RESUMEN
During gram-negative septicemia, interactions between platelets and neutrophils initiate a detrimental feedback loop that sustains neutrophil extracellular trap (NET) induction, disseminated intravascular coagulation, and inflammation. Understanding intracellular pathways that control platelet-neutrophil interactions is essential for identifying new therapeutic targets. Here, we found that thrombin signaling induced activation of the transcription factor NFAT in platelets. Using genetic and pharmacologic approaches, as well as iNFATuation, a newly developed mouse model in which NFAT activation can be abrogated in a cell-specific manner, we demonstrated that NFAT inhibition in activated murine and human platelets enhanced their activation and aggregation, as well as their interactions with neutrophils and NET induction. During gram-negative septicemia, NFAT inhibition in platelets promoted disease severity by increasing disseminated coagulation and NETosis. NFAT inhibition also partially restored coagulation ex vivo in patients with hypoactive platelets. Our results define non-transcriptional roles for NFAT that could be harnessed to address pressing clinical needs.
Asunto(s)
Plaquetas/efectos de los fármacos , Factores de Transcripción NFATC/antagonistas & inhibidores , Agregación Plaquetaria/efectos de los fármacos , Sepsis/patología , Animales , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/metabolismo , Comunicación Celular/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Modelos Animales de Enfermedad , Trampas Extracelulares/metabolismo , Humanos , Inflamación , Ratones , Factores de Transcripción NFATC/metabolismo , Neutrófilos/metabolismo , Receptores de Trombina/metabolismo , Sepsis/metabolismoRESUMEN
The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.
Asunto(s)
Antígenos Dermatofagoides/metabolismo , Proteínas de Artrópodos/metabolismo , Señalización del Calcio , Movimiento Celular , Mastocitos/metabolismo , Mucosa Nasal/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Pyroglyphidae/enzimología , Receptores de Trombina/metabolismo , Serina Endopeptidasas/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Animales , Antígenos Dermatofagoides/efectos adversos , Antígenos Dermatofagoides/genética , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/efectos adversos , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Asma/inmunología , Asma/metabolismo , Células HEK293 , Humanos , Exposición por Inhalación , Inositol 1,4,5-Trifosfato/metabolismo , Activación del Canal Iónico , Células Jurkat , Mastocitos/inmunología , Ratones Endogámicos C57BL , Mucosa Nasal/inmunología , Pyroglyphidae/genética , Pyroglyphidae/inmunología , Receptor PAR-2 , Receptores Acoplados a Proteínas G/metabolismo , Serina Endopeptidasas/efectos adversos , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunologíaRESUMEN
PURPOSE OF REVIEW: Cardiovascular disease is a major cause of death worldwide. Platelets play a key role in this pathological process. The serine protease thrombin is a critical regulator of platelet reactivity through protease activated receptors-1 (PAR1) and PAR4. Since targeting PAR4 comes with a low chance for bleeding, strategies blocking PAR4 function have great antithrombotic potential. Here, we reviewed the literature on platelet PAR4 with a particular focus on its role in thromboinflammation. RECENT FINDINGS: Functional PAR4 variants are associated with reduced venous thrombosis risk (rs2227376) and increased risk for ischemic stroke (rs773902). Recent advances have allowed for the creation of humanized mouse lines in which human PAR4 is express instead of murine PAR4. This has led to a better understanding of the discrepancies between human and murine PAR4. It also made it possible to introduce single nucleotide polymorphisms (SNPs) in mice allowing to directly test the in vivo functional effects of a specific SNP and to develop in vivo models to study mechanistic and pharmacologic alterations induced by a SNP. SUMMARY: PAR4 plays an important role in cardiovascular diseases including stroke, myocardial infarction and atherosclerosis. Targeting PAR4 hold great potential as a safe antithrombotic strategy.
