RESUMO
AIM: T cells are key immunomodulatory cells in periapical lesions. This study aimed to explore the roles of T cells in chronic apical periodontitis (CAP) using single-cell RNA sequencing and to further investigate Granzyme A (GZMA) in angiogenesis regulation. METHODOLOGY: A total of five CAP samples were collected for single-cell RNA sequencing. We performed subcluster and lineage-tracing analyses for T cells. According to differential gene expression, distinct biological functions enriched in T cells of CAP were presented by gene set enrichment analysis (GSEA) and compared with healthy gingiva (data obtained from the GEO database). CellChat was used to explore potential ligand-receptor interactions between T cells and endothelial cells in CAP. The coculture of primary human umbilical vein endothelial cells (HUVECs) and Jurkat T cells, as well as the addition of GZMA recombinant protein, was used to validate the predicted pair of GZMA and coagulation factor II thrombin receptor (F2R) by RT-PCR, angiogenesis and migration assays. RESULTS: A transcriptomic atlas of 44 746 individual cells was constructed from the periapical lesions of five patients with CAP by single-cell RNA-seq, and eight cell types were identified. We identified nine subsets of T cells and deciphered the cellular heterogeneity of T cells in CAP at the functional level by subclustering and GSEA. Lineage tracing revealed a distinct lineage of T cells in CAP and predicted the transition of the T cellular state upon CAP. GSEA revealed multiple biological processes and relevant angiogenesis genes upregulated in CAP T cells. GZMA-F2R pairs were predicted by cell-cell interactions in CAP. High expression of GZMA and F2R was observed in the coculture of HUVECs and Jurkat T cells, and the proangiogenic capacity of the GZMA recombinant protein was emphasized by in vitro experiments. CONCLUSIONS: Our study provides novel insights into the heterogeneity of T cells in periapical lesions and reveals the potential role of GZMA in T cells in regulating angiogenesis in HUVECs.
Assuntos
Linfócitos T , Humanos , Granzimas/genética , Granzimas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Linfócitos T/metabolismoRESUMO
Amyloid ß protein deposition in cerebral vessels, a characteristic of Alzheimer's disease, is a risk factor for intracerebral hemorrhage. Amyloid ß protein directly modulates human platelet function; however, the exact mechanism of action is unclear. Therefore, we investigated the effects of amyloid ß protein on human platelet activation using an aggregometer with laser scattering. Amyloid ß protein decreased platelet aggregation induced by thrombin receptor-activating protein, but not by collagen and ADP. Amyloid ß protein also suppressed platelet aggregation induced by SCP0237 and A3227. Platelet-derived growth factor-AB secretion and phosphorylated-heat shock protein 27 release by thrombin receptor-activating protein were inhibited by amyloid ß protein. Additionally, thrombin receptor-activating protein-induced phosphorylation of JNK and p38 MAP kinase was reduced by amyloid ß protein. Collectively, our results strongly suggest that amyloid ß protein negatively regulates protease-activated receptor-elicited human platelet activation. These findings may indicate a cause of intracerebral hemorrhage due to amyloid ß protein.
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Peptídeos beta-AmiloidesRESUMO
Type 2 DM is a risk factor for dementia, including Alzheimer's disease (AD), and is associated with brain atrophy. Amyloid ß protein (Aß) deposition in the brain parenchyma is implicated in the neurodegeneration that occurs in AD. Platelets, known as abundant storage of Aß, are recognized to play important roles in the onset and progression of AD. We recently showed that Aß negatively regulates platelet activation induced by thrombin receptor-activating protein (TRAP) in healthy people. In the present study, we investigated the effects of Aß on the TRAP-stimulated platelet activation in DM patients, and the relationship between the individual responsiveness to Aß and quantitative findings of MRI, the volume of white matter hyperintensity (WMH)/intracranial volume (IC) and the volume of parenchyma (PAR)/IC. In some DM patients, Aß reduced platelet aggregation induced by TRAP, while in others it was unchanged or rather enhanced. The TRAP-induced levels of phosphorylated-Akt and phosphorylated-HSP27, the levels of PDGF-AB and the released phosphorylated-HSP27 correlated with the degree of platelet aggregability. The individual levels of not WMH/IC but PAR/IC was correlated with those of TRAP-stimulated PDGF-AB release. Collectively, our results suggest that the reactivity of TRAP-stimulated platelet activation to Aß differs in DM patients from healthy people. The anti-suppressive feature of platelet activation to Aß might be protective for brain atrophy in DM patients.
