Mechanisms involved in regulation of cyclic nucleotide pathway : to better define the respective roles of the transporter MRP4 and of lipid rafts

Les plaquettes sanguines ont pour fonction principale le maintien de l'intégrité vasculaire. En situation physiologique, les nucléotides cycliques et notamment l'AMPc, permettent de maintenir un niveau basal d'inhibition plaquettaire afin d'éviter une activation inappropriée pouvant conduire à la formation d'un caillot à l'origine de thrombus pathologique. La concentration d'AMPc joue donc un rôle crucial dans la balance «activation/inhibition» plaquettaire. Cette concentration est contrôlée par l'activité de synthèse des cyclases, mais également par l'activité des phosphodiestérases (PDE) qui hydrolysent l'AMPc. Plus récemment, un rôle de la protéine d'efflux MRP4 (Multidrug Resistance Protein-4) dans le contrôle de la concentration cytosolique de l'AMPc a également été suggéré puis confirmé par nos travaux. MRP4 permet la déplétion de l'AMPc du cytosol et limite ainsi l'action de l'AMPc sur ses effecteurs. Cependant, il existe une controverse concernant la localisation de MRP4 qui selon les études serait au niveau des membranes des granules denses et/ou sur la membrane plasmique. Par ailleurs, depuis quelques années, de nouveaux mécanismes de régulation de l'AMPc émergent, concernant différents types cellulaires comme les cardiomyocytes, avec la notion de compartimentation de la voie de l'AMPc. Ce dernier ne serait pas réparti de façon homogène dans la cellule, mais aurait une distribution spatio-temporelle « compartimentée » dans la cellule. Une famille de protéines appelée les AKAPs (A-Kinase Anchoring Protein) permettrait notamment de localiser, et former des pools d'AMPc. Ces microdomaines AMPc sont très peu connus dans la plaquette. En revanche, les microdomaines membranaires lipidiques appelés radeaux lipidiques, ont fait l'objet de diverses études mais très peu se sont intéressées à leur rôle dans la compartimentation de la voie de l'AMPc. Les radeaux lipidiques sont des structures de la membrane plasmique plus ou moins enrichies en lipides spécifiques comme le cholestérol et les sphingolipides, ayant la capacité de contrôler et de contenir sélectivement certains acteurs de la signalisation cellulaire. L'objectif de ce travail a été de progresser dans la caractérisation des microdomaines AMPc plaquettaire, en particulier en étudiant leur localisation, ou non, au niveau des radeaux lipidiques, et en définissant le rôle de MRP4 dans ces structures afin de mieux définir le rôle de ce transporteur dans la compartimentation de l'AMPc autre que granulaire. Pour ce travail, nous avons utilisé des préparations de plaquettes lavées humaines ainsi que des plaquettes murines WT ou KO pour MRP4. Nos résultats sont en faveur d'un rôle des radeaux lipidiques sur la régulation de l'activation plaquettaire. En particulier, l'intégrité des radeaux lipidiques serait nécessaire pour l'homéostasie de la voie de signalisation de l'AMPc en maintenant un taux contrôlé d'AMPc. En effet, leur destruction augmente la concentration d'AMPc tout en induisant une inhibition de l'agrégation, mais l'absence de MRP4 n'interfère pas ou peu sur ce mécanisme. Les « microdomaines AMPc », formés notamment des AKAPs et les PDEs tels que décrits dans la littérature, seraient principalement en dehors des radeaux lipidiques. Le transporteur MRP4, retrouvé à l'état de traces dans les radeaux lipidiques, joue un rôle positif dans l'activation plaquettaire en participant à la diminution du niveau de l'AMPc cytosolique. Nous montrons qu'il existe une sécrétion basale d'AMPc, et que cette sécrétion est en partie sous la dépendance de MRP4. Ce résultat est donc en faveur d'une présence au moins partielle de MRP4 à la surface des plaquettes au repos. Bien que la voie de l'AMPc soit déjà ciblée par nombre de molécules antiplaquettaires comme les inhibiteurs de PDE ou ceux ciblant le récepteur P2Y12, MRP4 constituerait une cible potentielle pour un agent