RESUMO
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
BACKGROUND: The SAME device (i-SEP, France) is an innovative filtration-based autotransfusion device able to salvage and wash both red blood cells and platelets. This study evaluated the device performances using human whole blood with the hypothesis that the device will be able to salvage platelets while achieving a erythrocyte yield of 80% and removal ratios of 90% for heparin and 80% for major plasma proteins without inducing signification activation of salvaged cells. METHODS: Thirty healthy human whole blood units (median volume, 478 ml) were diluted, heparinized, and processed by the device in two consecutive treatment cycles. Samples from the collection reservoir and the concentrated blood were analyzed. Complete blood count was performed to measure blood cell recovery rates. Flow cytometry evaluated the activation state and function of platelets and leukocytes. Heparin and plasma proteins were measured to assess washing performance. RESULTS: The global erythrocyte yield was 88.1% (84.1 to 91.1%; median [25th to 75th]) with posttreatment hematocrits of 48.9% (44.8 to 51.4%) and 51.4% (48.4 to 53.2%) for the first and second cycles, respectively. Ektacytometry did not show evidence of erythrocyte alteration. Platelet recovery was 36.8% (26.3 to 43.4%), with posttreatment counts of 88 × 109/l (73 to 101 × 109/l) and 115 × 109/l (95 to 135 × 109/l) for the first and second cycles, respectively. Recovered platelets showed a low basal P-selectin expression at 10.8% (8.1 to 15.2%) and a strong response to thrombin-activating peptide. Leukocyte yield was 93.0% (90.1 to 95.7%) with no activation or cell death. Global removal ratios were 98.3% (97.8 to 98.9%), 98.2% (96.9 to 98.8%), and 88.3% (86.6 to 90.7%) for heparin, albumin, and fibrinogen, respectively. The processing times were 4.4 min (4.2 to 4.6 min) and 4.4 min (4.2 to 4.7 min) for the first and second cycles, respectively. CONCLUSIONS: This study demonstrated the performance of the SAME device. Platelets and red blood cells were salvaged without significant impact on cell integrity and function. In the meantime, leukocytes were not activated, and the washing quality of the device prevented reinfusion of high concentrations of heparin and plasma proteins.
Assuntos
Transfusão de Sangue Autóloga , Transfusão de Plaquetas , Humanos , Transfusão de Sangue Autóloga/instrumentação , Transfusão de Sangue Autóloga/métodos , Desenho de Equipamento , Transfusão de Eritrócitos/instrumentação , Filtração/instrumentação , Filtração/métodos , Citometria de Fluxo , França , Transfusão de Plaquetas/instrumentação , Transfusão de Plaquetas/métodosRESUMO
Over the last decades, antiplatelet agents, mainly aspirin and P2Y12 receptor antagonists, have significantly reduced morbidity and mortality associated with arterial thrombosis. Their pharmacological characteristics, including pharmacokinetic/pharmacodynamics profiles, have been extensively studied, and a significant number of clinical trials assessing their efficacy and safety in various clinical settings have established antithrombotic efficacy. Notwithstanding, antiplatelet agents carry an inherent risk of bleeding. Given that bleeding is associated with adverse cardiovascular outcomes and mortality, there is an unmet clinical need to develop novel antiplatelet therapies that inhibit thrombosis while maintaining hemostasis. In this review, we present the currently available antiplatelet agents, with a particular focus on their targets, pharmacological characteristics, and patterns of use. We will further discuss the novel antiplatelet therapies in the pipeline, with the goal of improved clinical outcomes among patients with atherothrombotic diseases.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Humanos , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/- mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFα and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFα-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/-, and MLEC+/- compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Permeabilidade da Membrana Celular , Endoglina/metabolismo , Células Endoteliais/patologia , Inflamação/patologia , Quinases Lim/metabolismo , Neovascularização Patológica/patologia , Fatores de Despolimerização de Actina/genética , Animais , Endoglina/genética , Células Endoteliais/metabolismo , Inflamação/genética , Inflamação/metabolismo , Quinases Lim/genética , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismoRESUMO
The P2Y12 receptor is a key player in platelet activation and a major target for antithrombotic drugs. The beneficial effects of P2Y12 receptor antagonists might, however, not be restricted to the primary and secondary prevention of arterial thrombosis. Indeed, it has been established that platelet activation also has an essential role in inflammation. Additionally, nonplatelet P2Y12 receptors present in immune cells and vascular smooth muscle cells might be effective players in the inflammatory response. This review will investigate the biological and clinical impact of P2Y12 receptor inhibition beyond its platelet-driven antithrombotic effects, focusing on its anti-inflammatory role. We will discuss the potential molecular and cellular mechanisms of P2Y12-mediated inflammation, including cytokine release, platelet-leukocyte interactions and neutrophil extracellular trap formation. Then we will summarize the current evidence on the beneficial effects of P2Y12 antagonists during various clinical inflammatory diseases, especially during sepsis, acute lung injury, asthma, atherosclerosis, and cancer.