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Peptídeos beta-Amiloides , Complicações do Diabetes , Ativação Plaquetária , Humanos , Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Atrofia/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Proteínas de Choque Térmico HSP27/metabolismo , Ativação Plaquetária/fisiologia , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Complicações do Diabetes/metabolismo , Complicações do Diabetes/patologiaRESUMO
Heptapeptide SFLLRNP is a receptor-tethered ligand of protease-activated receptor 1 (PAR-1), and its Phe at position 2 is essential for the aggregation of human platelets. To validate the structural elements of the Phe-phenyl group in receptor activation, we have synthesized a complete set of S/Phe/LLRNP peptides comprising different series of fluorophenylalanine isomers (Fn)Phe, where n = 1, 2, 3, and 5. Phe-2-phenyl was strongly suggested to be involved in the edge-to-face CH/π interaction with the receptor aromatic group. In the present study, to prove this receptor interaction definitively, we synthesized another series of peptide analogs containing (F4)Phe-isomers, with the phenyl group of each isomer possessing only one hydrogen atom at the ortho, meta, or para position. When the peptides were assayed for their platelet aggregation activity, S/(2,3,4,6-F4)Phe/LLRNP and S/(2,3,4,5-F4)Phe/LLRNP exhibited noticeable activity (34% and 6% intensities of the native peptide, respectively), whereas S/(2,3,5,6-F4)Phe/LLRNP was completely inactive. The results indicated that, at the ortho and meta positions but not at the para position, benzene-hydrogen atoms are required for the CH/π interaction to activate the receptor. The results provided a decisive evidence of the molecular recognition property of Phe, the phenyl benzene-hydrogen atom of which participates directly in the interaction with the receptor aromatic π plane.
Assuntos
Fragmentos de Peptídeos/farmacologia , Fenilalanina/farmacologia , Receptor PAR-1/antagonistas & inibidores , Relação Dose-Resposta a Droga , Voluntários Saudáveis , Humanos , Ligantes , Estrutura Molecular , Fragmentos de Peptídeos/química , Fenilalanina/química , Agregação Plaquetária/efeitos dos fármacos , Receptor PAR-1/metabolismo , Relação Estrutura-AtividadeRESUMO
Platelet-activating factor (PAF) is a potent mediator of inflammation that plays a crucial role in atherosclerosis. The purpose of this study was to evaluate the effect of a dietary supplement containing mainly plant extracts on PAF actions and metabolism in healthy volunteers. A double-blind, placebo-controlled, 8 weeks' duration study was performed. Healthy volunteers were randomly allocated into the supplement or the placebo group and fifty-eight of them completed the study. The supplement contained plant extracts (Aloe gel, grape juice, Polygonum cuspidatum) and vitamins. The activities of PAF metabolic enzymes: the two isoforms of acetyl-CoA:lyso-PAF acetyltransferase, cytidine 5'-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-cholinephosphotransferase) and platelet-activating factor-acetylhydrolase (PAF-AH) in leucocytes and lipoprotein associated phospholipase-A2 in plasma were measured along with several markers of endothelial function. Platelet aggregation against PAF, ADP and thrombin receptor activating peptide was measured in human platelet-rich plasma by light transmission aggregometry. No difference was observed on soluble vascular cell adhesion molecule-1, sP-selectin and IL-6 levels at the beginning or during the study period between the two groups. Concerning PAF metabolism enzymes' activity, no difference was observed at baseline between the groups. PAF-AH activity was only increased in the supplement group at 4 and 8 weeks compared with baseline levels. In addition, supplement consumption led to lower platelet sensitivity against PAF and ADP compared with baseline levels. However, a trial effect was only observed when platelets were stimulated by PAF. In conclusion, supplementation with plant extracts and vitamins ameliorates platelet aggregation primarily against PAF and secondarily against ADP and affects PAF catabolism by enhancing PAF-acetylhydrolase activity in healthy subjects.
