RESUMO
Blood platelets are produced by megakaryocytes through a complex program of differentiation and play a critical role in hemostasis and thrombosis. These anucleate cells are the target of antithrombotic drugs that prevent them from clumping in cardiovascular disease conditions. Platelets also significantly contribute to various aspects of physiopathology, including interorgan communications, healing, inflammation, and thromboinflammation. Their production and activation are strictly regulated by highly elaborated mechanisms. Among them, those involving inositol lipids have drawn the attention of researchers. Phosphoinositides represent the seven combinatorially phosphorylated forms of the inositol head group of inositol lipids. They play a crucial role in regulating intracellular mechanisms, such as signal transduction, actin cytoskeleton rearrangements, and membrane trafficking, either by generating second messengers or by directly binding to specific domains of effector proteins. In this review, we will explore how phosphoinositides are implicated in controlling platelet production by megakaryocytes and in platelet activation processes. We will also discuss the diversity of phosphoinositides in platelets, their role in granule biogenesis and maintenance, as well as in integrin signaling. Finally, we will address the discovery of a novel pool of phosphatidylinositol 3-monophosphate in the outerleaflet of the plasma membrane of human and mouse platelets.
Assuntos
Plaquetas , Trombose , Animais , Camundongos , Humanos , Plaquetas/patologia , Fosfatidilinositóis/metabolismo , Inflamação , Trombose/metabolismo , Inositol/metabolismoRESUMO
Background & Aims: The constitutive androstane receptor (CAR) is a nuclear receptor that binds diverse xenobiotics and whose activation leads to the modulation of the expression of target genes involved in xenobiotic detoxification and energy metabolism. Although CAR hepatic activity is considered to be higher in women than in men, its sex-dependent response to an acute pharmacological activation has seldom been investigated. Methods: The hepatic transcriptome, plasma markers, and hepatic metabolome, were analysed in Car+/+ and Car-/- male and female mice treated either with the CAR-specific agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or with vehicle. Results: Although 90% of TCPOBOP-sensitive genes were modulated in a sex-independent manner, the remaining 10% showed almost exclusive female liver specificity. These female-specific CAR-sensitive genes were mainly involved in xenobiotic metabolism, inflammation, and extracellular matrix organisation. CAR activation also induced higher hepatic oxidative stress and hepatocyte cytolysis in females than in males. Hepatic expression of flavin monooxygenase 3 (Fmo3) was almost abolished and was associated with a decrease in hepatic trimethylamine-N-oxide (TMAO) concentration in TCPOBOP-treated females. In line with a potential role in the control of TMAO homeostasis, CAR activation decreased platelet hyper-responsiveness in female mice supplemented with dietary choline. Conclusions: More than 10% of CAR-sensitive genes are sex-specific and influence hepatic and systemic responses such as platelet aggregation. CAR activation may be an important mechanism of sexually-dimorphic drug-induced liver injury. Impact and implications: CAR is activated by many drugs and pollutants. Its pharmacological activation had a stronger impact on hepatic gene expression and metabolism in females than in males, and had a specific impact on liver toxicity and trimethylamine metabolism. Sexual dimorphism should be considered when testing and/or prescribing xenobiotics known to activate CAR.
RESUMO
Phosphoinositides (PIs) play a crucial role in regulating intracellular signaling, actin cytoskeleton rearrangements, and membrane trafficking by binding to specific domains of effector proteins. They are primarily found in the membrane leaflets facing the cytosol. Our study demonstrates the presence of a pool of phosphatidylinositol 3-monophosphate (PI3P) in the outer leaflet of the plasma membrane of resting human and mouse platelets. This pool of PI3P is accessible to exogenous recombinant myotubularin 3-phosphatase and ABH phospholipase. Mouse platelets with loss of function of class III PI 3-kinase and class II PI 3-kinase α have a decreased level of external PI3P, suggesting a contribution of these kinases to this pool of PI3P. After injection in mouse, or incubation ex vivo in human blood, PI3P-binding proteins decorated the platelet surface as well as α-granules. Upon activation, these platelets were able to secrete the PI3P-binding proteins. These data sheds light on a previously unknown external pool of PI3P in the platelet plasma membrane that recognizes PI3P-binding proteins, leading to their uptake towards α-granules. This study raises questions about the potential function of this external PI3P in the communication of platelets with the extracellular environment, and its possible role in eliminating proteins from the plasma.
