RESUMO
The in vitro production of blood platelets for transfusion purposes is an important goal in the context of a sustained demand for controlled products free of infectious, immune and inflammatory risks. The aim of this study was to characterize human platelets derived from CD34+ progenitors and to evaluate their hemostatic properties. These cultured platelets exhibited a typical discoid morphology despite an enlarged size and expressed normal levels of the major surface glycoproteins. They aggregated in response to ADP and a thrombin receptor agonist peptide (TRAP). After infusion into NSG mice, cultured and native platelets circulated with a similar 24 h half-life. Notably, the level of circulating cultured platelets remained constant during the first two hours following infusion. During this period of time their size decreased to reach normal values, probably due to their remodeling in the pulmonary circulation, as evidenced by the presence of numerous twisted platelet elements in the lungs. Finally, cultured platelets were capable of limiting blood loss in a bleeding assay performed in thrombocytopenic mice. In conclusion, we show here that cultured platelets derived from human CD34+ cells display the properties required for use in transfusion, opening the way to clinical trials.
Assuntos
Antígenos CD34 , Plaquetas/fisiologia , Hemostasia , Agregação Plaquetária , Transfusão de Plaquetas , Células-Tronco , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/metabolismo , Células Cultivadas , Feminino , Glicoproteínas/metabolismo , Técnicas In Vitro , Camundongos Transgênicos , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacosRESUMO
OBJECTIVE: The platelet glycoprotein (GP)Ib-V-IX complex is a receptor required for normal hemostasis deficient in the Bernard-Soulier bleeding disorder. To evaluate the consequences of GPIb-V-IX deficiency in thrombosis we generated mouse models of the disease by targeting the GPIb beta subunit. METHODS AND RESULTS: Complete deletion (GPIb beta-/-) or an intracellular truncation (GPIb beta deltaIC-/-) reproduced typical and variant forms of Bernard-Soulier, with absent and partial (20%) expression of the complex on the platelet surface. Both strains exhibited thrombocytopenia and enlarged platelets with abnormal microtubular structures but normal granule composition. They exhibited prolonged tail bleeding times, which were less pronounced in GPIb beta deltaIC-/-. Decreased thrombus formation was observed after blood perfusion over a collagen coated surface at high shear. Resistance to vascular occlusion and an abnormal thrombus composition were observed in a model of FeCl3-induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIb beta-/- mice, while a more modest effect was observed in GPIb beta deltaIC-/- animals. Finally, the two strains were protected against death in a model of systemic thromboembolism. CONCLUSIONS: This study provides in vivo evidence of a decreased thrombotic tendency linked to defective platelet GPIb-V-IX in mouse models of Bernard-Soulier syndrome.
Assuntos
Síndrome de Bernard-Soulier/complicações , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombose/etiologia , Trombose/prevenção & controle , Animais , Síndrome de Bernard-Soulier/metabolismo , Síndrome de Bernard-Soulier/fisiopatologia , Plaquetas/patologia , Plaquetas/fisiologia , Colágeno , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hemostasia/genética , Hemostasia/fisiologia , Camundongos , Camundongos Knockout , Contagem de Plaquetas , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Trombocitopenia/patologia , Trombocitopenia/fisiopatologia , Trombose/metabolismo , Trombose/fisiopatologiaRESUMO
Platelets are produced upon profound reorganization of mature megakaryocytes (MK) leading to proplatelet elongation and release into the blood stream, a process termed thrombopoiesis. This highly dynamic process requires microtubules (MT) reorganization by mechanisms that are still incompletely understood. Adenomatous polyposis coli (APC) is a microtubule plus-end tracking protein involved in the regulation of MT in a number of cell systems and its inactivation has been reported to alter hematopoiesis. The aim of our study was to investigate the role of APC in megakaryopoiesis and the final steps of platelet formation. Down-regulation of APC in cultured human MK by RNA interference increased endomitosis and the proportion of cells able to extend proplatelets (68.8% (shAPC1) and 52.5% (shAPC2) vs 28.1% in the control). Similarly an increased ploidy and amplification of the proplatelet network were observed in MK differentiated from Lin- cells of mice with APC-deficiency in the MK lineage. In accordance, these mice exhibited increased platelet counts when compared to wild type mice (1,323 ± 111 vs 919 ± 52 platelets/µL; n = 12 p 0.0033**). Their platelets had a normal size, ultrastructure and number of microtubules coils and their main functions were also preserved. Loss of APC resulted in lower levels of acetylated tubulin and decreased activation of the Wnt signaling pathway. Thus, APC appears as an important regulator of proplatelet formation and overall thrombopoiesis.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Plaquetas/metabolismo , Microtúbulos/metabolismo , Acetilação , Proteína da Polipose Adenomatosa do Colo/deficiência , Animais , Plaquetas/ultraestrutura , Linhagem da Célula , Células Cultivadas , Megacariócitos/citologia , Megacariócitos/metabolismo , Megacariócitos/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/ultraestrutura , Contagem de Plaquetas , Ploidias , Via de Sinalização WntRESUMO
ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.