Asunto(s)
Receptores de Trombina , Humanos , Receptores de Trombina/metabolismo , Receptores de Trombina/genética , Animales , Ratones , Polimorfismo de Nucleótido Simple , Trombosis/metabolismo , Plaquetas/metabolismo , Plaquetas/patologíaRESUMEN
Protease-activated receptor 4 (PAR4) is a G protein-coupled receptor activated by thrombin. In the platelet, response to thrombin PAR4 contributes to the predominant procoagulant microparticle formation, increased fibrin deposition, and initiation of platelet-stimulated inflammation. In addition, PAR4 is expressed in other cell types, including endothelial cells. Under inflammatory conditions, PAR4 is overexpressed via epigenetic demethylation of the PAR4 gene, F2RL3. PAR4 knockout (KO) studies have determined a role for PAR4 in ischemia-reperfusion injury in the brain, and PAR4 KO mice display normal cardiac function but present less myocyte death and cardiac dysfunction in response to acute myocardial infarction. Although PAR4 has been reported to be expressed within the kidney, the contribution of PAR4 to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 KO mice are protected against kidney injury in two mouse models. First, PAR4 KO mice are protected against induction of markers of both fibrosis and inflammation in two different models of kidney injury: 1) 7 days following unilateral ureter obstruction (UUO) and 2) an AKI-CKD model of ischemia-reperfusion followed by 8 days of contralateral nephrectomy. We further show that PAR4 expression in the kidney is low in the control mouse kidney but induced over time following UUO. PAR4 KO mice are protected against blood urea nitrogen (BUN) and glomerular filtration rate (GFR) kidney function pathologies in the AKI-CKD model. Following the AKI-CKD model, PAR4 is expressed in the collecting duct colocalizing with Dolichos biflorus agglutinin (DBA), but not in the proximal tubule with Lotus tetragonolobus lectin (LTL). Collectively, the results reported in this study implicate PAR4 as contributing to the pathology in mouse models of acute and chronic kidney injury.NEW & NOTEWORTHY The contribution of the thrombin receptor protease-activated receptor 4 (PAR4) to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 expression is upregulated after kidney injury and PAR4 knockout (KO) mice are protected against fibrosis following kidney injury in two mouse models. First, PAR4 KO mice are protected against unilateral ureter obstruction. Second, PAR4 KO mice are protected against an AKI-CKD model of ischemia-reperfusion followed by contralateral nephrectomy.
Asunto(s)
Lesión Renal Aguda , Insuficiencia Renal Crónica , Animales , Ratones , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Células Endoteliales/metabolismo , Fibrosis , Inflamación/patología , Isquemia/patología , Riñón/metabolismo , Ratones Noqueados , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/patología , Daño por Reperfusión/patología , Trombina/metabolismo , Trombina/farmacologíaRESUMEN
BACKGROUND: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. METHODS: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. RESULTS: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. CONCLUSIONS: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.
Asunto(s)
Plaquetas/metabolismo , Epigénesis Genética , Infarto del Miocardio/genética , Receptores de Trombina/genética , Anciano , Metilación de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/epidemiología , Receptores de Trombina/metabolismo , Fumar/epidemiologíaRESUMEN
The protein Par-4 acts in the cytoplasm to trigger cell death signaling via caspase activation and the mitochondrial release of cytochrome c. Burikhanov et al. (2009) now provide surprising evidence that Par-4 can also promote apoptosis from outside the cell, after its secretion in response to endoplasmic reticulum stress.
Asunto(s)
Apoptosis , Receptores de Trombina/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Humanos , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
Prostate apoptosis response-4 (Par-4) is a proapoptotic protein with intracellular functions in the cytoplasm and nucleus. Unexpectedly, we noted Par-4 protein is spontaneously secreted by normal and cancer cells in culture, and by Par-4 transgenic mice that are resistant to spontaneous tumors. Short exposure to endoplasmic reticulum (ER) stress-inducing agents further increased cellular secretion of Par-4 by a brefeldin A-sensitive pathway. Secretion occurred independently of caspase activation and apoptosis. Interestingly, extracellular Par-4 induced apoptosis by binding to the stress response protein, glucose-regulated protein-78 (GRP78), expressed at the surface of cancer cells. The interaction of extracellular Par-4 and cell surface GRP78 led to apoptosis via ER stress and activation of the FADD/caspase-8/caspase-3 pathway. Moreover, apoptosis inducible by TRAIL, which also exerts cancer cell-specific effects, is dependent on extracellular Par-4 signaling via cell surface GRP78. Thus, Par-4 activates an extrinsic pathway involving cell surface GRP78 receptor for induction of apoptosis.
