dosagem , Cloridrato de Prasugrel/farmacocinética , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/genéticaRESUMO
Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/fisiologia , Neoplasias do Colo/sangue , Trombose/sangue , Plaquetas/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Estudos Transversais , Humanos , Estudos Retrospectivos , Trombose/patologiaRESUMO
C-type lectin-like receptor 2 (CLEC-2) has been identified on the surface of platelets as a receptor for a platelet activating snake venom, rhodocytin/aggretin. CLEC-2 belongs to a C-type lectin superfamily and binds to a sialoglycoprotein, podoplanin, in vivo. Platelets play a crucial role in hemostasis and thrombosis, but recent studies have uncovered multiple roles of platelets beyond hemostasis in physiology and pathology. The interaction between platelet CLEC-2 and podoplanin is the key to several roles of platelets beyond hemostasis. The spatial and temporal expression patterns of podoplanin regulate vascular/lymphatic development, maintenance of vascular integrity, tissue regeneration, and some pathological processes including tumor metastasis and thromboinflammation. CLEC-2 facilitates blood/lymphatic vessel separation during embryonic development by binding to podoplanin on lymphatic endothelial cells. The leakage of platelets from hyperpermeable vessels for maintaining vascular integrity during inflammation depends on CLEC-2. During wound healing, the expression of podoplanin in keratinocytes is upregulated, which helps in the process. Podoplanin is expressed on the surface of tumor cells and facilitates hematogenous metastasis by inducing platelet aggregation through CLEC-2. During thrombotic processes, such as development of deep vein thrombosis, podoplanin is upregulated on unknown cells in the vessel wall in the area of inflammation, facilitates thrombus formation, and promotes further inflammation by binding to CLEC-2. In this article, the roles of platelets beyond hemostasis are comprehensively reviewed.
Assuntos
Plaquetas/metabolismo , Homeostase/imunologia , Inflamação/imunologia , Lectinas Tipo C/sangue , Glicoproteínas de Membrana/sangue , Trombose/imunologia , HumanosRESUMO
Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.
Assuntos
Plaquetas/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Lectinas Tipo C/fisiologia , Megacariócitos/fisiologia , Glicoproteínas de Membrana/metabolismo , Células Estromais/fisiologia , Trombopoese/genética , Animais , Arteríolas/citologia , Arteríolas/fisiologia , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Lectinas Tipo C/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Estromais/metabolismoRESUMO
BACKGROUND: An iron overload status induces ferroptosis, an iron-dependent nonapoptotic cell death, in various pathological conditions. We previously reported that hemin (heme), protoporphyrin-IX with ferric iron, activates platelets via C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI/FcRγ, but protoporphyrin-IX alone blocks CLEC-2-dependent platelet activation. Therefore, we hypothesized that free iron has the ability to activate platelets. OBJECTIVES: This study aimed to elucidate platelet activation mechanisms of iron (ferric chloride), including the identification of signaling pathways and receptors, and to examine whether platelets regulate ferroptosis. METHODS: Platelet aggregometry, platelet activation marker expression, and protein phosphorylation were examined in ferric chloride-stimulated human and murine platelets. Inhibitors of platelet activation signaling pathways and receptor-deleted platelets were utilized to identify the responsible signaling pathway and receptor. The effect of platelets on ferroptosis of endothelial cells was investigated in vitro. RESULTS: Ferric chloride induced platelet activation dependent on Src family kinase pathways in humans and mice. Ferric chloride-induced platelet aggregation was almost lost in CLEC-2-depleted murine platelets and wild-type platelets preincubated with recombinant CLEC-2 proteins. Furthermore, coculture of wild-type platelets, but not CLEC-2-deficient platelets, attenuated ferroptosis of endothelial cells in vitro. CONCLUSION: Ferric chloride activates platelets via CLEC-2 and Src family kinase pathways, and platelets have a protective role in the ferroptosis of endothelial cells dependent on CLEC-2.
Assuntos
Plaquetas , Cloretos , Compostos Férricos , Ferroptose , Lectinas Tipo C , Camundongos Endogâmicos C57BL , Ativação Plaquetária , Agregação Plaquetária , Transdução de Sinais , Animais , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Compostos Férricos/farmacologia , Humanos , Ativação Plaquetária/efeitos dos fármacos , Lectinas Tipo C/metabolismo , Cloretos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Quinases da Família src/metabolismo , Fosforilação , Camundongos Knockout , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Camundongos , Células Endoteliais da Veia Umbilical Humana/metabolismoRESUMO
Thrombocytopenia, anasarca, fever, renal dysfunction, and organomegaly (TAFRO) syndrome is an inflammatory disorder with an unclear pathogenesis. We herein report a case of TAFRO syndrome in remission in a patient who experienced recurrent intracranial bleeding despite a normal platelet count and coagulation system. A further investigation suggested the presence of anti-glycoprotein VI (GPVI) autoantibodies in the plasma, which induced platelet dysfunction and bleeding tendency. No new bleeding or relapse of TAFRO syndrome occurred after immunosuppressive therapy was initiated. These findings may help elucidate the autoimmune pathogenesis of TAFRO syndrome.