Assuntos
Agregação Plaquetária , Receptores Purinérgicos P2/fisiologia , Trombose/prevenção & controle , Difosfato de Adenosina/farmacologia , Animais , Tempo de Sangramento , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ativação Plaquetária , Receptores Purinérgicos P2Y1 , Recombinação GenéticaRESUMO
BACKGROUND: Interaction between the platelet glycoprotein (GP)Ib-V-IX complex and von Willebrand factor (VWF) is critical for initiating platelet-vessel wall contacts, particularly under high shear conditions. This interaction also plays an important role in initiating platelet activation through the generation of intracellular signals resulting in platelet shape change and integrin alpha(IIb)beta3 activation. OBJECTIVE: A cell-penetrating peptide strategy was used to study the role of the intracellular domain of the GPIbalpha subunit in VWF/GPIb-V-IX-dependent adhesion and activation. METHODS: Peptides of 11-13 amino acids, covering the 557-610 region, were coupled to a nine-arginine permeating tag (R9) and the effects of their cell entry on VWF-dependent responses were analyzed. RESULTS: The R9alpha557 peptide corresponding to the 557-569 segment reduced platelet agglutination in response to VWF, while the other peptides had no effect. The decreased platelet agglutination appeared to be an indirect consequence of adenosine diphosphate release as a normal response was restored by apyrase or a P2Y1 receptor antagonist. A more direct effect of R9alpha557 on GPIb VWF-dependent functions was observed in adhesion studies on a VWF matrix, where it decreased platelet adhesion and profoundly inhibited filopodia formation. In addition, cell adhesion was reduced and shape change absent when Chinese hamster ovary cells expressing the GPIb-IX complex were incubated with R9alpha557. CONCLUSION: This study performed in intact platelets suggests a functional role of the 557-569 domain of GPIbalpha in controlling VWF-dependent adhesion and signaling.
Assuntos
Plaquetas/metabolismo , Proteínas de Membrana/metabolismo , Adesividade Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Transdução de Sinais , Difosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Plaquetas/citologia , Células CHO , Adesão Celular , Forma Celular , Cricetinae , Cricetulus , Citometria de Fluxo , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peptídeos/síntese química , Peptídeos/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Estresse Mecânico , Transfecção , Fator de von Willebrand/metabolismoRESUMO
The molecular defect of a new Bernard-Soulier patient, originating from Morocco and presenting thrombocytopenia with large platelets and an absence of ristocetin-induced platelet agglutination, has been identified and reproduced in transfected heterologous cells. Gene sequencing revealed insertion of a guanine in the domain coding for the transmembrane region of the glycoprotein (GP) Ib beta subunit. This mutation causes a translational frame shift, which creates putative novel transmembrane and cytoplasmic 37 and 125 amino acids domains, respectively. A 34 kDa immunoreactive GPIb beta band, instead of the normal 26 kDa subunit, was detected by Western blotting in lysates from the patient's platelets and from transfected cells and in immunoprecipitates of metabolically labeled cells. The abnormal subunit did not associate with GPIb alpha and was mainly intracellular, although a significant fraction could reach the cell surface. Cells expressing the mutant GPIb-IX complex adhered to a von Willebrand factor matrix but were unable to change shape, unlike cells expressing the wild-type receptor. These results strongly suggest a novel role of the GPIb beta subunit and its transmembrane-intracellular region in GPIb-VWF-dependent signaling, in addition to a role in correct assembly and cell surface targeting of the GPIb-V-IX complex.
Assuntos
Síndrome de Bernard-Soulier/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Transdução de Sinais , Sequência de Aminoácidos , Animais , Síndrome de Bernard-Soulier/sangue , Síndrome de Bernard-Soulier/complicações , Plaquetas/patologia , Células CHO , Membrana Celular , Forma Celular , Pré-Escolar , Cricetinae , Citoplasma , Feminino , Mutação da Fase de Leitura , Humanos , Fragmentos de Peptídeos , Transdução de Sinais/genética , Trombocitopenia/etiologia , Transfecção , Fator de von Willebrand/metabolismoRESUMO
UNLABELLED: Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis. SUMMARY: Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Förster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.