pharmacologique antiplaquettaire avec des propriétés élargies au niveau du système cardiovasculaire.Platelets play a central role in haemostasis and thrombosis. In physiological situation, blood platelets are maintained in a circulating inactive state to avoid any inappropriate activation. This resting state is mostly dependent on cyclic nucleotide homeostasis, and notably cAMP. Platelet cytosolic cAMP level results from a balance between its synthesis and its degradation by PDEs and, potentially, its transport out of cytosol to dense granules or to extracellular medium. An additional means of regulating cAMP homeostasis is the transporter MRP4 (multi drug resistant protein-4). Several groups, including our, have shown that MRP4 regulates platelet aggregation by modulating the cAMP-protein kinase A signaling pathway. According to different studies, MRP4 expression has been described on platelet dense granule membrane, on plasmatic membrane, or both. Although MRP4 localization on resting platelets remains a matter of debate, it is obvious that MRP4 plays also an important role in cyclic nucleotide homeostasis in platelets by transporting cAMP out of the cytosol. The localization of MRP4 underlines the emerging concept of cAMP compartmentalization in platelets, which is known as a major regulatory mechanism in other cells. Specificity of cAMP activity results from the nature of stimulus and from its localization in the cell. The so-called "cAMP-dependent microdomains" maintain in restricted zones some of the players of the cAMP pathway, leading to the concept of "cAMP compartmentation ". Notably, A-kinase anchoring proteins (AKAPs) play a major role by assembling proteins (PDE, phosphatase, AC and receptors) in these restricted microdomains. In platelets, little is known on this mechanism but it is likely that same features exist. Some membrane microdomains have been identified in various cell types as well as in platelets. These so-called lipid rafts are dynamic assemblies of sphingolipids and cholesterol, characterized by insolubility in soft detergents as well as a low density. They play a critical role in maintaining multiprotein complexes at the cytoplasmic side of the plasma membrane. In platelets, the role of lipid rafts has been described in numerous activation pathways. However, only one group has linked rafts to the cAMP pathway. Data are in favor of the existence in platelets of structures regulating the cAMP signaling pathway. Thus, cAMP microdomains, whether or not associated with rafts, could regulate the availability and / or distribution of cAMP in platelets. The aim of this work was to better characterize the role of MRP4 in the compartmentalization of non-granular cAMP. We analyzed whether the cAMP microdomains belonged to lipid rafts on one hand, and tried to define the role of MRP4 in these structures on the other hand. Methods used washed platelets from healthy volunteers and from WT or MRP4-deleted mice. Our results argue for a role of lipid rafts in the regulation of platelet activation upon cAMP pathway. Disruption of rafts induced an increase of cAMP level and a concomitant inhibition of platelet aggregation in response to agonists. However, this effect was independent of the presence or the absence of MRP4. We also found that the proteins forming the so-called "microdomains AMPc-dependent", such as AKAPs and PDEs, were situated in majority outside lipid rafts. We know the role of MRP4 in depleting cAMP from cytosol to dense granules. In the present work, we evidenced a basal secretion of cAMP partly dependent on MRP4, suggesting its presence on plasmatic membrane of resting platelets, in addition to dense granule membrane. Since MRP4 was found only as traces in the fractions containing lipid rafts, we suggest its presence outside rafts. Although the cAMP pathway is already targeted by numerous antiplatelet agents (antiP2Y12 or PDE inhibitors), MRP4 may represent a target for a future antiplatelet agent, given its properties on cardiovascular