Asunto(s)
Apoptosis , Receptores de Trombina/metabolismo , Animales , Brefeldino A/farmacología , Línea Celular , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Transgénicos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Receptores de Trombina/químicaRESUMEN
BACKGROUND: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. METHODS: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. RESULTS: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1ß/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). CONCLUSIONS: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.
Asunto(s)
Enfermedades Cardiovasculares , Receptor PAR-1 , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Anciano , Plaquetas/metabolismo , Calcio/metabolismo , Enfermedades Cardiovasculares/metabolismo , Humanos , Inflamación/metabolismo , Interleucina-6/metabolismo , Selectina-P/metabolismo , Fenotipo , Activación Plaquetaria , Agregación Plaquetaria , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Protease-activated receptor 1 (PAR1) is crucial in individuals with acute myocardial infarction (AMI). The continuous and prompt PAR1 activation mainly dependent on PAR1 trafficking is essential for the role of PAR1 during AMI in which cardiomyocytes are in hypoxia. However, the PAR1 trafficking in cardiomyocytes specially during the hypoxia is still unclear. METHODS AND RESULT: A rat AMI model was created. PAR1 activation with thrombin-receptor activated peptide (TRAP) had a transient effect on cardiac function in normal rats but persistent improvement in rats with AMI. Cardiomyocytes from neonatal rats were cultured in a normal CO2 incubator and a hypoxic modular incubator chamber. The cells were then subjected to western blot for the total protein expression and staining with fluorescent reagent and antibody for PAR1 localization. No change in total PAR1 expression following TRAP stimulation was observed; however, it led to increased PAR1 expression in the early endosomes in normoxic cells and decreased expression in the early endosomes in hypoxic cells. Under hypoxic conditions, TRAP restored the PAR1 expression on both cell and endosomal surfaces within an hour by decreasing Rab11A (8.5-fold; 179.93 ± 9.82% of the normoxic control group, n = 5) and increasing Rab11B (15.5-fold) expression after 4 h of hypoxia. Similarly, Rab11A knockdown upregulated PAR1 expression under normoxia, and Rab11B knockdown downregulated PAR1 expression under both normoxic and hypoxic conditions. Cardiomyocytes knocked out of both Rab11A, and Rad11B lost the TRAP-induced PAR1 expression but still exhibited the early endosomal TRAP-induced PAR1 expression under hypoxia. CONCLUSIONS: TRAP-mediated activation of PAR1 in cardiomyocytes did not alter the total PAR1 expression under normoxic conditions. Instead, it triggers a redistribution of PAR1 levels under normoxic and hypoxic conditions. TRAP reverses the hypoxia-inhibited PAR1 expression in cardiomyocytes by downregulating Rab11A expression and upregulating Rab11B expression.
Asunto(s)
Infarto del Miocardio , Receptor PAR-1 , Animales , Ratas , Hipoxia/metabolismo , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismo , Trombina/farmacologíaRESUMEN
Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.
Asunto(s)
Receptores Proteinasa-Activados , Receptores de Trombina , Humanos , Ratones , Animales , Receptores Proteinasa-Activados/metabolismo , Receptores de Trombina/metabolismo , Especificidad de la Especie , Plaquetas/metabolismo , Trombina/metabolismoRESUMEN
Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.