Assuntos
Actinas/metabolismo , Megacariócitos/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Plaquetas/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Imageamento Tridimensional , Lentivirus , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Transdução de Sinais , Trombopoese , Quinases Ativadas por p21/metabolismoRESUMO
UNLABELLED: Essentials A signaling role of glycoprotein (GP)Ibß is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbß function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. SUMMARY: Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbß subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbß in hemostasis and thrombosis. Methods GPIbß(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbß were transplanted into lethally irradiated GPIbß(-/-) recipient mice. Results hGPIbß transplanted into the bone marrow of GPIbß(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbß(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbß intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbß in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.
Assuntos
Síndrome de Bernard-Soulier/genética , Técnicas de Transferência de Genes , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Animais , Aorta/metabolismo , Tempo de Sangramento , Plaquetas/metabolismo , Células da Medula Óssea/citologia , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Vetores Genéticos , Células HEK293 , Hemorragia , Hemostasia , Humanos , Lentivirus , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Domínios Proteicos , Trombose/metabolismo , Transgenes , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: At the end of maturation, megakaryocytes (MKs) form long cytoplasmic extensions called proplatelets (PPT). Enormous changes in cytoskeletal structures cause PPT to extend further, to re-localize organelles such as mitochondria and to fragment, leading to platelet release. Two non-muscle myosin IIs (NMIIs) are expressed in MKs; however, only NMII-A (MYH9), but not NMII-B (MYH10), is expressed in mature MKs and is implicated in PPT formation. OBJECTIVES: To provide in vivo evidence on the specific role of NMII-A and IIB in MK PPT formation. METHODS: We studied two transgenic mouse models in which non-muscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. RESULTS AND CONCLUSIONS: This work demonstrates that the kinetic properties of NM-IIA, depending on the N-terminal domain, render NMII-A the better NMII candidate to control PPT formation. Furthermore, the carboxyl-terminal domain determines myosin II localization in the constriction region of PPT and is responsible for the specific role of NMII in platelet release.
Assuntos
Plaquetas/metabolismo , Miosina Tipo II/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Transgenic mice expressing cre recombinase under the control of the platelet factor 4 (Pf4) promoter, in the context of a 100-kb bacterial artificial chromosome, have become a valuable tool with which to study genetic modifications in the platelet lineage. However, the specificity of cre expression has recently been questioned, and the time of its onset during megakaryopoiesis remains unknown. OBJECTIVES/METHODS: To characterize the expression of this transgene, we used double-fluorescent cre reporter mice. RESULTS: In the bone marrow, Pf4-cre-mediated recombination had occurred in all CD42-positive megakaryocytes as early as stage I of maturation, and in rare CD42-negative cells. In circulating blood, all platelets had recombined, along with only a minor fraction of CD45-positive cells. However, we found that all tissues contained recombined cells of monocyte/macrophage origin. When recombined, these cells might potentially modify the function of the tissues under particular conditions, especially inflammatory conditions, which further increase recombination in immune cells. Unexpectedly, a subset of epithelial cells from the distal colon showed signs of recombination resulting from endogenous Pf4-cre expression. This is probably the basis of the unexplained colon tumors developed by Apc(flox/flox) ;Pf4-cre mice, generated in a separate study on the role of Apc in platelet formation. CONCLUSION: Altogether, our results indicate early recombination with full penetrance in megakaryopoiesis, and confirm the value of Pf4-cre mice for the genetic engineering of megakaryocytes and platelets. However, care must be taken when investigating the role of platelets in processes outside hemostasis, especially when immune cells might be involved.
Assuntos
Linhagem da Célula , Integrases/genética , Megacariócitos/metabolismo , Fator Plaquetário 4/metabolismo , Animais , Plaquetas/metabolismo , Células Cultivadas , Cromossomos Artificiais Bacterianos , Colo/citologia , Colo/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Leucócitos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Fenótipo , Fator Plaquetário 4/genética , Recombinação Genética , Transdução de Sinais , TrombopoeseRESUMO
High concentrations of adenosine-5'-diphosphate ADP are able to induce partial aggregation without shape change of P2Y(1) receptor-deficient mouse platelets through activation of the P2Y(12) receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse alpha(IIb)beta(3) integrin, revealed a low level activation of alpha(IIb)beta(3) in P2Y(1) receptor-deficient platelets in response to 100 microM ADP or 1 microM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of (32)P-labeled platelets showed that P2Y(12)-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P(20)) or pleckstrin (P(47)) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y(12) receptor is able to trigger a P2Y(1) receptor-independent inside-out signal leading to alpha(IIb)beta(3) integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the alpha(IIb)beta(3) integrin, and (iii) the transduction pathway triggered by the P2Y(12) receptor is independent of PKC but dependent on phosphoinositide 3-kinase.
