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1.
Circ Res ; 127(7): e166-e183, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32588751

RESUMO

RATIONALE: Ca2+ signaling is a key and ubiquitous actor of cell organization and its modulation controls many cellular responses. SERCAs (sarco-endoplasmic reticulum Ca2+-ATPases) pump Ca2+ into internal stores that play a major role in the cytosolic Ca2+ concentration rise upon cell activation. Platelets exhibit 2 types of SERCAs, SERCA2b and SERCA3 (SERCA3 deficient mice), which may exert specific roles, yet ill-defined. We have recently shown that Ca2+ mobilization from SERCA3-dependent stores was required for full platelet activation in weak stimulation conditions. OBJECTIVE: To uncover the signaling mechanisms associated with Ca2+ mobilization from SERCA3-dependent stores leading to ADP secretion. METHODS AND RESULTS: Using platelets from wild-type or Serca3-deficient mice, we demonstrated that an early (within 5-10 s following stimulation) secretion of ADP specifically dependent on SERCA3 stored Ca2+ is exclusively mobilized by nicotinic acid adenosine dinucleotide-phosphate (NAADP): both Ca2+ mobilization from SERCA3-dependent stores and primary ADP secretion are blocked by the NAADP receptor antagonist Ned-19, and reciprocally both are stimulated by permeant NAADP. In contrast, Ca2+ mobilization from SERCA3-dependent stores and primary ADP secretion were unaffected by inhibition of the production of IP3 (inositol-1,4,5-trisphosphate) by phospholipase-C and accordingly were not stimulated by permeant IP3. CONCLUSIONS: Upon activation, an NAADP/SERCA3 Ca2+ mobilization pathway initiates an early ADP secretion, potentiating platelet activation, and a secondary wave of ADP secretion driven by both an IP3/SERCA2b-dependent Ca2+ stores pathway and the NAADP/SERCA3 pathway. This does not exclude that Ca2+ mobilized from SERCA3 stores may also enhance platelet global reactivity to agonists. Because of its modulating effect on platelet activation, this NAADP-SERCA3 pathway may be a relevant target for anti-thrombotic therapy. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Difosfato de Adenosina/sangue , Comunicação Autócrina , Plaquetas/enzimologia , Sinalização do Cálcio , NADP/análogos & derivados , Ativação Plaquetária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangue , Animais , Comunicação Autócrina/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Inositol 1,4,5-Trifosfato/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADP/sangue , Ativação Plaquetária/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Via Secretória , Trombina/farmacologia , Tromboxano A2/sangue , Fatores de Tempo
2.
Blood ; 132(19): 2067-2077, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30213874

RESUMO

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbß3 activation, and granule secretion induced by G-protein-coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbß3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet-platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin-mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.


Assuntos
Plaquetas/patologia , Mutação de Sentido Incorreto , Ativação Plaquetária , Receptor EphB2/genética , Adolescente , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Criança , Feminino , Humanos , Masculino , Linhagem , Adesividade Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptor EphB2/metabolismo , Transdução de Sinais , Adulto Jovem
3.
Blood ; 128(8): 1129-38, 2016 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-27301859

RESUMO

The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbß3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbß3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent of αIIbß3.


Assuntos
Difosfato de Adenosina/metabolismo , Plaquetas/enzimologia , Cálcio/metabolismo , Ativação Plaquetária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Tempo de Sangramento , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Deleção de Genes , Hemorreologia/efeitos dos fármacos , Hemostasia/efeitos dos fármacos , Cavalos , Camundongos Endogâmicos C57BL , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/deficiência , Serotonina/farmacologia , Trombose/patologia
4.
Blood ; 124(16): 2554-63, 2014 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-25061177

RESUMO

Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG, a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy.