Asunto(s)
Diabetes Mellitus Experimental , Receptor PAR-1 , Trombina , Animales , Ratones , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Modelos Animales de Enfermedad , Inflamación/metabolismo , Ratones Endogámicos C57BL , Protrombina/metabolismo , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , ARN Mensajero/metabolismo , Estreptozocina , Trombina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Brain death (BD) induces a systemic inflammatory response that influences donor liver quality. Protease-activated receptor 4 (PAR4) is a thrombin receptor that mediates platelet activation and is involved in inflammatory and apoptotic processes. Therefore, we investigated the role of PAR4 blockade in liver injury induced by BD and its associated mechanisms. In this study, we constructed a BD rat model and treated rats with TcY-NH2, a selective PAR4 antagonist, to block PAR4 signaling at the onset of BD induction. Our results revealed that PAR4 protein expression increased in the livers of rats with BD. PAR4 blockade alleviated liver injury induced by BD, as indicated by lower serum ALT/AST levels and an improvement in histomorphology. Blood platelet activation and hepatic platelet accumulation in BD rats were reduced by PAR4 blockade. Additionally, PAR4 blockade attenuated the inflammatory response and apoptosis in the livers of BD rats. Moreover, the activation of NF-κB and MAPK pathways induced by BD was inhibited by PAR4 blockade. Thus, our results suggest that PAR4 contributes to liver injury induced by BD by regulating inflammation and apoptosis through the NF-κB and MAPK pathways. Thus, PAR4 blockade may provide a feasible approach to improve the quality of organs from BD donors.
Asunto(s)
Muerte Encefálica/metabolismo , Hígado/efectos de los fármacos , Oligopéptidos/farmacología , Receptores de Trombina/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Muerte Encefálica/fisiopatología , Citocinas/genética , Citocinas/metabolismo , Expresión Génica/efectos de los fármacos , Inflamación/genética , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Hígado/metabolismo , Hígado/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , FN-kappa B/metabolismo , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Ratas Sprague-Dawley , Receptores de Trombina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Protease-activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling, and signaling studies, we determined the molecular mechanism for tethered ligand-mediated PAR4 activation. H/D exchange identified that the LBS is composed of transmembrane 3 (TM3) domain and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and 2 experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the International Network Against Venous Thrombosis (INVENT) consortium multi-ancestry genome-wide association study (GWAS) meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% reduction in relative risk for VTE (odds ratio, 0.85; 95% confidence interval, 0.77-0.94) compared with the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE.
Asunto(s)
Membrana Celular/química , Prolina/metabolismo , Receptores de Trombina/metabolismo , Treonina/metabolismo , Trombosis de la Vena/patología , Membrana Celular/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Prolina/química , Prolina/genética , Conformación Proteica , Dominios Proteicos , Receptores de Trombina/química , Receptores de Trombina/genética , Treonina/química , Treonina/genética , Trombosis de la Vena/genética , Trombosis de la Vena/metabolismoRESUMEN
Studies of platelet function in surgical patients often involve both arterial and venous sampling. Possible effects of different sampling sites could be important, but have not been thoroughly investigated. We aimed to compare platelet function in arterial and venous blood samples using a novel flow cytometry protocol and impedance aggregometry. Arterial and venous blood was collected before anesthesia in 10 patients undergoing cardiac surgery of which nine was treated with acetylsalicylic acid until the day before surgery. Flow cytometry included simultaneous analysis of phosphatidylserine exposure, active conformation of the fibrinogen receptor (PAC-1 binding), α-granule and lysosomal release (P-selectin and LAMP-1 exposure) and mitochondrial membrane integrity. Platelets were activated with ADP or peptides activating thrombin receptors (PAR1-AP/PAR4-AP) or collagen receptor GPVI (CRP-XL). Leukocyte-platelet conjugates and P-selectin exposure were evaluated immediately in fixated samples. For impedance aggregometry (Multiplate®), ADP, arachidonic acid, collagen and PAR1-AP (TRAP) were used as activators. Using impedance aggregometry and in 27 out of 37 parameters studied with flow cytometry there was no significant difference between venous and arterial blood sampling. Arterial blood showed more PAC-1 positive platelets when activated with PAR1-AP or PAR4-AP and venous blood showed more monocyte-platelet and neutrophil-platelet conjugates and higher phosphatidylserine exposure with CRP-XL alone and combined with PAR1-AP or PAR4-AP. We found no differences using impedance aggregometry. In conclusion, testing of platelet function by flow cytometry and impedance aggregometry gave comparable results for most of the studied parameters in venous and arterial samples. Flow cytometry identified differences in PAC-1 binding when activated with PAR1-AP, exposure of phosphatidyl serine and monocyte/neutrophil-platelet conjugates, which might reflect differences in blood sampling technique or in flow conditions in this patient cohort with coronary artery disease. These differences might be considered when comparing data from different sample sites, but caution should be exercised if a different protocol is used or another patient group is studied.