Assuntos
Proteínas de Membrana , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores Purinérgicos P2/fisiologia , Androstadienos/farmacologia , Animais , Proteínas Sanguíneas/metabolismo , Cromonas/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Epinefrina/farmacologia , Fibrinogênio/metabolismo , Citometria de Fluxo , Cinética , Camundongos , Microscopia Eletrônica de Varredura , Morfolinas/farmacologia , Cadeias Leves de Miosina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Transdução de Sinais , Estaurosporina/farmacologia , Fatores de Tempo , WortmaninaRESUMO
The role of the phospholipase C (PLC)gamma 2 isotype in platelet activation was evaluated by studying PLC gamma 2 -/- mice. These mice have a prolonged bleeding time but their platelets respond normally to non-collagenous agonists. PLC gamma 2-null platelets show residual aggregation response to collagen fibres (6% versus 74% for wild-type) with minimal granule secretion and no shape change. A delayed shape change is observed at later aggregation times. Specific activation by glycoprotein (GP)VI agonists (convulxin, collagen-related peptide and GPVI crosslinking) is, however, abolished. Antibodies against integrin alpha(2)beta(1) and GPVI each inhibit the residual collagen response, implying a role of alpha(2)beta(1) in platelet activation and a functional association with GPVI. These responses are also prevented by blocking integrin alpha(IIb)beta(3) and phosphoinositide 3-kinase, whereas aspirin treatment and ADP receptor blockade only inhibit shape change. These results provide evidence for a PLC gamma 2-independent collagen activation pathway requiring cooperation between GPVI and alpha(2)beta(1) leading to alpha(IIb)beta(3)-dependent aggregation and shape change by released ADP and thromboxane A(2).
Assuntos
Colágeno/farmacologia , Integrina alfa2beta1/fisiologia , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/fisiologia , Fosfolipases Tipo C/fisiologia , Animais , Tempo de Sangramento , Plaquetas/citologia , Plaquetas/enzimologia , Plaquetas/metabolismo , Plaquetas/fisiologia , Integrina alfa2beta1/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Vesículas Secretórias/metabolismo , Fosfolipases Tipo C/genéticaRESUMO
ADP and TxA2 are secondary agonists which play an important role as cofactors when platelets are activated by agonists such as collagen or thrombin. The aim of the present study was to characterize the role of the ADP receptor P2Y(1) in collagen-induced activation of washed platelets. Inhibition of P2Y(1) alone with the selective antagonist MRS2179 prolonged the lag phase preceding aggregation in response to low or high concentrations of fibrillar collagen, without affecting the maximum amplitude of aggregation or secretion. A combination of MRS2179 and aspirin resulted in complete inhibition of platelet shape change at low and high collagen concentrations, together with a profound decrease in aggregation and secretion. Scanning electron microscopy showed that these platelets had conserved the discoid morphology typical of the resting state. A lack of shape change was also observed in aspirin-treated P2Y(1)- and G(alphaq)-deficient mouse platelets and in delta-storage pool-deficient platelets from Fawn Hooded rats. In contrast, when the second ADP receptor P2Y(12) was inhibited with AR-C69931MX, aspirin-treated platelets were still able to change shape and displayed only a moderate decrease in aggregation and secretion. In conclusion, this study provides evidence that collagen requires not only the TxA2 receptor Tpalpha, but also P2Y(1), to induce platelet shape change.
Assuntos
Difosfato de Adenosina/análogos & derivados , Plaquetas/citologia , Colágeno/farmacologia , Receptores Purinérgicos P2/fisiologia , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Aspirina/farmacologia , Plaquetas/metabolismo , Plaquetas/fisiologia , Forma Celular/efeitos dos fármacos , Sinergismo Farmacológico , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Cinética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Ativação Plaquetária , Antagonistas do Receptor Purinérgico P2 , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Transdução de Sinais , Tromboxano A2/antagonistas & inibidores , Tromboxano A2/biossínteseRESUMO
In order to investigate the role of the platelet P2Y1 receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [33P]2MeSADP in the presence or absence of the selective P2Y1 antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 +/- 31 P2Y1 receptors and TG platelets 276 +/- 34, representing an 84% increase in P2Y1 receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5'-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y1 receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y1 expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y1 receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y1 receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed.