Assuntos
Plaquetas , Proteínas de Transporte , Camundongos , Humanos , Animais , Plaquetas/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte/metabolismo , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
BACKGROUND: Purinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step. RESULTS: Here, we investigated the pharmacology of P2Y1-R signaling through Gq and ß-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y1-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y1-R is constitutively active in physiological conditions. We showed that P2Y1-R also promotes constitutive recruitment of ß-arrestin 2 in HEK293T cells. Moreover, the P2Y1-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists. CONCLUSIONS: This study sheds new light on P2Y1-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y1-R population in human platelets. Given the recent interest of P2Y12-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y1-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y1-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.
Assuntos
Agonismo Inverso de Drogas , Proteínas de Ligação ao GTP , Receptores Purinérgicos P2Y1 , Transdução de Sinais , beta-Arrestina 2 , Animais , Humanos , Camundongos , beta-Arrestina 2/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Receptores Purinérgicos P2Y1/metabolismo , PlaquetasRESUMO
The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is known to dephosphorylate PtdIns(3,4,5)P3 into PtdIns(3,4)P2 and to interact with several signaling proteins though its docking functions. It has been shown to negatively regulate platelet adhesion and spreading on a fibrinogen surface and to positively regulate thrombus growth. In the present study, we have investigated its role during the early phase of platelet activation. Using confocal-based morphometric analysis, we found that SHIP1 is involved in the regulation of cytoskeletal organization and internal contractile activity in thrombin-activated platelets. The absence of SHIP1 has no significant impact on thrombin-induced Akt or Erk1/2 activation, but it selectively affects the RhoA/Rho-kinase pathway and myosin IIA relocalization to the cytoskeleton. SHIP1 interacts with the spectrin-based membrane skeleton, and its absence induces a loss of sustained association of integrins to this network together with a decrease in αIIbß3 integrin clustering following thrombin stimulation. This αIIbß3 integrin dynamics requires the contractile cytoskeleton under the control of SHIP1. RhoA activation, internal platelet contraction, and membrane skeleton integrin association were insensitive to the inhibition of PtdIns(3,4,5)P3 synthesis or SHIP1 phosphatase activity, indicating a role for the docking properties of SHIP1 in these processes. Altogether, our data reveal a lipid-independent function for SHIP1 in the regulation of the contractile cytoskeleton and integrin dynamics in platelets.
Assuntos
Integrina alfa2 , Integrina beta3 , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Ativação Plaquetária , Plaquetas/metabolismo , Integrina beta3/metabolismo , Fosfatidilinositóis/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombina/farmacologia , Trombina/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Integrina alfa2/metabolismoRESUMO
Phosphatidylinositol 3-kinase catalytic subunit p110ß is involved in tumorigenesis and hemostasis. However, it remains unclear if p110ß also regulates platelet-mediated immune responses, which could have important consequences for immune modulation during anti-cancer treatment with p110ß inhibitors. Thus, we investigate how platelet p110ß affects inflammation and infection. Using a mouse model of Streptococcus pneumoniae-induced pneumonia, we find that both platelet-specific p110ß deficiency and pharmacologic inhibition of p110ß with TGX-221 exacerbate disease pathogenesis by preventing platelet-monocyte and neutrophil interactions, diminishing their infiltration and enhancing bacterial dissemination. Platelet p110ß mediates neutrophil phagocytosis of S. pneumoniae in vitro and curtails bacteremia in vivo. Genetic deficiency or inhibition of platelet p110ß also impairs macrophage recruitment in an independent model of sterile peritonitis. Our results demonstrate that platelet p110ß dysfunction exacerbates pulmonary infection by impeding leukocyte functions. Thereby, our findings provide important insights into the immunomodulatory potential of PI3K inhibitors in bacterial infection.