Assuntos
Autoanticorpos , Recidiva , Humanos , Autoanticorpos/sangue , Autoanticorpos/imunologia , Síndrome , Glicoproteínas da Membrana de Plaquetas/imunologia , Hemorragia Cerebral/imunologia , Hemorragia Cerebral/etiologia , Hemorragia Cerebral/sangue , Trombocitopenia/imunologia , Trombocitopenia/sangue , Febre/imunologia , Febre/etiologia , Feminino , Pessoa de Meia-Idade , Masculino , Transtornos Plaquetários/imunologia , Transtornos Plaquetários/complicações , Transtornos Plaquetários/sangueRESUMO
The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-ß and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.
Assuntos
Plaquetas/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Lectinas Tipo C/fisiologia , Vasos Linfáticos/metabolismo , Glicoproteínas de Membrana/fisiologia , Ativação Plaquetária , Animais , Movimento Celular , Proliferação de Células , Cruzamentos Genéticos , Células Endoteliais/citologia , Éxons , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Cancer-associated thrombosis (CAT) is the leading cause of morbidity and mortality. Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment that contributes to cancer progression through direct cell-cell interactions and the release of extracellular vesicles (EVs). However, the role of CAFs in CAT remains unclear. OBJECTIVE: This study aims to investigate whether CAFs aggravate CAT and the underlying molecular mechanism using a preclinical mouse lung cancer model. METHODS: We designed a Lewis lung carcinoma (LLC) tumor-bearing mouse model. CAFs were characterized using fluorescence immunohistostaining. The presence of podoplanin, a platelet-activating membrane protein through C-type lectin-like receptor 2 (CLEC-2), in EVs isolated from primary CAFs or LLC tumor tissues was assessed by immunoblotting. The platelet activation and aggregation abilities of the EVs were quantified using flow cytometry. Podoplanin plasma levels were measured by enzyme-linked immunosorbent assay. Venous thrombosis was induced in the femoral vein using 2.5% ferric chloride. The anti-CLEC-2 monoclonal antibody 2A2B10 was used to deplete CLEC-2 on the surface of the platelets. RESULTS: CAFs expressing CD90, PDGFRß, HSP47, CD34, and vimentin, co-expressed podoplanin and induced platelet activation and aggregation in a CLEC-2-dependent manner. Tumor-bearing mice showed elevated podoplanin plasma levels. CAF-EV injection and tumor-bearing mice showed shorter occlusion time in the venous thrombosis model. Although tumor growth was not altered, antibody-induced CLEC-2 depletion suppressed venous thrombosis in the tumor-bearing state but not in the healthy condition. CONCLUSION: CAFs and CAF-derived EVs induce CLEC-2-dependent platelet aggregation and aggravate venous thrombosis.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pulmonares , Trombose , Trombose Venosa , Camundongos , Animais , Fibroblastos Associados a Câncer/metabolismo , Agregação Plaquetária , Plaquetas/metabolismo , Neoplasias Pulmonares/metabolismo , Trombose Venosa/metabolismo , Trombose/metabolismo , Lectinas Tipo C/metabolismo , Microambiente TumoralRESUMO
Platelets form complexes with gastric cancer (GC) cells via direct contact, enhancing their malignant behavior. In the present study, the molecules responsible for GC cell-platelet interactions were examined and their therapeutic application in inhibiting the peritoneal dissemination of GC was investigated. First, the inhibitory effects of various candidate surface molecules were investigated on platelets and GC cells, such as C-type lectin-like receptor 2 (CLEC-2), glycoprotein VI (GPVI) and integrin αIIbß3, in the platelet-induced enhancement of GC cell malignant potential. Second, the therapeutic effects of molecules responsible for the development and progression of GC were investigated in a mouse model of peritoneal dissemination. Platelet-induced enhancement of the migratory ability of GC cells was markedly inhibited by an anti-GPVI antibody and inhibitor of galectin-3, a GPVI ligand. However, neither the CLEC-2 inhibitor nor the integrin-blocking peptide significantly suppressed this enhanced migratory ability. In experiments using mouse GC cells and platelets, the migratory and invasive abilities enhanced by platelets were significantly suppressed by the anti-GPVI antibody and galectin-3 inhibitor. Furthermore, in vivo analyses demonstrated that the platelet-induced enhancement of peritoneal dissemination was significantly suppressed by the coadministration of anti-GPVI antibody and galectin-3 inhibitor, and was nearly eliminated by the combined treatment. The inhibition of adhesion resulting from GPVI-galectin-3 interaction may be a promising therapeutic strategy for preventing peritoneal dissemination in patients with GC.