Assuntos
Plaquetas/metabolismo , Inflamação/metabolismo , Neoplasias/metabolismo , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/metabolismo , Sepse/metabolismo , Trombose/metabolismo , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Asma/tratamento farmacológico , Asma/metabolismo , Aterosclerose/dietoterapia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Plaquetas/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genética , Sepse/tratamento farmacológico , Sepse/imunologia , Sepse/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Trombose/tratamento farmacológicoRESUMO
Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbß3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann's thrombasthenia patients lacking the αIIbß3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng +/-) mice compared to Eng +/+ animals. These results suggest a new role for endoglin in αIIbß3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.
Assuntos
Plaquetas/metabolismo , Comunicação Celular , Endoglina/metabolismo , Células Endoteliais/metabolismo , Animais , Plaquetas/citologia , Células CHO , Adesão Celular , Linhagem Celular , Células Cultivadas , Cricetinae , Cricetulus , Endoglina/genética , Células Endoteliais/citologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismoRESUMO
RATIONALE: Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear. OBJECTIVE: To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature. METHODS AND RESULTS: S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus-response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P2 receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P1 receptor was dispensable for survival despite its importance for maintaining vascular integrity. CONCLUSIONS: Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock.
Assuntos
Anafilaxia/metabolismo , Plaquetas/metabolismo , Endotélio Vascular/metabolismo , Eritrócitos/metabolismo , Homeostase/fisiologia , Lisofosfolipídeos/deficiência , Esfingosina/análogos & derivados , Anafilaxia/patologia , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esfingosina/deficiênciaRESUMO
αIIbß3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbß3 changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence 1000LEEDDEEGE1008 of αIIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313-320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIb and ß3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets' surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbß3 activation.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Sinergismo Farmacológico , Fosfatase 2 de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Selectina-P/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Ligação ProteicaRESUMO
Molecules that reduce the level of cyclic adenosine 5'-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5'-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4(-/-) platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbß3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents.
Assuntos
Plaquetas/patologia , AMP Cíclico/metabolismo , Homeostase/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Ativação Plaquetária , Trombose/patologia , Animais , Transporte Biológico , Plaquetas/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Agregação Plaquetária , Testes de Função Plaquetária , Transdução de Sinais , Trombose/metabolismoRESUMO
This review focuses on multidrug resistance protein 4 (MRP4 or ABCC4) that has recently been shown to play a role in cAMP homeostasis, a key-pathway in vascular biology and in platelet functions. In vascular system, recent data provide evidence that inhibition of MRP4 prevents human coronary artery smooth muscle cell proliferation in vitro and in vivo, as well as human pulmonary artery smooth muscle cell proliferation in vitro and pulmonary hypertension in mice in vivo. In the heart, MRP4 silencing in adult rat ventricular myocytes results in an increase in intracellular cAMP levels leading to enhanced cardiomyocyte contractility. However, a prolonged inhibition of MRP4 can promote cardiac hypertrophy. In addition, secreted cAMP, through its metabolite adenosine, prevents adrenergically induced cardiac hypertrophy and fibrosis. Finally, MRP4 inhibition in platelets induces a moderate thrombopathy. The localization of MRP4 underlines the emerging concept of cAMP compartmentalization in platelets, which is a major regulatory mechanism in other cells. cAMP storage in platelet dense granules might limit the cAMP cytosolic concentration upon adenylate cyclase activation, a necessary step to induce platelet activation. In this review, we discuss the therapeutic potential of direct pharmacological inhibition of MRP4 in atherothrombotic disease, via its vasodilating and antiplatelet effects.