Assuntos
Pneumonia Pneumocócica , Humanos , Fosfatidilinositol 3-Quinases/genética , Plaquetas , Leucócitos , Inibidores de Fosfoinositídeo-3 Quinase , Streptococcus pneumoniaeRESUMO
Platelets are mainly known for their key role in hemostasis and thrombosis. However, studies over the last two decades have shown their strong implication in mechanisms associated with inflammation, thrombosis, and the immune system in various neoplastic, inflammatory, autoimmune, and infectious diseases. During sepsis, platelets amplify the recruitment and activation of innate immune cells at the site of infection and contribute to the elimination of pathogens. In certain conditions, these mechanisms can lead to thromboinflammation resulting in severe organ dysfunction. Here, we discuss the interactions of platelets with leukocytes, neutrophil extracellular traps (NETs), and endothelial cells during sepsis. The intrinsic properties of platelets that generate an inflammatory signal through the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome are discussed. As an example of immunothrombosis, the implication of platelets in vaccine-induced immune thrombotic thrombocytopenia is documented. Finally, we discuss the role of megakaryocytes (MKs) in thromboinflammation and their adaptive responses.
Assuntos
Sepse , Trombose , Plaquetas , Células Endoteliais , Humanos , Inflamação , Megacariócitos , TromboinflamaçãoRESUMO
During severe sepsis, platelet activation may induce disseminate microvascular thrombosis, which play a key role in critical organ failure. Crucially, most of the studies in this field have explored platelet-leukocyte interactions in animal models, or explored platelets under the spectrum of thrombocytopenia or disseminated intravascular coagulation and have not taken into account the complex interplay that might exist between platelets and leukocytes during human septic shock nor the kinetics of platelet activation. Here, we assessed platelet activation parameters at the admission of patients with sepsis to the intensive care unit (ICU) and 48 hours later. Twenty-two patients were enrolled in the study, thirteen (59.1%) of whom were thrombocytopenic. The control group was composed of twelve infection-free patients admitted during the study period. The activation parameters studied included platelet-leukocyte interactions, assessed by flow cytometry in whole blood, as well as membrane surface and soluble platelet activation markers measured by flow cytometry and dedicated ELISA kits. We also investigated platelet aggregation and secretion responses of patients with sepsis following stimulation, compared to controls. At admission, the level of circulating monocyte-platelet and neutrophil-platelet heterotypic aggregates was significantly higher in sepsis patients compared to controls and returned to a level comparable to controls or even below 48 hours later. Basal levels of CD62P and CD63 platelet membrane exposure at admission and 48 hours later were low and similar to controls. In contrast, plasma level of soluble GPVI and soluble CD40 ligand was significantly increased in septic patients, at the two times of analysis, reflecting previous platelet activation. Platelet aggregation and secretion responses induced by specific agonists were significantly decreased in septic conditions, particularly 48 hours after admission. Hence, we have observed for the first time that critically ill septic patients compared to controls have both an early and durable platelet activation while their circulating platelets are less responsive to different agonists.
Assuntos
Sepse , Choque Séptico , Animais , Plaquetas/fisiologia , Humanos , Unidades de Terapia Intensiva , Ativação Plaquetária/fisiologiaRESUMO
Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2ß) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2ß in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2ß showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2ß in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2ß as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.
Assuntos
Células Endoteliais , Fosfatidilinositol 3-Quinases , Junções Aderentes/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
PI3Ks are important lipid kinases that produce phosphoinositides phosphorylated in position 3 of the inositol ring. There are three classes of PI3Ks: class I PI3Ks produce PIP3 at plasma membrane level. Although D. melanogaster and C. elegans have only one form of class I PI3K, vertebrates have four class I PI3Ks called isoforms despite being encoded by four different genes. Hence, duplication of these genes coincides with the acquisition of coordinated multi-organ development. Of the class I PI3Ks, PI3Kα and PI3Kß, encoded by PIK3CA and PIK3CB, are ubiquitously expressed. They present similar putative protein domains and share PI(4,5)P2 lipid substrate specificity. Fifteen years after publication of their first isoform-selective pharmacological inhibitors and genetically engineered mouse models (GEMMs) that mimic their complete and specific pharmacological inhibition, we review the knowledge gathered in relation to the redundant and selective roles of PI3Kα and PI3Kß. Recent data suggest that, further to their redundancy, they cooperate for the integration of organ-specific and context-specific signal cues, to orchestrate organ development, physiology, and disease. This knowledge reinforces the importance of isoform-selective inhibitors in clinical settings.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Humanos , Fosforilação , Transdução de Sinais , Especificidade por SubstratoRESUMO
Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.