RESUMO
Coronavirus disease 2019 is diagnosed based on the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal swabs or saliva samples using reverse-transcription quantitative polymerase chain reaction. Nasopharyngeal swabs should be collected by medical professionals who are covered with full personal protective equipment (PPE), while saliva samples can be collected by patients themselves without any PPE. However, collecting saliva is difficult for people who are unable to follow instructions, including infants or unconscious patients. Owing to the high viscosity of saliva, special attention is required to handle saliva samples in laboratories. To solve these problems, we compared lingual and buccal mucosal swabs (oral swabs) with nasopharyngeal swabs and saliva samples. Among 13 patients who had a positive result for SARS-CoV-2 RNA in their nasopharyngeal swabs, 8 and 10 patients had a positive result for SARS-CoV-2 RNA in their saliva (concordance rate, 61.5%) and oral swabs (76.9%), respectively. Among the eight patients with a positive result for SARS-CoV-2 RNA in saliva, seven (87.5%) had SARS-CoV-2 detected in their oral swabs. We could not obtain saliva samples from four patients, but we found perfect concordance of SARS-CoV-2 positivity between the nasopharyngeal and oral swabs. Therefore, oral swabs can be used for SARS-CoV-2 RNA detection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Nasofaringe , RNA Viral/genética , Saliva , Manejo de EspécimesRESUMO
BACKGROUND: Erythropoiesis is a complex multistep process by which erythrocytes are produced. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor almost exclusively expressed on the surface of platelets and megakaryocytes. Deletion of megakaryocyte/platelet CLEC-2 was reported to cause anemia along with thrombocytopenia in mice. PDPN-expressing stromal cells in the bone marrow (BM) were also reported to facilitate megakaryocyte expansion and maturation depending on the CLEC-2/PDPN interaction. OBJECTIVES: We investigated how specific deletion of CLEC-2 in megakaryocytes/platelets leads to anemia. METHODS: We used flow cytometry to analyze maturation of erythroblasts, apoptotic cell death, and cell cycle distribution. CLEC-2 stimulated PDPN-expressing stromal cell-conditioned medium was analyzed by cytokine array and ELISA, and co-cultured with immature erythroblasts. Cytokine levels in serum and BM extracellular fluid were quantified by ELISA. RESULTS: We observed increased apoptosis of BM erythroblasts in megakaryocyte/platelet-specific CLEC-2 conditional knockout (Clec1bΔPLT ) mice. Moreover, PDPN-expressing stromal cells in the BM secreted insulin-like growth factor 1 (IGF-1) depending on the CLEC-2/PDPN interaction. Pretreatment with IGF-1 receptor inhibitor increased apoptosis rate and decreased the proliferation of erythroblasts in vitro. Furthermore, in Clec1bΔPLT mice, IGF-1 concentrations in serum and BM extracellular fluid were decreased, and IGF-1 replacement in Clec1bΔPLT mice attenuated anemia. CONCLUSIONS: Our findings suggest that IGF-1 secretion from PDPN-expressing stromal cells by CLEC-2 stimulation positively regulates erythroblasts. This novel mechanism of erythropoiesis regulation indicates that a microenvironment consisting of megakaryocytes and PDPN-expressing stromal cells supports erythropoiesis.
Assuntos
Eritropoese , Fator de Crescimento Insulin-Like I , Animais , Plaquetas , Lectinas Tipo C , Glicoproteínas de Membrana/genética , Camundongos , Células EstromaisRESUMO
There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2-depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2-depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.
Assuntos
Injúria Renal Aguda , Rabdomiólise , Injúria Renal Aguda/etiologia , Animais , Plaquetas , Feminino , Heme , Lectinas Tipo C/genética , Camundongos , Glicoproteínas da Membrana de Plaquetas/genética , GravidezRESUMO
AIMS: The efficacy of antiplatelet therapy may vary among different disease subtypes. Prasugrel is generally a more potent, consistent, and fast-acting platelet inhibitor than clopidogrel. This sub-analysis of the phase III comparison of PRAsugrel and clopidogrel in Japanese patients with ischemic STROke (PRASTRO-I) trial aimed to assess the differences in efficacy of these treatments for each stroke subtype. METHODS: In the PRASTRO-I trial, a total of 3,753 patients with ischemic stroke were recruited from 224 centers throughout Japan and randomized (1:1) to prasugrel (3.75 mg/day) or clopidogrel (75 mg/day) for 96 weeks. For the sub-analysis, strokes were classified as large-artery atherosclerosis, small-artery occlusion (lacunar), stroke of other etiology, and stroke of undetermined etiology. The cumulative incidence of primary events (ischemic stroke, myocardial infarction, and death from other vascular cause) and hazard ratios (HRs) were calculated for each subgroup. RESULTS: For patients with large-artery atherosclerosis, the primary event incidence was 3.8% in the prasugrel group and 4.8% in the clopidogrel group (HR 0.79; 95% confidence interval [CI] 0.45-1.41). For patients with small-artery occlusion, the incidence was 3.3% in the prasugrel group and 3.9% in the clopidogrel group (HR 0.82; 95% CI 0.45-1.50). For patients with stroke of undetermined etiology, the incidence was 4.6% in the prasugrel group and 3.0% in the clopidogrel group (HR 1.56; 95% CI 0.90-2.72). The incidence of bleeding was similar across subtypes. CONCLUSIONS: Although statistical significance was not reached, the efficacy of prasugrel was potentially different between stroke subtypes, warranting further studies.