Asunto(s)
Selectina-P , Activación Plaquetaria , Adenosina Difosfato/farmacología , Plaquetas/metabolismo , Citometría de Flujo , Humanos , Selectina-P/metabolismo , Fosfatidilserinas/metabolismo , Agregación Plaquetaria , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismoRESUMEN
Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of megakaryocytes and platelets. Upon platelet activation TLT-1 is rapidly relocated to the surface of platelets. In plasma, a soluble form of TLT-1 (sTLT-1) is present. Plasma levels of sTLT-1 are significantly elevated in thrombotic diseases. In the present study, we investigated to whether TLT-1 reflects platelet activation in pregnant women with preeclampsia. We studied 30 preeclamptic patients who were matched with 30 normotensive pregnant women and 30 non-pregnant controls. Basal TLT-1, P-selectin, and CD63 expressions on platelets were analyzed with the use of flow-cytometry (FCM). Platelet reactivity was induced by thrombin receptor activation peptide and determined by FCM. Plasma concentrations of sTLT-1 and soluble P-selectin (sP-selectin) were measured by an enzyme-linked immunosorbent assay. Results show that basal platelet expression of TLT-1, P-selectin and CD63 were increased in women with preeclampsia (PE) compared with normotensive pregnant women (NP). Platelets from PE women and NP women were more responsive compared to from nonpregnant women controls (NC), and which was demonstrated by increased expression of TLT-1, P-selectin, and CD63 upon stimulation in vitro. Plasma concentration of sTLT-1 was greater in PE women compared to NP women and NC women. Plasma sP-selectin level was higher in pregnant women than in nonpregnant women, but there were no significant differences between PE and NP women. In summary, our results revealed that platelet activation is prominent in preeclampsia, TLT-1 reflects platelet activation and may be a useful indicator for preeclampsia.
Asunto(s)
Selectina-P , Preeclampsia , Plaquetas/metabolismo , Femenino , Humanos , Células Mieloides/metabolismo , Selectina-P/metabolismo , Péptidos , Activación Plaquetaria , Embarazo , Receptores Inmunológicos , Receptores de Trombina/metabolismoRESUMEN
Thrombin is a potent platelet activator, acting through proteinase-activated receptors -1 and -4 (PAR1 and PAR4). Of these, PAR-1 is activated more rapidly and by lower thrombin concentrations. Consequently, PAR-1 has been extensively investigated as a target for anti-platelet drugs to prevent myocardial infarction. Q94 has been reported to act as an allosteric modulator of PAR1, potently and selectively inhibiting PAR1-Gαq coupling in multiple cell lines, but its effects on human platelet activation have not been previously studied. Platelet Ca2+ signaling, integrin αIIbß3 activation and α-granule secretion were monitored following stimulation by a PAR1-activating peptide (PAR1-AP). Although Q94 inhibited these responses, its potency was low compared to other PAR1 antagonists. In addition, αIIbß3 activation and α-granule secretion in response to other platelet activators were also inhibited with similar potency. Finally, in endothelial cells, Q94 did not inhibit PAR1-dependent Ca2+ signaling. Our data suggest that Q94 may have PAR1-independent off-target effects in platelets, precluding its use as a selective PAR1 allosteric modulator.