Assuntos
Doenças Cardiovasculares/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Plaquetas/metabolismo , HumanosRESUMO
BACKGROUND: Management of ticagrelor-induced bleeding is challenging, as no antidote is currently available. Platelet transfusion, usually proposed to reverse antiplatelet drugs, has been suggested to be ineffective but few data are available. OBJECTIVE: To assess the efficacy of platelet supplementation to restore platelet aggregation inhibited by ticagrelor. DESIGN: In vitro study. SETTING: Blood samples were obtained from the French Blood Bank Institute. PARTICIPANTS: Healthy blood donors. INTERVENTIONS: Whole blood from healthy donors was spiked with ticagrelor or aspirin (used as a positive control). MAIN OUTCOME MEASURES: Platelet aggregation was investigated with impedance aggregometry on whole blood [expressed in ohms (V)] and light transmission aggregometry (expressed in %) on platelet-rich plasma using ADP or arachidonic acid as agonists for ticagrelor or aspirin, respectively. Platelet supplementation was defined as the addition of washed platelet suspension increasing at least 60% of whole blood platelet count. RESULTS: Ticagrelor (3.25âmM) inhibited ADP-induced platelet aggregation compared with control either in whole blood (2 vs. 13âV, Pâ<â0.05) or in platelet-rich plasma (15 vs. 75% Pâ<â0.05). Aspirin (25âmM) inhibited arachidonic acid-induced aggregation (1 vs. 7.5âV, Pâ<â0.05 in whole blood and 5 vs. 77.5%, Pâ=â0.01 in platelet-rich plasma). Platelet supplementation completely restored arachidonic acid-induced platelet aggregation in whole blood (10 vs. 1âV, Pâ=â0.008) and platelet-rich plasma (73 vs. 5%, Pâ<â0.01) in aspirin-treated samples, whereas it failed to correct ADP-induced aggregation (2 vs. 2âV in whole blood and 13.5 vs. 15% in platelet-rich plasma, Pâ>â0.05) in ticagrelor-treated samples. We also report a case of a ticagrelor-treated patient in whom platelet transfusion failed to restore ADP-induced platelet aggregation. CONCLUSION: Platelet supplementation restored platelet aggregation in aspirin-spiked but not in ticagrelor-spiked samples. These results do not support the use of platelet transfusion to reverse the effects of ticagrelor.
Assuntos
Adenosina/análogos & derivados , Inibidores da Agregação Plaquetária/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Transfusão de Plaquetas , Adenosina/toxicidade , Difosfato de Adenosina/farmacologia , Ácido Araquidônico/farmacologia , Aspirina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Função Plaquetária , TicagrelorRESUMO
The αIIb cytoplasmic domain of platelet integrin αIIbß3 contains an unorganized acidic membrane-distal (1000)LEEDDEEGE(1008) region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain (989)KVGFFKR(995). We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-(1000)LEEDDE(1005) (pal-K-1000-1005) and palmitoyl-K-(1005)EEGE(1008) (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbß3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbß3.