Assuntos
Plaquetas/metabolismo , Linhagem da Célula/genética , Ativação Plaquetária/fisiologia , Receptores Purinérgicos P2/biossíntese , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Tempo de Sangramento , Cálcio/análise , Cálcio/química , Cálcio/metabolismo , Colágeno/farmacologia , AMP Cíclico/análise , Epinefrina/farmacologia , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Plasmídeos/genética , Agregação Plaquetária/fisiologia , Fator Plaquetário 4/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1RESUMO
The relative contributions of the P2Y1 and P2YT receptors to the morphological changes induced in platelets by ADP or ADP-releasing agonists were assessed using two P2 antagonists, A2P5P and AR-C67085, selective for P2Y1 and P2YT, respectively. The P2Y1 receptor was found to be involved in i) the centralization of secretory granules elicited by ADP, ii) the formation of filopodia induced by released ADP in weakly activated platelets and iii) actin polymerization and the cytoskeletal translocation of cdc42, rac1 and rhoA, in an integrin alphaIIbbeta3 dependent manner, in ADP-stimulated platelets. In contrast, the P2YT receptor was shown i) to be essential for the formation of stable macroaggregates, ii) to enhance actin polymerization and the cytoskeletal translocation of small GTPases, probably through amplification of platelet aggregation, and iii) not to be involved in the early steps of platelet activation since its blockade did not affect the cytoskeletal translocation of rhoA.
Assuntos
Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Plaquetas/ultraestrutura , Proteínas de Membrana , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/fisiologia , Receptores Purinérgicos P2/fisiologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Actinas/análise , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Humanos , Microscopia Eletrônica , Agregação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2Y1 , Receptores Purinérgicos P2Y12 , Trombina/farmacologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Platelets activated by ADP become refractory to restimulation, but the mechanism of this process is not well understood. A normal platelet response to ADP requires coactivation of the P2Y(1) receptor responsible for shape change and the P2cyc receptor, responsible for completion and amplification of the response. The aim of the present study was to characterize the desensitization of platelets to ADP and to determine whether or not these two receptors are desensitized simultaneously through identical pathways when platelets become refractory to ADP. It was found that full inhibition of platelet aggregation in response to restimulation by ADP required the presence of ADP in the medium or use of a high concentration (1 mM) of its non-hydrolysable analogue ADPbetaS. Platelets incubated for 1 h at 37 degrees C with 1 mM ADPbetaS and resuspended in Tyrode's buffer containing apyrase displayed a stable refractory state characterized by the inability to aggregate or change shape in response to ADP. ADPbetaS treated platelets loaded with fura-2/AM showed complete blockade of the calcium signal in response to ADP, whereas the capacity of ADP to inhibit PGE1 stimulated cAMP accumulation in these platelets was only diminished. Consequently, serotonin was able to promote ADP induced aggregation through activation of the Gq coupled 5HT(2A) receptor while adrenaline had no such effect. These results suggested that the refractory state of ADPbetaS treated platelets was entirely due to desensitization of the P2Y(1) receptor, the P2cyc receptor remaining functional. Binding studies were performed to determine whether the P2Y(1) and/or P2cyc binding sites were modified in refractory platelets. Using selective P2Y(1) and P2cyc antagonists (A3P5P and AR-C66096 respectively), we could demonstrate that the decrease in [33P]2MeSADP binding sites on refractory platelets corresponded to disappearance of the P2Y(1) sites with no change in the number of P2cyc sites, suggesting internalization of the P2Y(1) receptor. This was confirmed by flow cytometric analysis of Jurkat cells expressing an epitope-tagged P2Y(1) receptor, where ADPbetaS treatment resulted in complete loss of the receptor from the cell surface. We conclude that the P2Y(1) and P2cyc receptors are differently regulated during platelet activation.
