Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome.

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENT Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.

Distinct P2Y Receptors Mediate Extension and Retraction of Microglial Processes in Epileptic and Peritumoral Human Tissue.

Microglia exhibit multiple, phenotype-dependent motility patterns often triggered by purinergic stimuli. However, little data exist on motility of human microglia in pathological situations. Here we examine motility of microglia stained with a fluorescent lectin in tissue slices from female and male epileptic patients diagnosed with mesial temporal lobe epilepsy or cortical glioma (peritumoral cortex). Microglial shape varied from ramified to amoeboid cells predominantly in regions of high neuronal loss or closer to a tumor. Live imaging revealed unstimulated or purine-induced microglial motilities, including surveillance movements, membrane ruffling, and process extension or retraction. At different concentrations, ADP triggered opposing motilities. Low doses triggered process extension. It was suppressed by P2Y12 receptor antagonists, which also reduced process length and surveillance movements. Higher purine doses caused process retraction and membrane ruffling, which were blocked by joint application of P2Y1 and P2Y13 receptor antagonists. Purinergic effects on motility were similar for all microglia tested. Both amoeboid and ramified cells from mesial temporal lobe epilepsy or peritumoral cortex tissue expressed P2Y12 receptors. A minority of microglia expressed the adenosine A2A receptor, which has been linked with process withdrawal of rodent cells. Laser-mediated tissue damage let us test the functional significance of these effects. Moderate damage induced microglial process extension, which was blocked by P2Y12 receptor antagonists. Overall, the purine-induced motility of human microglia in epileptic tissue is similar to that of rodent microglia in that the P2Y12 receptor initiates process extension. It differs in that retraction is triggered by joint activation of P2Y1/P2Y13 receptors.SIGNIFICANCE STATEMENT Microglial cells are brain-resident immune cells with multiple functions in healthy or diseased brains. These diverse functions are associated with distinct phenotypes, including different microglial shapes. In the rodent, purinergic signaling is associated with changes in cell shape, such as process extension toward tissue damage. However, there are little data on living human microglia, especially in diseased states. We developed a reliable technique to stain microglia from epileptic and glioma patients to examine responses to purines. Low-intensity purinergic stimuli induced process extension, as in rodents. In contrast, high-intensity stimuli triggered a process withdrawal mediated by both P2Y1 and P2Y13 receptors. P2Y1/P2Y13 receptor activation has not previously been linked to microglial morphological changes.

sLRP1 (Soluble Low-Density Lipoprotein Receptor-Related Protein 1): A Novel Biomarker for P2Y12 (P2Y Purinoceptor 12) Receptor Expression in Atherosclerotic Plaques.

OBJECTIVE: Recent studies suggest that the P2Y12 (P2Y purinoceptor 12) receptor of vascular smooth muscle cells in atherosclerotic plaques aggravates atherosclerosis, and P2Y12 receptor inhibitors such as CDL (clopidogrel) may effectively treat atherosclerosis. It is imperative to identify an effective biomarker for reflecting the P2Y12 receptor expression on vascular smooth muscle cells in plaques. Approach and Results: We found that there was a positive correlation between the level of circulating sLRP1 (soluble low-density lipoprotein receptor-related protein 1) and the number of LRP1+ α-SMA+ (α-smooth muscle actin), P2Y12+, or P2Y12+ LRP1+ cells in plaques from apoE-/- mice fed a high-fat diet. Furthermore, activation of the P2Y12 receptor increased the expression and shedding of LRP1 in vascular smooth muscle cells by inhibiting cAMP (3'-5'-cyclic adenosine monophosphate)/PKA (protein kinase A)/SREBP-2 (sterol regulatory element binding transcription factor 2). Conversely, genetic knockdown or pharmacological inhibition of the P2Y12 receptor had the opposite effects. Additionally, CDL decreased the number of lesional LRP1+ α-SMA+ cells and the levels of circulating sLRP1 by activating cAMP/PKA/SREBP-2 in apoE-/- mice fed a high-fat diet. CONCLUSIONS: Our study suggests that sLRP1 may be a biomarker that reflects the P2Y12 receptor level in plaques and has the potential to be an indicator for administering P2Y12 receptor inhibitors for patients with atherosclerosis.

ADP receptor P2y12 prevents excessive primitive hematopoiesis in zebrafish by inhibiting Gata1.