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Suplementos Nutricionais , Extratos Vegetais/farmacologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Adulto , Biomarcadores/sangue , Plaquetas/metabolismo , Método Duplo-Cego , Feminino , Voluntários Saudáveis , Humanos , Leucócitos/metabolismo , MasculinoRESUMO
BACKGROUND/AIMS: Thrombin induces the activation of human platelets through protease-activated receptor (PAR) 1 and PAR4, and Rac, a member of the Rho family of small GTPases, is implicated in PAR activation. We previously reported that phosphorylated-heat shock protein 27 (HSP27) is released from the thrombin receptor-activating peptide (TRAP)-stimulated platelets of diabetic patients. In the present study, we investigated the role of Rac in the TRAP-elicited release of phosphorylated-HSP27 from human platelets. METHODS: Platelet aggregation was measured using an aggregometer with laser scattering. Protein phosphorylation was analyzed by Western blotting. The levels of phosphorylated-HSP27 and platelet-derived growth factor-AB (PDGF-AB) were measured by enzyme-linked immunosorbent assays. RESULTS: NSC23766, an inhibitor of Rac-guanine nucleotide exchange factor interaction, suppressed the TRAP-elicited release of phosphorylated-HSP27 as well as platelet aggregation. The TRAP-induced phosphorylation of HSP27, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was attenuated by NSC23766. SB203580, a p38 MAPK inhibitor, but not SP600125, a JNK inhibitor, suppressed the release of phosphorylated-HSP27 in addition to HSP27 phosphorylation. On the other hand, both SB203580 and SP600125 reduced the TRAP-stimulated secretion of PDGF-AB. CONCLUSION: Our results strongly suggest that Rac acts as a positive regulator of the PAR-elicited release of phosphorylated-HSP27 from human platelets via p38 MAPK but not JNK.
Assuntos
Plaquetas/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Aminoquinolinas/farmacologia , Plaquetas/citologia , Plaquetas/metabolismo , Células Cultivadas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/antagonistas & inibidoresRESUMO
Protease-activated receptor (PAR)4 is a low affinity thrombin receptor with less understood function relative to PAR1. PAR4 is involved in platelet activation and hemostasis, but its specific actions on myocyte growth and cardiac function remain unknown. This study examined the role of PAR4 deficiency on cardioprotection after myocardial ischemia-reperfusion (IR) injury in mice. When challenged by in vivo or ex vivo IR, PAR4 knockout (KO) mice exhibited increased tolerance to injury, which was manifest as reduced infarct size and a more robust functional recovery compared to wild-type mice. PAR4 KO mice also showed reduced cardiomyocyte apoptosis and putative signaling shifts in survival pathways in response to IR. Inhibition of PAR4 expression in isolated cardiomyocytes by shRNA offered protection against thrombin and PAR4-agonist peptide-induced apoptosis, while overexpression of wild-type PAR4 significantly enhanced the susceptibility of cardiomyocytes to apoptosis, even under low thrombin concentrations. Further studies implicate Src- and epidermal growth factor receptor-dependent activation of JNK on the proapoptotic effect of PAR4 in cardiomyocytes. These findings reveal a pivotal role for PAR4 as a regulator of cardiomyocyte survival and point to PAR4 inhibition as a therapeutic target offering cardioprotection after acute IR injury.