Assuntos
Peptídeos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Talina/metabolismo , HumanosRESUMO
Background: High on-treatment platelet reactivity has been reported in 30% of patients on clopidogrel and 50% in elderly patients; however, little is known about the mechanisms of this biological resistance. One hypothesis is an age-related impaired hepatic metabolism of the prodrug clopidogrel, leading to a lower formation of its active metabolite (clopidogrel-AM). Objectives: To compare the levels of clopidogrel-AM formed in vitro using "old" and "young" human liver microsomes (HLMs) and their consequences on platelet functions. Methods: We developed an in vitro model using "old" (73.6 ± 2.3 years) and "young" (51.2 ± 8.5 years) HLMs, added to platelet-rich plasma from 21 healthy donors with or without clopidogrel (50 µM) and incubated at 37 °C for 30 (T30) and 45 minutes (T45). Clopidogrel-AM was quantified by liquid chromatography-mass spectrometry/mass spectrometry method. Platelet aggregation was performed by light transmission aggregometry. Results: The generation of clopidogrel-AM increased over time and reached concentrations comparable with those reported in treated patients. At T30, mean clopidogrel-AM concentrations were significantly higher with "young" (8.56 µg/L; 95% CI, 5.87-11.24) than with "old" HLMs (7.64 µg/L; 95% CI, 5.14-10.14; P = .002); and at T45, 11.40 µg/L; 95% CI (7.57-15.22) vs 10.63 µg/L, 95% CI (7.10-14.15), P = .02 (n = 21). Despite a significant inhibition of platelet aggregation, no significant difference was found in light transmission aggregometry (adenosine diphosphate, 10 µM) after clopidogrel metabolism by "old" or "young" HLMs, probably because of low sensitivity of the method to small variations of clopidogrel-AM. Conclusion: In this original model combining metabolic and functional approaches, less clopidogrel-AM was produced with HLMs from older patients. This provides support for a decreased CYP450 activity that may contribute to high on-treatment platelet reactivity in elderly patients.
RESUMO
Bothrops venoms are rich in enzymes acting on platelets and coagulation. This action is dependent on two major co-factors, i.e., calcium and phospholipids, while antivenoms variably neutralize venom-related coagulopathy effects. Our aims were (i) to describe the composition of B. atrox and B. lanceolatus venoms; (ii) to study their activity on the whole blood using rotational thromboelastometry (ROTEM); (iii) to evaluate the contribution of calcium and phospholipids in their activity; and (iv) to compare the effectiveness of four antivenoms (Bothrofav™, Inoserp™ South America, Antivipmyn™ TRI, and PoliVal-ICP™) on the procoagulant activity of these two venoms. Venom composition was comparable. Both venoms exhibited hypercoagulant effects. B. lanceolatus venom was completely dependent on calcium but less dependent on phospholipids than B. atrox venom to induce in vitro coagulation. The four antivenoms neutralized the procoagulant activity of the two venoms; however, with quantitative differences. Bothrofav™ was more effective against both venoms than the three other antivenoms. The relatively similar venom-induced effects in vitro were unexpected considering the opposite clinical manifestations resulting from envenomation (i.e., systemic bleeding with B. atrox and thrombosis with B. lanceolatus). In vivo studies are warranted to better understand the pathophysiology of systemic bleeding and thrombosis associated with Bothrops bites.
Assuntos
Bothrops , Venenos de Crotalídeos , Mordeduras de Serpentes , Trombose , Animais , Antivenenos/farmacologia , Cálcio , Hemorragia/induzido quimicamente , Hemorragia/tratamento farmacológico , Venenos de Crotalídeos/toxicidadeRESUMO
Anthracyclines, such as doxorubicin (Dox), are widely used chemotherapeutic agents for the treatment of solid tumors and hematologic malignancies. However, they frequently induce cardiotoxicity leading to dilated cardiomyopathy and heart failure. This study sought to investigate the role of the exchange protein directly activated by cAMP (EPAC) in Dox-induced cardiotoxicity and the potential cardioprotective effects of EPAC inhibition. We show that Dox induces DNA damage and cardiomyocyte cell death with apoptotic features. Dox also led to an increase in both cAMP concentration and EPAC1 activity. The pharmacological inhibition of EPAC1 (with CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations. When administered in vivo, Dox-treated WT mice developed a dilated cardiomyopathy which was totally prevented in EPAC1 knock-out (KO) mice. Moreover, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Thus, EPAC1 inhibition appears as a potential therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.