Evaluation of commonly used tests to measure the effect of single-dose aspirin on mouse hemostasis

PG and CBL share equal senior authorship.International audienceDiscrepancies in preclinical studies of aspirin (ASA) antiplatelet activity in mouse models of bleeding and arterial thrombosis led us to evaluate commonly reported methods in order to propose a procedure for reliably measuring the effects of single dose ASA on mouse hemostasis. FVB and C57Bl6 mice received 100 mg/kg of ASA or vehicle orally 30 min or 3 h prior to investigate either hemostasis using the tail bleeding assay or carotid thrombosis induced by FeCl3, or to blood sampling for isolated platelet aggregation and TXB2 generation. Expected inhibition of COX1 by ASA was ascertained by a strong decrease in TXB2 production, and its effect on platelet function and hemostasis, by decreased collagen-induced aggregation and increased bleeding time, respectively. Strikingly, we determined that anti-hemostatic effects of ASA were more predictable 30 min after administration than 3 h later. Conversely, ASA did not alter time to arterial occlusion of the carotid upon FeCl3-induced thrombosis, suggesting ASA not to be used as reference inhibitor drug in this model of arterial thrombosis

Comparative In Vitro Study of Various α2-Adrenoreceptor Agonist Drugs for Ticagrelor Reversal

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

Pain assessment and factors influencing pain during bone marrow aspiration: A prospective study

International audienceAlthough bone marrow aspiration (BMA) is still considered a painful procedure, pain level remains poorly documented. We therefore conducted a prospective study intended to evaluate pain level in adult patients undergoing BMA at the sternal or iliac crest site to identify factors associated with pain. We enrolled a total of 448 patients who underwent 461 BMA and asked those patients to score their pain intensity after BMA using numerical pain rating scale (NPRS). The following factors: level of anxiety, quality of the information given to the patient, operator's experience, and bone texture were recorded using a standardized questionnaire. The median NPRS score was 3.5 (IQR [2.0; 5.0]) the sternal site (n = 405) was associated with an increased median NPRS score (3.5 [2.0; 5.0]) compared to the iliac crest (n = 56, 2.5 [1.0; 4.0]; p<0.0001). For those patients who underwent sternal BMA, the median NPRS score was significantly lower when using lidocaine infiltration (p = 0.0159) as compared with no anesthetic use. Additionally there was no significant effect of anesthetic cream found. After multivariate analysis, the model of NPRS score at the sternal site included patient anxiety (p<0.0001) and the use of lidocaine infiltration (0.0378). This study underlines the usefulness of a comprehensive management including pain relief and efforts to reduce anxiety including appropriate information given to the patient during BMA

Epinephrine restores platelet functions inhibited by ticagrelor: A mechanistic approach

International audienceTicagrelor, an antagonist of the platelet adenosine diphosphate (ADP)-P2Y(12) receptor is recommended for patients with acute coronary syndromes. However, ticagrelor exposes to a risk of bleeding, the management of which is challenging because platelet transfusion is ineffective, and no antidote is yet available. We hypothesized that the vasopressor drug epinephrine could counter the antiplatelet effects of ticagrelor and restore platelet functions. We assessed in vitro the efficiency of epinephrine in restoring platelet aggregation inhibited by ticagrelor and investigated the underlying mechanisms. Washed platelet aggregation and secretion were measured upon stimulation by epinephrine alone or in combination with ADP, in the presence or absence of ticagrelor. Mechanistic investigations used P2Y(1) and phosphoinositide 3-kinase (PI3K) inhibitors and included vasodilator-stimulated phosphoprotein (VASP) and Akt phosphorylation assays as well as measurement of Ca2+ mobilisation. We found that epinephrine restored ADP-induced platelet aggregation, but not dense granule release. Epinephrine alone failed to induce aggregation whereas it fully induced VASP dephosphorylation and Akt phosphorylation regardless of the presence of ticagrelor. In the presence of ticagrelor, blockage of the P2Y(1) receptor prevented restoration of platelet aggregation by the combination of epinephrine and ADP, as well as intracellular Ca2+ mobilisation. In combination with ADP, epinephrine induced platelet aggregation of ticagrelor-treated platelets through inhibition of the CAMP pathway and activation of the PI3K pathway, thus enabling the P2Y(1) receptor signalling and subsequent Ca2+ mobilisation. This proof-of-concept study needs to be challenged in vivo for the management of bleeding in ticagrelor-treated patients