Assuntos
Plaquetas/citologia , Megacariócitos/citologia , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/fisiologia , Trombopoese/fisiologia , Actomiosina/análise , Adolescente , Adulto , Anemia/etiologia , Coagulação Sanguínea , Plaquetas/ultraestrutura , Estudos de Casos e Controles , Forma Celular , Criança , Colágeno , Citoesqueleto/ultraestrutura , Feminino , Inativação Gênica , Humanos , Masculino , Megacariócitos/ultraestrutura , Pessoa de Meia-Idade , Mutação , Cadeias Leves de Miosina/metabolismo , Síndrome Oculocerebrorrenal/sangue , Síndrome Oculocerebrorrenal/patologia , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Domínios Proteicos , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , Transdução de Sinais , Trombocitopenia/etiologia , Adulto JovemRESUMO
Our knowledge on the expression, regulation and roles of the different phosphoinositide 3-kinases (PI3Ks) in platelet signaling and functions has greatly expanded these last twenty years. Much progress has been made in understanding the roles and regulations of class I PI3Ks which produce the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3). Selective pharmacological inhibitors and genetic approaches have allowed researchers to generate an impressive amount of data on the role of class I PI3Kα, ß, δ and γ in platelet activation and in thrombosis. Furthermore, platelets do also express two class II PI3Ks (PI3KC2α and PI3KC2ß), thought to generate PtdIns(3,4)P2 and PtdIns3P, and the sole class III PI3K (Vps34), known to synthesize PtdIns3P. Recent studies have started to reveal the importance of PI3KC2α and Vps34 in megakaryocytes and platelets, opening new perspective in our comprehension of platelet biology and thrombosis. In this review, we will summarize previous and recent advances on platelet PI3Ks isoforms. The implication of these kinases and their lipid products in fundamental platelet biological processes and thrombosis will be discussed. Finally, the relevance of developing potential antithrombotic strategies by targeting PI3Ks will be examined.
Assuntos
Plaquetas/enzimologia , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Trombose/enzimologia , Trombose/terapia , Animais , Plaquetas/patologia , Humanos , Isoenzimas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Trombose/patologiaRESUMO
Blood platelets, produced by the fragmentation of megakaryocytes, play a key role in hemostasis and thrombosis. Being implicated in atherothrombosis and other thromboembolic disorders, they represent a major therapeutic target for antithrombotic drug development. Several recent studies have highlighted an important role for the lipid phosphatidylinositol 3 monophosphate (PtdIns3P) in megakaryocytes and platelets. PtdIns3P, present in small amounts in mammalian cells, is involved in the control of endocytic trafficking and autophagy. Its metabolism is finely regulated by specific kinases and phosphatases. Class II (α, ß and γ) and III (Vps34) phosphoinositide-3-kinases (PI3Ks), INPP4 and Fig4 are involved in the production of PtdIns3P whereas PIKFyve, myotubularins (MTMs) and type II PIPK metabolize PtdIns3P. By regulating the turnover of different pools of PtdIns3P, class II (PI3KC2α) and class III (Vps34) PI3Ks have been recently involved in the regulation of platelet production and functions. These pools of PtdIns3P appear to modulate membrane organization and intracellular trafficking. Moreover, PIKFyve and INPP4 have been recently implicated in arterial thrombosis. In this review, we will discuss the role of PtdIns3P metabolizing enzymes in platelet production and function. Potential new anti-thrombotic therapeutic perspectives based on inhibitors targeting specifically PtdIns3P metabolizing enzymes will also be commented.