Asunto(s)
Receptor PAR-1 , Trombina , Plaquetas/metabolismo , Células Endoteliales/metabolismo , Humanos , Activación Plaquetaria , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismo , Trombina/farmacologíaRESUMEN
Cirrhotic patients have an increased risk of bleeding and thromboembolic events, with platelets being involved as key players in both situations. The impact of peripheral versus central blood sampling on platelet activation remains unclear. In 33 cirrhotic patients, we thus analyzed platelet function in peripheral (P) and central (C) blood samples. Platelet surface expression of P-selectin, activated glycoprotein (GP) IIb/IIIa, and leukocyte-platelet aggregate formation were measured by flow cytometry in response to different agonists: thrombin receptor-activating peptide-6, adenosine diphosphate, collagen-related peptide (CrP), epinephrine, AYPGKF, Pam3CSK4, and lipopolysaccharide. Unstimulated platelet surface expression of P-selectin (p = .850) and activated GPIIb/IIIa (p = .625) were similar in peripheral and central blood samples. Stimulation with various agonists yielded similar results of platelet surface expression of P-selectin and activated GPIIb/IIIa in peripheral and central samples, except for CrP-inducible expression of activated GPIIb/IIIa (median fluorescence intensity, MFI in P: 7.61 [0.00-24.66] vs. C: 4.12 [0.00-19.04], p < .001). The formation of leukocyte-platelet aggregate was similar in central and peripheral blood samples, both unstimulated and after stimulation with all above-mentioned agonists. In conclusion, peripheral vs. central venous blood sampling does not influence the assessment of platelet activation by flow cytometry in cirrhosis.Abbreviations: ACLD: advanced chronic liver disease; ADP: adenosine diphosphate; ALD: alcoholic liver disease; AYPGKF: PAR-4 agonist AYPGKF; CrP: collagen related protein; EPI: epinephrine; FACS: fluorescence-activated cell sorting; GP: glycoprotein; HVPG: hepatic venous pressure gradient; IQR: interquartile range; LPS: lipopolysaccharide; LSM: liver stiffness measurement; MFI: median fluorescence intensity; NAFLD: nonalcoholic fatty liver disease; PAM: lipopeptide Pam3CSK4; PAR: protease-activated receptor; PBS: phosphate-buffered saline; PH: portal hypertension; TIPS: transjugular intrahepatic portosystemic stent shunt; TLR: toll-like receptor; TRAP-6: thrombin receptor-activator peptide-6; vWF: von Willebrand factor.
Asunto(s)
Selectina-P , Inhibidores de Agregación Plaquetaria , Adenosina Difosfato/farmacología , Plaquetas/metabolismo , Epinefrina/farmacología , Citometría de Flujo , Humanos , Lipopolisacáridos/metabolismo , Cirrosis Hepática/metabolismo , Selectina-P/metabolismo , Activación Plaquetaria , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de Trombina/metabolismoRESUMEN
We aimed to investigate the effects of integrin αIIbß3 inhibitor tirofiban on hallmarks of platelet activation, degranulation, and aggregation during its use to analyze activated but non-complexed platelets via flow cytometry. To do so, we used washed platelets from healthy human donors. We combined aggregometry, an assay of platelet functionality, with flow cytometry and ELISA to detect and correlate, respectively, platelet aggregation, activation, and granule release. While tirofiban effectively inhibited agonist-induced platelet aggregation (thrombin receptor-activating peptide 6 (TRAP), convulxin (CVX), U46619 and IV.3), the surface expression of P-selectin and CD63 and granule release of RANTES were significantly increased, indicating that tirofiban enhances degranulation, uncoupled from aggregation. The results show that tirofiban alters the activation phenotype of platelets, something that should be considered when using tirofiban to enable flow cytometric analysis of activated but unaggregated platelet suspensions.
Asunto(s)
Selectina-P , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Plaquetas/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacología , Humanos , Selectina-P/metabolismo , Activación Plaquetaria , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de Trombina/metabolismo , Tirofibán/farmacología , Tirosina/metabolismo , Tirosina/farmacologíaRESUMEN
Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Trombina/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Plaquetas/metabolismo , Calcio/metabolismo , Línea Celular , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Megacariocitos/citología , Megacariocitos/metabolismo , Modelos Biológicos , Trombina/metabolismoRESUMEN
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.