Assuntos
Difosfato de Adenosina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Sítios de Ligação , Ligação Competitiva , Plaquetas/metabolismo , Plaquetas/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Citometria de Fluxo , Corantes Fluorescentes , Fura-2 , Humanos , Células Jurkat , Agregação Plaquetária/fisiologia , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2Y1 , Proteínas Recombinantes/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
1. The effect on cyclic nucleotide contents of selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) isoforms III and IV (respectively SK&F 94120 and rolipram) and their interactions with endothelium and NO have been studied in rat aorta in the presence of indomethacin (10 microM). The participation of NO was assessed by using either NG-nitro-L-arginine methyl ester (L-NAME) (NO synthase inhibitor: 30 microM) or 3-morpholinosydnonimine (SIN-1, NO donor: 10 microM with SOD 100 units ml-1). 2. The presence of endothelium significantly increased both adenosine 3':5'-cyclic monophosphate (cyclic AMP, 1.7 fold) and guanosine 3':5'-cyclic monophosphate (cyclic GMP, 2.2 fold) contents. Cyclic GMP was largely affected by L-NAME or SIN-1 treatment, this was not the case for cyclic AMP suggesting that the presence of endothelium modified cyclic AMP content in aorta independently of the NO production. 3. In the presence or absence of endothelium, neither SK&F 94120 nor rolipram, alone or combined, significantly modified cyclic GMP content. 4. The PDE III inhibitor significantly affected cyclic AMP content only in non treated aorta without endothelium. In contrast, the PDE IV inhibitor increased cyclic AMP in all conditions. These increases were generally about 2 fold but markedly higher in aorta treated with SIN-1 and superoxide dismutase (SOD, 6 fold). Association of a low concentration of the PDE III inhibitor (5 microM) with the PDE IV inhibitor (30 microM) potentiated the effect of the PDE IV inhibitor on cyclic AMP content, except for aorta without endothelium treated with SIN-1 plus SOD. 5. These data indicate that the presence of the endothelium could increase cyclic AMP content independently of NO and prostacyclin (PGI2) production. Furthermore, an increase in cyclic GMP content (modulated by NO production) could enhance the cyclic AMP accumulation induced by the PDE IV inhibitor. This result supports the hypothesis that PDE III inhibition by endogenous cyclic GMP may potentiate the effect of PDE IV inhibition on cyclic AMP content. Taken together with our previous studies on relaxation, these results suggest that the NO/cyclic GMP pathway could induce PDE IV-dependent regulation of cyclic AMP via PDE III inhibition.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/enzimologia , Aorta Torácica/metabolismo , Cardiotônicos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , DNA/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Inibidores de Fosfodiesterase/farmacologia , Pirazinas/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Wistar , RolipramRESUMO
The role of endothelium in isoprenaline-induced relaxation was investigated in aortic rings brought to different levels of pre-contraction. Relaxation elicited by isoprenaline decreased with increasing pre-contraction. However, relaxation was identical in rings with and without endothelium brought to the same initial tension by adjusting the noradrenaline concentration. Furthermore, isoprenaline increased cAMP and cGMP contents to the same levels whether endothelium was present or not. These results do not support an obligatory role for the endothelium in isoprenaline-induced relaxation in rat aorta. They indicate that relaxation induced by isoprenaline can be enhanced by the endothelium as a consequence of its effect on the precontraction level of the aorta.
Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Endotélio Vascular/fisiologia , Isoproterenol/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: The FeCl(3)-induced vascular injury model is widely used to study thrombogenesis in vivo, but the processes leading to vascular injury and thrombosis are poorly defined. OBJECTIVES: The aim of our study was to better characterize the mechanisms of FeCl(3)-induced vascular injury and thrombus formation, in order to evaluate the pathophysiological relevance of this model. METHODS: FeCl(3) was applied at different concentrations (from 7.5% to 20%) and for different time periods (up to 5 min) to mouse carotid or mesenteric arteries. RESULTS: Under all the conditions tested, ultrastructural analysis revealed that FeCl(3) diffused through the vessel wall, resulting in endothelial cell denudation without exposure of the inner layers. Hence, only the basement membrane components were exposed to circulating blood cells and might have contributed to thrombus formation. Shortly after FeCl(3) application, numerous ferric ion-filled spherical bodies appeared on the endothelial cells. Interestingly, platelets could adhere to these spheres and form aggregates. Immunogold labeling revealed important amounts of tissue factor at their surface, suggesting that these spheres may play a role in thrombin generation. In vitro experiments indicated that FeCl(3) altered the ability of adhesive proteins, including collagen, fibrinogen and von Willebrand factor, to support platelet adhesion. Finally, real-time intravital microscopy showed no protection against thrombosis in GPVI-immunodepleted and ß(1)(-/-) mice, suggesting that GPVI and ß(1) integrins, known to be involved in initial platelet adhesion and activation, do not play a critical role in FeCl(3)-induced thrombus formation. CONCLUSION: This model should be used cautiously, in particular to study the earliest stage of thrombus formation.