Assuntos
Plaquetas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais , Trombopoese , Trombose/metabolismo , Animais , Plaquetas/patologia , Humanos , Transporte Proteico , Trombose/patologiaRESUMO
INTRODUCTION: Thrombocytopenia is well recognized as a poor prognosis sign associated with increased mortality and prolonged Intensive Care Unit (ICU) stay, particularly in septic patients. Mean platelet volume (MPV) could represent a relevant predictive marker of mortality. Here we investigated whether MPV kinetics during the first 15 days after hospital admission has a potential prognostic value for clinical outcome in septic shock. METHODS: We performed a retrospectively analysis of a cohort of 301 septic patients admitted in ICU. Three-month mortality was the primary endpoint. The prognostic value of the covariates of interest was ascertained by multidimensional analysis. We proposed a classification and regression trees analysis to predict survival probability. RESULTS: MPV kinetics was significantly different between 90-day survivors and non-survivors when followed during 15 days (except on day 3). 10-day MPV >11.6fL was an independent predictive factor of 90-day mortality (Hazard Ratio (HR) 3.796, 95% Confidence Interval (CI) [1.96-7.35], p = 0.0001) in multivariate analysis. Base excess on day 4 <1.9mmol/L was also a predictive factor of mortality (HR 2.972, 95%CI [1.38-6.40], p = 0.0054. CONCLUSION: MPV increase during the first 15 days after ICU admission in non-survivors was observed during septic shock and 10-day MPV >11.6fL was an independent predictive factor of 90-day mortality. This could be explained by the emergent response to acute platelet loss during septic shock, leading to megakaryocyte rupture to produce new but potentially immature platelets in the circulation. Therefore, continuous monitoring of MPV may be a useful parameter to stratify mortality risk in septic shock.
Assuntos
Plaquetas/metabolismo , Volume Plaquetário Médio , Choque Séptico/sangue , Choque Séptico/mortalidade , Idoso , Biomarcadores , Feminino , Humanos , Estimativa de Kaplan-Meier , Cinética , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Modelos de Riscos Proporcionais , Curva ROC , Estudos Retrospectivos , Choque Séptico/diagnóstico , Choque Séptico/etiologiaRESUMO
Host defense against infection is based on two crucial mechanisms: the inflammatory response and the activation of coagulation. Platelets are involved in both hemostasis and immune response. These mechanisms work together in a complex and synchronous manner making the contribution of platelets of major importance in sepsis. This is a summary of the pathophysiology of sepsis-induced thrombocytopenia, microvascular consequences, platelet-endothelial cells and platelet-pathogens interactions. The critical role of platelets during sepsis and the therapeutic implications are also reviewed.
Assuntos
Plaquetas/imunologia , Sepse/sangue , Trombocitopenia/sangue , Animais , Toxinas Bacterianas/toxicidade , Plaquetas/patologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Humanos , Sepse/complicações , Trombocitopenia/etiologiaAssuntos
Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Tromboembolia/enzimologia , Tromboembolia/patologia , Trombose/enzimologia , Trombose/patologia , Anticoagulantes/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/genética , Fatores de RiscoRESUMO
Objective- PI3Kα (phosphoinositide 3-kinase alpha) is a therapeutic target in oncology, but its role in platelets and thrombosis remains ill characterized. In this study, we have analyzed the role of PI3Kα in vitro, ex vivo, and in vivo in 2 models of arterial thrombosis. Approach and Results- Using mice selectively deficient in p110α in the megakaryocyte lineage and isoform-selective inhibitors, we confirm that PI3Kα is not mandatory but participates to thrombus growth over a collagen matrix at arterial shear rate. Our data uncover a role for PI3Kα in low-level activation of the GP (glycoprotein) VI-collagen receptor by contributing to ADP secretion and in turn full activation of PI3Kß and Akt/PKB (protein kinase B). This effect was no longer observed at high level of GP VI agonist concentration. Our study also reveals that over a vWF (von Willebrand factor) matrix, PI3Kα regulates platelet stationary adhesion contacts under arterial flow through its involvement in the outside-in signaling of vWF-engaged αIIbß3 integrin. In vivo, absence or inhibition of PI3Kα resulted in a modest but significant decrease in thrombus size after superficial injuries of mouse mesenteric arteries and an increased time to arterial occlusion after carotid lesion, without modification in the tail bleeding time. Considering the more discrete and nonredundant role of PI3Kα compared with PI3Kß, selective PI3Kα inhibitors are unlikely to increase the bleeding risk at least in the absence of combination with antiplatelet drugs or thrombopenia. Conclusions- This study provides mechanistic insight into the role of PI3Kα in platelet activation and arterial thrombosis.
