RESUMO
BACKGROUND: Cigarette smoking plays a major role in cardiovascular diseases. The acute effects of cigarette smoking produce central nervous system-mediated activation of the sympathetic nervous system. The overactive sympathetic nervous system stimulates the secretion of serotonin (5-HT) and catecholamine into blood at supraphysiological levels. The correlation between these pathological conditions induced by smoking and the increased risk of thrombosis has not been thoroughly investigated. The goal of our study was to explore cigarette smoking-associated changes in platelet biology mediated by elevated 5-HT and catecholamine levels in blood plasma. METHODS AND RESULTS: Using blood samples collected from healthy nonsmokers and smokers (15 minutes after smoking), we determined that cigarette smoking increased the plasma 5-HT/catecholamine concentration by several fold and the percent aggregation of platelets 2-fold. Liquid chromatography-tandem mass spectrometry analysis of proteins eluted from platelet plasma membranes of smokers and nonsmokers demonstrated that GTPase-activating proteins and proteins participating in the actin cytoskeletal network were differentially and significantly elevated in smokers' platelet membranes compared with those of nonsmokers. Interestingly, Matrix-assisted laser desorption/ionization-mass spectrometry analyses of the glycans eluted from platelet plasma membranes of the smokers demonstrated that the level and structures of glycans are different from the nonsmokers' platelet surface glycans. Pharmacological blockade of 5-HT or catecholamine receptors counteracted the 5-HT/catecholamine-mediated aggregation and altered the level and composition of glycan on platelet surfaces. CONCLUSIONS: Based on our findings, we propose that smoking-associated 5-HT/catecholamine signaling accelerates the trafficking dynamics of platelets, and this remodels the surface proteins and glycans and predisposes platelets to hyperactive levels. Smokers' platelets also had correspondingly higher resting concentrations of intracellular calcium and transglutaminase activity. These findings suggest a link among smoking, platelet 5-HT, catecholamine signaling, and their downstream effectors-including phospholipase C and inositol-1,4,5-triphosphate pathways-resulting in an increased tonic level of platelet activation in smokers.
Assuntos
Plaquetas/metabolismo , Membrana Celular/metabolismo , Epinefrina/sangue , Ativação Plaquetária , Serotonina/sangue , Transdução de Sinais , Fumar/sangue , Antagonistas Adrenérgicos beta/farmacologia , Adulto , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Estudos de Casos e Controles , Membrana Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Humanos , Masculino , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Polissacarídeos/sangue , Transporte Proteico , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fumar/efeitos adversos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Fatores de Tempo , Transglutaminases/sangue , Regulação para CimaRESUMO
In this issue of Blood, Darbousset et al define opposing roles for adenosine triphosphate (ATP) and adenosine in regulating polymorphonuclear neutrophil (PMN) activation, fibrin formation, and thrombus growth following vascular injury.
Assuntos
Plaquetas/metabolismo , Neutrófilos/metabolismo , Receptores Purinérgicos P2X1/genética , Trombose/genética , Animais , HumanosRESUMO
In vivo visualization of thrombopoiesis suggests an important role for shear flow in platelet biogenesis. In vitro, shear stress was shown to accelerate proplatelet formation from mature megakaryocytes (Mks). Yet, the role of biomechanical forces on Mk biology and platelet biogenesis remains largely unexplored. In this study, we investigated the impact of shear stress on Mk maturation and formation of platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs), and furthermore, we explored a physiological role for MkMPs. We found that shear accelerated DNA synthesis of immature Mks in an exposure time- and shear stress level-dependent manner. Both phosphatidylserine exposure and caspase-3 activation were enhanced by shear stress. Exposure to physiological shear dramatically increased generation of PLPs/PPTs and MkMPs by up to 10.8 and 47-fold, respectively. Caspase-3 inhibition reduced shear-induced PLP/PPT and MkMP formation. PLPs generated under shear flow displayed improved functionality as assessed by CD62P exposure and fibrinogen binding. Significantly, coculture of MkMPs with hematopoietic stem and progenitor cells promoted hematopoietic stem and progenitor cell differentiation to mature Mks synthesizing α- and dense-granules, and forming PPTs without exogenous thrombopoietin, thus identifying a novel and unexplored potential physiological role for MkMPs.
Assuntos
Diferenciação Celular , Micropartículas Derivadas de Células/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Resistência ao Cisalhamento , Estresse Fisiológico , Trombopoese/fisiologia , Antígenos CD34/metabolismo , Caspase 3/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Replicação do DNA , Ativação Enzimática , Humanos , PoliploidiaRESUMO
It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi.