Assuntos
Plaquetas/patologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Mutação em Linhagem Germinativa , Megacariócitos/patologia , Trombocitopenia/genética , Adulto , Plaquetas/metabolismo , Pré-Escolar , Citoesqueleto/genética , Citoesqueleto/patologia , Humanos , Lactente , Masculino , Megacariócitos/metabolismo , Linhagem , Contagem de Plaquetas , Trombocitopenia/complicações , Trombocitopenia/patologia , Adulto Jovem
5.
Adv Exp Med Biol ; 898: 333-52, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27161235

RESUMO

Calcium ions (Ca(2+)) are versatile messengers that need to be tidily regulated in time and space in order to create a large number of signals. The coupling between Ca(2+) entry and Ca(2+) refilling is playing a central role in this Ca(2+) homeostasis. Since the capacitative Ca(2+) entry has been described, different mechanisms have been proposed in order to explain how the Ca(2+) entry could be under control of intracellular store Ca(2+) depletion. Today, in addition of STIM1 and Orai1, the two major elements of SOCe, increasing attention is put on the role of the transient receptor potential canonical (TRPC), that can form protein clusters with Orai1, and Sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs), that refill the stores and are also located in the same environment than SOC clusters. Altogether, these proteins elaborate either Ca(2+) microdomains in the vicinity of the membrane or larger Ca(2+) increases overtaking the whole cell. The coupling between Ca(2+) entry and Ca(2+) refilling can possibly act much further away from the plasma membrane. Ca(2+), uptaken by SERCAs, have been described to move faster and further in the ER than in the cytosol and to create specific signal that depends on Ca(2+) entry but at longer distance from it. The complexity of such created Ca(2+) currents resides in the heteromeric nature of channels as well as the presence of different intracellular stores controlled by SERCA2b and SERCA3, respectively. A role for mitochondria has also been explored. To date, mitochondria are other crucial compartments that play an important role in Ca(2+) homeostasis. Although mitochondria mostly interact with intracellular stores, coupling of Ca(2+) entry and mitochondria cannot be completely rule out.


Assuntos
Cálcio/metabolismo , Animais , Humanos , Transporte de Íons , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Canais de Cátion TRPC/metabolismo
6.
Biochim Biophys Acta ; 1843(11): 2705-18, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25110346

RESUMO

UNLABELLED: The sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis). Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~91% of total SERCA and SERCA2b for ~5%. Among SERCA3, SERCA3b was the most expressed (~3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b. In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively. IN CONCLUSION: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation-contraction coupling in both CMs and contractile VSMCs.

7.
Thromb Haemost ; 119(3): 384-396, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30650444

RESUMO

In obesity, platelets are described as hyperactive, mainly based on increased platelet size and presence of pro-thrombotic plasmatic molecules. We explored platelet functions, including calcium signalling in obesity, and the effect of weight loss. We included 40 obese patients (women with body mass index [BMI] of ≥ 35 kg/m2) who were to undergo gastric bypass surgery and 40 healthy lean subjects (women with BMI of < 25 kg/m2) as a control group. Approximately 1 year after surgery, the obese patients lost weight (75% had a BMI < 35 kg/m2). They were explored a second time with the same healthy control for the same platelet experiments. Compared with controls, obese patients' platelets displayed reduced sensitivity to thrombin (aggregation EC50 increased by 1.9 ± 0.3-fold, p = 0.005) and a lower Ca2+ response (70 ± 7% decrease, p < 10-4). In 17 pairs of patients, we performed additional experiments: in obese patients' platelets, thrombin-induced αIIbß3 activation was significantly lower (p = 0.003) and sarco-endoplasmic reticulum Ca2+ATPase (SERCA3) expression was decreased (48 ± 6% decrease, p < 10-4). These differences were abolished after weight loss. Interestingly, pharmacological inhibition of SERCA3 activity in control group's platelets mimicked similar alterations than in obese patients' platelets and was associated with defective adenosine diphosphate (ADP) secretion. Addition of ADP to agonist restored platelet functions in obese patients and in SERCA3-inhibited control platelets (five experiments) confirming the direct involvement of the SERCA3-dependent ADP secretion pathway. This is the first study demonstrating that platelets from obese patients are hypo-reactive, due to a deficiency of SERCA3-dependent ADP secretion. Weight loss restores SERCA3 activity and subsequent calcium signalling, αIIbß3 activation, platelet aggregation and ADP secretion.


Assuntos
Difosfato de Adenosina/sangue , Plaquetas/metabolismo , Derivação Gástrica , Obesidade/cirurgia , Ativação Plaquetária , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangue , Redução de Peso , Adulto , Sinalização do Cálcio , Feminino , Humanos , Obesidade/sangue , Obesidade/diagnóstico , Obesidade/fisiopatologia , Paris , Agregação Plaquetária , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Via Secretória , Fatores de Tempo , Resultado do Tratamento
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