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Traumatismo por Reperfusão Miocárdica/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Receptores de Trombina/genética , Animais , Apoptose/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Peptídeos/farmacologia , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Trombina/agonistas , Receptores de Trombina/antagonistas & inibidores , Receptores de Trombina/deficiência , Transdução de Sinais , Trombina/farmacologia , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
OBJECTIVE: Patients requiring extracorporeal membrane oxygenation (ECMO) have a well-known bleeding risk and the potential for experiencing possibly fatal thromboembolic complications. Risk factors and predictors of transfusion requirements during ECMO support remain uncertain. The authors hypothesized that compromised organ function immediately before ECMO support will influence transfusion requirements. DESIGN: A prospective observational study. SETTING: A tertiary, single-institutional university hospital. PARTICIPANTS: The study included 40 adult patients requiring ECMO for intractable cardiac and respiratory failure between July 2010 and December 2012. Blood samples were taken before initiation of ECMO (baseline), after 24 and 48 hours on ECMO, and 24 hours after termination of ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Independent of veno-arterial or veno-venous support, 26% of patients required≥2 packed red blood cells per day (PRBC/d) and 74% of patients required<2 PRBC/d during ECMO. Requirements of≥2 PRBC/d during ECMO support were associated with higher creatinine levels and lower prothrombin times (PT, %) at baseline and with impaired platelet function after 24 hours on ECMO. Platelet function, activated by thrombin receptor-activating peptide stimulation, decreased by 30% to 40% over time on ECMO. Receiver operating characteristic curve analysis showed cut-off values for creatinine of 1.49 mg/dL (sensitivity 70%, specificity 70%; area under the curve [AUC] 0.76, 95% confidence interval [CI] 0.58-0.94), for PT of 48% (sensitivity 80%, specificity 59%; AUC 0.69, 95% CI 0.50-0.87), and for thrombin receptor-activating peptide (TRAP) 32 U (sensitivity 90%, specificity 68%; AUC 0.76, 95% CI 0.59-0.93). CONCLUSIONS: The results of this study demonstrated that increased creatinine levels and lower PT before ECMO and secondary impaired platelet function significantly increased transfusion requirement.
Assuntos
Transfusão de Sangue , Oxigenação por Membrana Extracorpórea , Adulto , Idoso , Área Sob a Curva , Humanos , Pessoa de Meia-Idade , Ativação Plaquetária , Estudos Prospectivos , Trombina/biossínteseRESUMO
BACKGROUND: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.
Assuntos
Plaquetas , Camundongos Endogâmicos C57BL , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Receptores de Trombina , Trombina , Animais , Plaquetas/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Trombina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Modelos Animais de Doenças , Venenos de Crotalídeos/farmacologia , Venenos de Crotalídeos/toxicidade , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Selectina-P/metabolismo , Selectina-P/genética , Mutação Puntual , Técnicas de Introdução de Genes , Transdução de Sinais , Trombose/genética , Trombose/sangue , Masculino , Cloretos , Camundongos , Ativação Plaquetária , Sistemas CRISPR-Cas , Humanos , Fenótipo , Compostos Férricos , Oligopeptídeos , Lectinas Tipo C , Receptores Ativados por ProteinaseRESUMO
P4 is a hormone with diverse functions that include roles in reproduction, growth, and development. The objectives of this study were to examine the effects of P4 on androgen production in the mature teleost testis and to identify molecular signaling cascades regulated by P4 to improve understanding of its role in male reproduction. Fathead minnow (FHM) testis explants were treated in vitro with two concentrations of P4 (10(-8) and 10(-6) M) for 6 and 12 h. P4 significantly increased testosterone (T) production in the FHM testis but did not affect 11-ketotestosterone. Gene network analysis revealed that insulin growth factor (Igf1) and tumor necrosis factor receptor (Tnfr) signaling was significantly depressed with P4 treatment after 12h. There was also a 20% increase in a gene network for follicle-stimulating hormone secretion and an 18% decrease in genes involved in vasopressin signaling. Genes in steroid metabolism (e.g. star, cyp19a, 11bhsd) were not significantly affected by P4 treatments in this study, and it is hypothesized that pre-existing molecular machinery may be more involved in the increased production of T rather than the de novo expression of steroid-related transcripts and receptors. There was a significant decrease in prostaglandin E synthase 3b (cytosolic) (ptges3b) after treatment with P4, suggesting that there is cross talk between P4 and prostaglandin pathways in the reproductive testis. P4 has a role in regulating steroid production in the male testis and may do so by modulating gene networks related to endocrine pathways, such as Igf1, Tnfr, and vasopressin.