Assuntos
Hemostasia , Fosfatidilinositol 3-Quinase/fisiologia , Adesividade Plaquetária , Agregação Plaquetária , Trombose/fisiopatologia , Animais , Ativação Enzimática , Feminino , Humanos , Masculino , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de von Willebrand/metabolismoRESUMO
Sepsis is associated with thrombocytopenia and microvascular thrombosis. Studies have described platelets implication in this pathology but their kinetics of activation and behavior remain poorly known. We show in a mouse model of peritonitis, the appearance of platelet-rich thrombi in organ microvessels and organ damage. Complementary methods are necessary to characterize platelet activation during sepsis as circulating soluble markers and platelet-monocyte aggregates revealed early platelet activation, while surface activation markers were detected at later stage. A microfluidic based ex-vivo thrombosis assay demonstrated that platelets from septic mice have a prothrombotic behavior at shear rate encountered in microvessels. Interestingly, we found that even though phosphoinositide-3-kinase ß-deficient platelet mice formed less thrombi in liver microcirculation, peritoneal sepsis activates a platelet alternative pathway to compensate the otherwise mandatory role of this lipid-kinase to form stable thrombi at high shear rate. Platelets are rapidly activated during sepsis. Thrombocytopenia can be attributed in part to platelet-rich thrombi formation in capillaries and platelet-leukocytes interactions. Platelets from septic mice have a prothrombotic phenotype at a shear rate encountered in arterioles. Further studies are necessary to unravel molecular mechanisms leading to this prothrombotic state of platelets in order to guide the development of future treatments of polymicrobial sepsis.
Assuntos
Plaquetas/patologia , Peritonite/fisiopatologia , Ativação Plaquetária , Sepse/fisiopatologia , Trombocitopenia/fisiopatologia , Trombose/fisiopatologia , Animais , Arteríolas/patologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peritonite/sangue , Peritonite/microbiologia , Fator Plaquetário 4/genética , Sepse/sangue , Sepse/microbiologia , Trombocitopenia/sangue , Trombocitopenia/microbiologia , Trombose/sangue , Trombose/microbiologiaRESUMO
Our knowledge on the role of the different lipid messengers produced by phosphoinositide 3-kinases (PI3Ks) in normal and cancer cells as well as in platelets during arterial thrombosis has greatly expanded these last 15 years. PI3Ks are a family of lipid kinases that catalyze the phosphorylation of the D3 position of the inositol ring of phosphoinositides to produce phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol-3,4,5 trisphosphate (PtdIns(3,4,5)P3). These D3-phosphoinositides act as intracellular messengers recruiting effector proteins involved in the control of diverse cellular functions including survival, proliferation, migration, membrane trafficking, and cytoskeleton dynamics. The current idea is that the different isoforms of PI3Ks produce specific pools of lipids that regulate in time and space, at the membrane/cytosol interface, the formation of appropriate functional protein complexes. Dysregulation of PI3K-dependent pathways is directly involved in the etiology of several pathologies including cancers where the PI3K/AKT/mTORC1 axis is frequently aberrantly activated. Moreover, PtdIns(3,4,5)P3 production has been shown to play an essential role in platelet functions, particularly in the formation of a stable platelet thrombus at high shear rate. Therefore, PI3Ks are attractive therapeutic targets in the treatment of cancer and arterial thrombosis. In this review, we will discuss the role of the different lipid products of PI3K isoforms in the context of cancer and thrombosis and the development of selective PI3Ks inhibitors in the treatment of these diseases.
Assuntos
Metabolismo dos Lipídeos , Neoplasias/etiologia , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Trombose/etiologia , Trombose/metabolismo , Animais , Plaquetas/metabolismo , Membrana Celular/metabolismo , Homeostase , Humanos , Isoenzimas , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP2 and PIP3 molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110ß deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110ß and the more subtle role of p110α in the production of PIP3 molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP2 during stimulation, opening new perspectives in phosphoinositide signaling in platelets.