Assuntos
Cardiomiopatias , Cardiomiopatia Dilatada , Camundongos , Humanos , Animais , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Cardiotoxicidade , Cardiomiopatia Dilatada/patologia , Doxorrubicina/metabolismo , Cardiomiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Camundongos Knockout , ApoptoseRESUMO
BACKGROUND: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbß3, thereby compromising platelet binding to fibrinogen and thrombus stability. METHODS: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. RESULTS: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbß3 and sEng and molecular modeling showed a good fitting between αIIbß3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbß3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. CONCLUSIONS: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbß3, suggesting its involvement in primary hemostasis control.
Assuntos
Agregação Plaquetária , Trombose , Humanos , Animais , Camundongos , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Endoglina/metabolismo , Células Endoteliais/metabolismo , Plaquetas/metabolismo , Fibrinogênio/metabolismoRESUMO
BACKGROUND: Platelet cytosolic cyclic adenosine monophosphate (cAMP) levels are balanced by synthesis, degradation, and efflux. Efflux can occur via multidrug resistant protein-4 (MRP4; ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux. METHODS: Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-ß-cyclodextrin (MßCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation. RESULTS: MßCD dose dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MßCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with prostaglandin E1 (PGE1) was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts. CONCLUSION: Our results are in favour of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.
Assuntos
Plaquetas/metabolismo , AMP Cíclico/sangue , Microdomínios da Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/sangue , Agregação Plaquetária , Sistemas do Segundo Mensageiro , Alprostadil/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/genética , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Agregação Plaquetária/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
Lowe syndrome (LS) is a rare X-linked disorder caused by mutations in the oculocerebrorenal gene (OCRL), encoding OCRL, a phosphatidylinositol 5-phosphatase with a RhoGAP domain. An abnormal rate of haemorrhagic events was found in a retrospective clinical survey. Herein, we report the results of exploration of haemostasis in six LS patients. All patients had normal coagulation tests but prolonged closure times (CTs) in the PFA-100 system. Healthy donors' blood samples incubated with a RhoA kinase inhibitor had prolonged CTs. This suggests that an aberrant RhoA pathway in platelets contributes to CT prolongation and primary haemostasis disorders in LS.
Assuntos
Transtornos Hemostáticos/etiologia , Mutação , Síndrome Oculocerebrorrenal/complicações , Monoéster Fosfórico Hidrolases/genética , Adolescente , Testes de Coagulação Sanguínea , Criança , Pré-Escolar , Proteínas Ativadoras de GTPase/genética , Predisposição Genética para Doença , Transtornos Hemostáticos/genética , Humanos , Lactente , Masculino , Síndrome Oculocerebrorrenal/genética , Estudos RetrospectivosRESUMO
Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y12 receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor through stimulation of α2A-adrenoreceptors. It subsequently inhibits cyclic adenosine monophosphate (cAMP) pathway and PI3K signaling. However, since epinephrine may expose a patient to deleterious hemodynamic effects, we hypothesized that other α2-adrenoreceptor agonist drugs used in clinical practice with fewer side effects could reverse the antiplatelet effects of ticagrelor. We compared in vitro the efficacy of clonidine, dexmedetomidine, brimonidine, and norepinephrine with epinephrine to restore ADP- and PAR-1-AP-induced washed platelet aggregation inhibited by ticagrelor, as well as resulting platelet cAMP levels. In ticagrelor-free samples, none of the α2-adrenoreceptor agonists induced aggregation by itself but all of them potentiated ADP-induced aggregation. Compared with epinephrine, norepinephrine, and brimonidine partially restored ADP- and fully restored PAR-1-AP-induced aggregation inhibited by ticagrelor while clonidine and dexmedetomidine were ineffective. Indeed, this lack of effect resulted from a lower decrease in cAMP concentration elicited by these partial α2-adrenoreceptor agonists, clonidine, and dexmedetomidine, compared with full α2-agonists. Our results support the development of specific full and systemic α2-adrenoreceptor agonists for ticagrelor reversal.