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Cyprinidae/genética , Cyprinidae/metabolismo , Progesterona/genética , Testículo/metabolismo , Animais , Masculino , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Testosterona/análogos & derivados , Testosterona/metabolismoRESUMO
BACKGROUND: The involvement of thrombin receptor PAR1 in blood vessel development has been largely demonstrated in knockout mice; however, its implication in adult mouse angiogenesis seems very moderate. OBJECTIVES: We aimed to explore the potential relationships between PAR1, stemness, and angiogenic properties of human endothelial colony-forming cells (ECFCs). METHODS AND RESULTS: PAR1 activation on ECFCs using the selective PAR1-activating peptide induced a significant decrease in CD133 expression (RTQ-PCR analysis). In line, silencing of PAR1 gene expression with siRNA increased CD133 mRNA as well as intracellular CD133 protein expression. To confirm the link between CD133 and PAR1, we explored the association between PAR1 and CD133 levels in fast and slow fibroblasts prone to reprogramming. An imbalance between PAR1 and CD133 levels was evidenced, with a decreased expression of PAR1 in fast reprogramming fibroblasts expressing a high CD133 level. Regarding in vitro ECFC angiogenic properties, PAR1 silencing with specific siRNA induced cell proliferation evidenced by the overexpression of Ki67. However, it did not impact migration properties nor ECFC adhesion on smooth muscle cells or human arterial endothelial cells. In a mouse model of hind-limb ischemia, PAR1 silencing in ECFCs significantly increased postischemic revascularization compared to siCtrl-ECFCs along with a significant increase in cutaneous blood flows (P < .0001), microvessel density (P = .02), myofiber regeneration (P < .0001), and human endothelial cell incorporation in muscle (P < .0001). CONCLUSION: In conclusion, our work describes for the first time a link between PAR1, stemness, and vasculogenesis in human ECFCs.
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Células Endoteliais , Receptor PAR-1 , Humanos , Células Cultivadas , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Background: Protease activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated a single nucleotide polymorphism in extracellular loop 3 (ECL3), PAR4-P310L (rs2227376) leads to a hypo-reactive receptor. Objectives: The goal of this study was to determine how the hypo-reactive PAR4 variant in ECL3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). Methods: A point mutation was introduced into the PAR4 gene, F2rl3, via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbß3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. Results: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbß3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. Conclusions: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.
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Background: The platelet population is heterogeneous, with different subsets that differ on the basis of their function and reactivity. An intrinsic factor participating in this difference of reactivity could be the platelet age. The lack of relevant tools allowing a formal identification of young platelets prevents so far to draw solid conclusions regarding platelet reactivity. We recently reported that human leukocyte antigen-I (HLA-I) molecules are more expressed on human young platelets. Objectives: The aim of this study was to assess platelet reactivity according to their age based on HLA-I expression level. Methods: Platelet activation was assessed by flow cytometry (FC) for different platelet subsets based on their HLA-I expression. These populations were further cell sorted and their intrinsic properties were determined by FC and electron microscopy (EM). Statistical analyses were performed with GraphPad Prism 5.02 software using two-way ANOVA followed by a Tukey post hoc test. Results: HLA-I expression level allowed the identification of 3 platelet subpopulations regarding to their age (HLA low, dim, and high). HLA-I was reliable to guide platelet cell sorting and highlighted the features of young platelets in the HLA-Ihigh population. In response to different soluble agonists, HLA-Ihigh platelets were the most reactive subset as shown by the level of P-selectin secretion and fibrinogen binding assessed by flow cytometry. Moreover, the highest capacity of HLA-Ihigh platelets to simultaneously express annexin-V and von Willebrand factor or activated αIIbß3 after coactivation with TRAP and CRP indicated that the procoagulant feature of platelets was age-related. Conclusion: The young HLA-Ihigh population is the most reactive and prone to become procoagulant. These results open up new perspectives to investigate deeply the role of young and old platelets.