Assuntos
Arrestinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores de Trombina/metabolismo , Cálcio/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Ativação Plaquetária/fisiologia , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Trombina/metabolismoRESUMO
The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on in vivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53(-/-) megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets in vitro. Culture of p53(-/-) marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53(+/+) megakaryocytes. This was due to 30% greater DNA synthesis in p53(-/-) megakaryocytes and 31% greater apoptosis in p53(+/+) megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53(+/+) and p53(-/-) mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53(-/-) mice. Although their platelet counts were normal, p53(-/-) mice exhibited significantly longer bleeding times and p53(-/-) platelets were less sensitive than p53(+/+) platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our in vivo and ex vivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.
Assuntos
Plaquetas/fisiologia , Trombopoese/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Cruzamento , Tamanho Celular , Hemostasia , Masculino , Camundongos , Selectina-P/metabolismo , Receptores Ativados por Proteinase/agonistasRESUMO
Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/ß subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.
Assuntos
Arrestinas/fisiologia , Plaquetas/metabolismo , Receptores Ativados por Proteinase/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico , Trombose/etiologia , beta-ArrestinasRESUMO
Two major pathways contribute to Ras-proximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) mediates the rapid but reversible activation of integrin αIIbß3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI(-/-) blood, even in the presence of exogenous adenosine diphosphate and thromboxane A(2). In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI(-/-) mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro- and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from athero-thrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Trombose/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Clopidogrel , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/sangue , Fatores de Troca do Nucleotídeo Guanina/genética , Hemostasia , Cinética , Masculino , Mesentério/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/irrigação sanguínea , Inibidores da Agregação Plaquetária/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Trombose/tratamento farmacológico , Trombose/genética , Ticlopidina/análogos & derivados , Ticlopidina/uso terapêuticoRESUMO
Platelet G protein-coupled receptors (GPCRs) initiate and reinforce platelet activation and thrombus formation. The clinical utility of antagonists of the P2Y(12) receptor for ADP suggests that other GPCRs and their intracellular signaling pathways may represent viable targets for novel antiplatelet agents. For example, thrombin stimulation of platelets is mediated by 2 protease-activated receptors (PARs), PAR-1 and PAR-4. Signaling downstream of PAR-1 or PAR-4 activates phospholipase C and protein kinase C and causes autoamplification by production of thromboxane A(2), release of ADP, and generation of more thrombin. In addition to ADP receptors, thrombin and thromboxane A(2) receptors and their downstream effectors-including phosphoinositol-3 kinase, Rap1b, talin, and kindlin-are promising targets for new antiplatelet agents. The mechanistic rationale and available clinical data for drugs targeting disruption of these signaling pathways are discussed. The identification and development of new agents directed against specific platelet signaling pathways may offer an advantage in preventing thrombotic events while minimizing bleeding risk.
Assuntos
Plaquetas/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Animais , Plaquetas/enzimologia , Plaquetas/metabolismo , Desenho de Fármacos , Hemorragia/induzido quimicamente , Humanos , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/química , Antagonistas do Receptor Purinérgico P2 , Receptor PAR-1/antagonistas & inibidores , Receptores Acoplados a Proteínas G/sangue , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Receptores de Trombina/antagonistas & inibidores , Receptores de Tromboxanos/antagonistas & inibidores , Trombina/metabolismo , Tromboxanos/metabolismoRESUMO
Glycogen synthase kinase (GSK)3beta is a ser-thr kinase that is phosphorylated by the kinase Akt. Although Akt has been shown to regulate platelet function and arterial thrombosis, its effectors in platelets remain unknown. We show here that agonist-dependent phosphorylation of GSK3beta in platelets is Akt dependent. To determine whether GSK3beta regulates platelet function, platelets from mice lacking a single allele of GSK3beta were compared with those of wild-type (WT) controls. GSK3beta+/- platelets demonstrated enhanced agonist-dependent aggregation, dense granule secretion, and fibrinogen binding, compared with WT platelets. Treatment of human platelets with GSK3 inhibitors renders them more sensitive to agonist-induced aggregation, suggesting that GSK3 suppresses platelet function in vitro. Finally, the effect of GSK3beta on platelet function in vivo was evaluated using 2 thrombosis models in mice. In the first, 80% of GSK3beta+/- mice (n=10) formed stable occlusive thrombi after ferric chloride carotid artery injury, whereas the majority of wild-type mice (67%) formed no thrombi (n=15). In a disseminated thrombosis model, deletion of a single allele of GSK3beta in mice conferred enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3beta acts as a negative regulator of platelet function in vitro and in vivo.
Assuntos
Plaquetas/enzimologia , Quinase 3 da Glicogênio Sintase/metabolismo , Agregação Plaquetária , Trombose/etnologia , Animais , Plaquetas/patologia , Lesões das Artérias Carótidas , Cloretos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Compostos Férricos/toxicidade , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Knockout , Noxas/toxicidade , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trombose/induzido quimicamente , Trombose/genética , Trombose/patologiaRESUMO
Serglycin (SG), the hematopoietic cell secretory granule proteoglycan, is crucial for storage of specific secretory proteins in mast cells, neutrophils, and cytotoxic T lymphocytes. We addressed the role of SG in platelets using SG-/- mice. Wild-type (WT) but not SG-/- platelets contained chondroitin sulfate proteoglycans. Electron microscopy revealed normal alpha-granule structure in SG-/- platelets. However, SG-/- platelets and megakaryocytes contained unusual scroll-like membranous inclusions, and SG-/- megakaryocytes showed extensive emperipolesis of neutrophils. SG-/- platelets had reduced ability to aggregate in response to low concentrations of collagen or PAR4 thrombin receptor agonist AYPGKF, and reduced fibrinogen binding after AYPGKF, but aggregated normally to ADP. 3H-serotonin and ATP secretion were greatly reduced in SG-/- platelets. The alpha-granule proteins platelet factor 4, beta-thromboglobulin, and platelet-derived growth factor were profoundly reduced in SG-/- platelets. Exposure of P-selectin and alphaIIb after thrombin treatment was similar in WT and SG-/- platelets. SG-/- mice exhibited reduced carotid artery thrombus formation after exposure to FeCl3. This study demonstrates that SG is crucial for platelet function and thrombus formation. We propose that SG-/- platelet function deficiencies are related to inadequate packaging and secretion of selected alpha-granule proteins and reduced secretion of dense granule contents critical for platelet activation.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , Agregação Plaquetária , Proteoglicanas/metabolismo , Vesículas Secretórias/metabolismo , Trombose/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/ultraestrutura , Cloretos , Compostos Férricos/farmacologia , Fibrinogênio/genética , Fibrinogênio/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Mastócitos/metabolismo , Mastócitos/patologia , Megacariócitos/ultraestrutura , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Neutrófilos/patologia , Noxas/farmacologia , Oligopeptídeos/farmacologia , Selectina-P/genética , Selectina-P/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Fator Plaquetário 4/genética , Fator Plaquetário 4/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Proteoglicanas/genética , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Vesículas Secretórias/genética , Vesículas Secretórias/ultraestrutura , Serotonina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia , Trombina/genética , Trombina/metabolismo , Trombose/genética , Trombose/patologia , Proteínas de Transporte Vesicular/genética , beta-Tromboglobulina/genética , beta-Tromboglobulina/metabolismoRESUMO
We have previously shown that platelets express 2 receptor tyrosine kinases, EphA4 and EphB1, and the Eph kinase ligand, ephrinB1, and proposed that transcellular Eph/ephrin interactions made possible by the onset of platelet aggregation promote the further growth and stability of the hemostatic plug. The present study examines how this might occur. The results show that clustering of either ephrinB1 or EphA4 causes platelets to adhere to immobilized fibrinogen via alpha(IIb)beta(3). Adhesion occurs more slowly than with adenosine diphosphate (ADP) and requires phosphatidylinositol 3 (PI3)-kinase and protein kinase C activity but not ephrinB1 phosphorylation. By itself, Eph and ephrin signaling is insufficient to cause aggregation or the binding of soluble fibrinogen, but it can potentiate aggregation initiated by a Ca(++) ionophore or by agonists for thrombin and thromboxane receptors. It also enhances Rap1 activation without requiring ADP secretion, ephrinB1 phosphorylation, or the activation of PI3-kinase and Src. From this we conclude that (1) Eph/ephrin signaling enhances the ability of platelet agonists to cause aggregation provided that those agonists can increase cytosolic Ca(++); (2) this is accomplished in part by activating Rap1; and (3) these effects require oligomerization of ephrinB1 but not phosphotyrosine-based interactions with the ephrinB1 cytoplasmic domain.