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Purpose: To select individuals and families with a low genetic burden for age-related macular degeneration (AMD), to inform the clinical diagnosis of macular disorders, and to find novel genetic variants associated with maculopathies. Design: Genetic association study based on targeted and whole-exome sequencing. Participants: A total of 758 subjects (481 individuals with maculopathy and 277 controls), including 316 individuals in 72 families. Methods: We focused on 150 genes involved in the complement, coagulation, and inflammatory pathways. Single-variant tests were performed on 7755 variants shared among ≥ 5 subjects using logistic regression. Gene-based tests were used to evaluate aggregate effects from rare and low-frequency variants (at minor allele frequency [MAF] ≤ 5% or ≤ 1%) in a gene using burden tests. For families whose affected members had a low burden of genetic risk based on known common and rare variants related to AMD, we searched for rare variants (MAF < 0.001) whose risk alleles occurred in ≥ 80% of affected individuals but not in controls. Immunohistochemistry was performed to determine the protein expression of a novel gene (coagulation factor II thrombin receptor-like 2 [F2RL2]) in retinal tissues. Main Outcome Measures: Genotypes and phenotypes of macular degeneration. Results: We confirmed the association of a synonymous variant in complement factor H (Ala473, rs2274700, proxy to intronic rs1410996, r 2 = 1) with maculopathy (odds ratio, 0.64; P = 4.5 × 10-4). Higher AMD polygenic risk scores (PRSs) were associated with intermediate and advanced AMD. Among families with low PRSs and no known rare variants for maculopathy, we identified 2 novel, highly penetrant missense rare variants in ADAM15, A disintegrin and metalloprotease, metallopeptidase domain 15 (p.Arg288Cys) and F2RL2 (p.Leu289Arg). Immunohistochemistry analyses revealed F2RL2 protein expression in cone photoreceptor outer segments and Müller glia cells of human and pig retinas. Coagulation factor II thrombin receptor-like 2 expression appeared increased in fibrotic areas in advanced AMD samples with neovascularization, suggesting that F2RL2 may play a role in the progression to advanced macular disease. Conclusions: New missense rare variants in the genes ADAM15 and F2RL2 were associated with maculopathies. Results suggest that novel genes related to the coagulation and immune pathways may be involved in the pathogenesis of macular diseases.
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The glycoprotein (GP) Ib-IX complex is a platelet receptor that mediates the initial interaction with subendothelial von Willebrand factor (VWF) causing platelet arrest at sites of vascular injury even under conditions of high shear. GPIb-IX dysfunction or deficiency is the reason for the rare but severe Bernard-Soulier syndrome (BSS), a congenital bleeding disorder. Although knowledge on GPIb-IX structure, its basic functions, ligands, and intracellular signaling cascades have been well established, several advances in GPIb-IX biology have been made in the recent years. Thus, two mechanosensitive domains and a trigger sequence in GPIb were characterized and its role as a thrombin receptor was deciphered. Furthermore, it became clear that GPIb-IX is involved in the regulation of platelet production, clearance and thrombopoietin secretion. GPIb is deemed to contribute to liver cancer development and metastasis. This review recapitulates these novel findings highlighting GPIb-IX in its multiple functions as a key for immune regulation, host defense, and liver cancer development.
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Genetic predisposition through F11R-single-nucleotide variation (SNV) influences circulatory soluble junctional adhesion molecule-A (sJAM-A) levels in coronary artery disease (CAD) patients. Homozygous carriers of the minor alleles (F11R-SNVs rs2774276, rs790056) show enhanced levels of thrombo-inflammatory sJAM-A. Both F11R-SNVs and sJAM-A are associated with worse prognosis for recurrent myocardial infarction in CAD patients. Platelet surface-associated JAM-A correlate with platelet activation markers in CAD patients. Activated platelets shed transmembrane-JAM-A, generating proinflammatory sJAM-A and JAM-A-bearing microparticles. Platelet transmembrane-JAM-A and sJAM-A as homophilic interaction partners exaggerate thrombotic and thrombo-inflammatory platelet monocyte interactions. Therapeutic strategies interfering with this homophilic interface may regulate thrombotic and thrombo-inflammatory platelet response in cardiovascular pathologies where circulatory sJAM-A levels are elevated.
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Background: The current post-procedure antithrombotic recommendation for left atrial appendage closure (LAAC) remains empiric. This study was designed to compare variations in platelet activation biomarkers and device-related thrombosis (DRT) under different antithrombotic regimens following LAAC. Methods: This study enrolled 105 consecutive patients with atrial fibrillation who underwent LAAC successfully and received post-procedure anticoagulation with either dabigatran (N = 33) or rivaroxaban (N = 72). After 3 months of anticoagulation treatment, thromboelastogram was used to evaluate thrombin receptor-activating peptide (TRAP)-induced platelet aggregation (PA). Measurements of platelet activation biomarkers, including thrombin-antithrombin complex (TAT), P-selectin, von Willebrand disease (vWF), and CD40L, were performed immediately before the LAAC procedure and after 3 months of post-procedure anticoagulation. Repeated transesophageal echocardiography was performed to evaluate DRT during follow-ups. Results: Three (4.2%) patients in the rivaroxaban and 4 (12.1%) patients in the dabigatran group experienced DRT events (odds ratio (OR) = 0.315, 95% confidence interval (95%CI): 0.066-1.489, p = 0.129) during follow-ups. The TRAP-induced PA was statistically significantly higher in the dabigatran group (62.9% vs 59.7%, p = 0.028*). Statistically significant increases in plasma concentration of TAT, P-selectin, and vWF were observed after 3 months of exposure to dabigatran when compared with rivaroxaban. An increased expression of platelet activation biomarkers was observed in DRT subjects compared with non-DRT subjects in terms of P-selectin and vWF (65.28 ± 13.93 ng/L vs 32.14 ± 12.11 ng/L, p = 0.037; 501.92 ± 106.48 U/L vs 280.98 ± 54.10 U/L, p = 0.045; respectively). Multivariate regression analysis indicated that the use of dabigatran might be an independent predictor of DRT (p = 0.022; OR = 4.366, 95%CI: 0.434-10.839). Furthermore, the CHA2DS2-VASc score (OR = 2.076, p = 0.016) and CD40L levels (OR = 1.015, p = 0.021) were independent predictors of increased D-dimer levels. Conclusions: Post-LAAC anticoagulation with dabigatran may increase the risk of DRT by enhancing platelet reactivity. In light of this potential increased risk in DRT, the authors recommend against using dabigatran for post-procedural anticoagulation in patients who have undergone LAAC.
RESUMO
Protease-activated receptors (PARs) are a family of highly conserved G protein-coupled receptors (GPCRs) that respond to extracellular proteases via a unique proteolysis-dependent activation mechanism. Protease-activated receptor 1 (PAR1) was the first identified member of the receptor family and plays important roles in hemostasis, inflammation and malignancy. The biology underlying PAR1 signaling by its canonical agonist thrombin is well characterized; however, definition of the mechanistic basis of PAR1 signaling by other proteases, including matrix metalloproteases, activated protein C, plasmin, and activated factors VII and X, remains incompletely understood. In this review, we discuss emerging insights into the molecular bases for "biased" PAR1 signaling, including atypical PAR1 proteolysis, PAR1 heterodimer and coreceptor interactions, PAR1 translocation on the membrane surface, and interactions with different G-proteins and ß-arrestins upon receptor activation. Moreover, we consider how these new insights into PAR1 signaling have acted to spur development of novel PAR1-targeted therapeutics that act to inhibit, redirect, or fine-tune PAR1 signaling output to treat cardiovascular and inflammatory disease. Finally, we discuss some of the key unanswered questions relating to PAR1 biology, in particular how differences in PAR1 proteolysis, signaling intermediate coupling, and engagement with coreceptors and GPCRs combine to mediate the diversity of identified PAR1 signaling outputs.
Assuntos
Receptor PAR-1 , Transdução de Sinais , Proteínas de Ligação ao GTP , Humanos , Inflamação , Receptor PAR-1/metabolismo , Trombina/metabolismoRESUMO
AIMS: Pulmonary hypertension (PH) is characterized by progressive increases in pulmonary vascular resistance (PVR). Thrombotic lesions are common pathological findings. The pulmonary artery has a unique property regarding the vasoconstrictive response to thrombin, which is mediated by proteinase-activated receptor 1 (PAR1). We aim to elucidate the role of PAR1 in the development and progression of PH. METHODS AND RESULTS: A rat model of monocrotaline-induced PH and a mouse model of hypoxia (Hx)-induced PH were used to investigate the effects of atopaxar (a PAR1 antagonist) and PAR1 knockout on haemodynamic parameters, right ventricular hypertrophy (RVH), vascular remodelling and survival. In perfused lung preparations, the pressor response to PAR1 agonist was significantly augmented in monocrotaline-induced PH. Both the preventive and therapeutic administration of atopaxar significantly inhibited the increase in PVR and the development of RVH and prolonged survival. A real-time PCR revealed that the level of PAR1 mRNA in the pulmonary artery was significantly higher than that in any of the systemic arteries examined in control rats, and the level was significantly up-regulated in monocrotaline-induced PH. PAR1 gene knockout significantly attenuated the haemodynamic and histological findings in the mouse model of Hx-induced PH. CONCLUSION: The specific expression of PAR1 in the pulmonary artery and its up-regulation were suggested to play a critical role in the development and progression of experimental PH in murine models. PAR1 is a potential therapeutic target for the treatment of PH.
Assuntos
Anti-Hipertensivos/farmacologia , Pressão Arterial/efeitos dos fármacos , Hipertensão Pulmonar/prevenção & controle , Iminas/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Piridinas/farmacologia , Receptor PAR-1/metabolismo , Animais , Modelos Animais de Doenças , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/prevenção & controle , Masculino , Camundongos Knockout , Monocrotalina , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley , Receptor PAR-1/deficiência , Receptor PAR-1/genética , Trombina/metabolismo , Remodelação Vascular/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
It is challenging for traditional hemostatic sponges to meet the clinic demand for both uncontrolled and noncompressible hemorrhage. With the aim to develop a rapid shape recovery material with both active and passive hemostatic performance, a dual-functional hemostatic sponge (TRAP-Sp) with a macroporous structure and good mechanical properties for controlling massive and noncompressible hemorrhage was prepared by chemically immobilizing thrombin-receptor-agonist-peptide (TRAP) onto a starch/polyethylene glycol (PEG) sponge. The TRAP2-Sp1 showed the best hemostatic performance among all samples in both rat artery uncontrollable hemorrhage and liver defect noncompressible hemorrhage models. When analyzing the hemostatic mechanism of TRAP-Sp, the high water absorption capacity of the sponge contributed to absorbing plasma, concentrating blood cells, and enhancing blood coagulation. After absorbing water, the shape-fixed TRAP-Sp with sufficient mechanical strength and high resilience can rapidly expand and apply pressure to the wound. TRAP immobilized on the sponge could activate the adhered platelets in an active pathway. Additionally, evaluation of cytotoxicity, hemolysis, and histology further highlighted the adequate biocompatibility of TRAP-Sp. With excellent hemostatic performance and biosafety, this sponge could be a potential candidate as a topical hemostatic agent for uncontrolled and noncompressible hemorrhage. STATEMENT OF SIGNIFICANCE: There is a need for innovative hemostatic materials for both uncontrolled and noncompressible hemorrhage. This manuscript describes a rapid shape recovery hemostatic sponge with both active and passive hemostatic performances synthesized by foaming technique, cross-linking reaction, and chemical immobilization of thrombin-receptor-agonist-peptide (TRAP). On contact with blood, the shape-fixed sponge can not only rapidly recover its original shape and concentrate platelets and RBCs but also activate the adhered platelets efficiently. The dual-functional sponge has excellent hemostatic efficacy in rat femoral artery hemorrhage and can control noncompressible hemorrhage in penetrating liver wound. Thus, we believe that this sponge could be a potential candidate as a topical hemostatic agent for uncontrolled and noncompressible hemorrhage.