In the past two decades, purinergic signaling has emerged as a key regulator of hematopoiesis in physiological and pathological conditions. ADP receptor P2y12 is a crucial component of this signaling, but whether it is involved in primitive hematopoiesis remains unknown. To elucidate the function of P2y12 and provide new insights for drug development, we established a zebrafish P2y12 mutant by CRISPR/Cas 9-based genetic modification system, and investigated whether P2y12 acted as an important regulator for primitive hematopoiesis. By using mass spectrometry (MS) combined with RNA sequencing, we showed that absence of P2y12 induced excessive erythropoiesis, evidenced by significantly increased expression of mature erythrocytes marker α-globin (Hbae1 and Hbae3), ß-globin (Hbbe1 and Hbbe3). Expression pattern analysis showed that P2y12 was mainly expressed in red blood cells and endothelial cells of early zebrafish embryos. Further studies revealed that primitive erythroid progenitor marker Gata1 was markedly up-regulated. Remarkably, inhibition of Gata1 by injection of Gata1 morpholino could rescue the erythroid abnormality in P2y12 mutants. The present study demonstrates the essential role of purinergic signaling in differentiation of proerythrocytes during primitive hematopoiesis, and provides potential targets for treatment of blood-related disease and drug development.

P2Y12 Purinergic Receptor and Brain Tumors: Implications on Glioma Microenvironment.

Gliomas are the most common malignant brain tumors in adults, characterized by a high proliferation and invasion. The tumor microenvironment is rich in growth-promoting signals and immunomodulatory pathways, which increase the tumor's aggressiveness. In response to hypoxia and glioma therapy, the amounts of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) strongly increase in the extracellular space, and the purinergic signaling is triggered by nucleotides' interaction in P2 receptors. Several cell types are present in the tumor microenvironment and can facilitate tumor growth. In fact, tumor cells can activate platelets by the ADP-P2Y12 engagement, which plays an essential role in the cancer context, protecting tumors from the immune attack and providing molecules that contribute to the growth and maintenance of a rich environment to sustain the protumor cycle. Besides platelets, the P2Y12 receptor is expressed by some tumors, such as renal carcinoma, colon carcinoma, and gliomas, being related to tumor progression. In this context, this review aims to depict the glioma microenvironment, focusing on the relationship between platelets and tumor malignancy.

Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier.

Microglia are integral functional elements of the central nervous system, but the contribution of these cells to the structural integrity of the neurovascular unit has not hitherto been assessed. We show here that following blood-brain barrier (BBB) breakdown, P2RY12 (purinergic receptor P2Y, G-protein coupled, 12)-mediated chemotaxis of microglia processes is required for the rapid closure of the BBB. Mice treated with the P2RY12 inhibitor clopidogrel, as well as those in which P2RY12 was genetically ablated, exhibited significantly diminished movement of juxtavascular microglial processes and failed to close laser-induced openings of the BBB. Thus, microglial cells play a previously unrecognized protective role in the maintenance of BBB integrity following cerebrovascular damage. Because clopidogrel antagonizes the platelet P2Y12 receptor, it is widely prescribed for patients with coronary artery and cerebrovascular disease. As such, these observations suggest the need for caution in the postincident continuation of P2RY12-targeted platelet inhibition.

Modulation of microglial process convergence toward neuronal dendrites by extracellular calcium.

Extracellular calcium concentrations in the brain fluctuate during neuronal activities and may affect the behavior of brain cells. Microglia are highly dynamic immune cells of the brain. However, the effects of extracellular calcium concentrations on microglial dynamics have not been investigated. Here, we addressed this question in mouse brain slices and in vivo using two-photon microscopy. We serendipitously found that extracellular calcium reduction induced microglial processes to converge at distinct sites, a phenomenon we termed microglial process convergence (MPCs). Our studies revealed that MPCs target neuronal dendrites independent of neuronal action potential firing and is mediated by ATP release and microglial P2Y12 receptors. These results indicate that microglia monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain.

Modulation and pre-amplification of PAR1 signaling by ADP acting via the P2Y12 receptor during platelet subpopulation formation.

BACKGROUND: Two major soluble blood platelet activators are thrombin and ADP. Of these two, only thrombin can induce mitochondrial collapse and programmed cell death leading to phosphatidylserine (PS) exposure required for blood clotting reactions acceleration. Thrombin can also greatly potentiate collagen-induced PS exposure. However, ADP acting through the P2Y12 receptor was shown to increase the PS-exposing (PS+) platelets fraction produced by thrombin or thrombin-plus-collagen via an unknown mechanism. METHODS: We developed a comprehensive multicompartmental computational model of platelet PAR1-and-P2Y12 calcium signal transduction that included cytoplasmic signaling, dense tubular system and mitochondria. To test model predictions, flow cytometry experiments with washed, annexin V-labeled platelets were performed. RESULTS: Stimulation of thrombin receptor PAR1 in the model induced cytoplasmic calcium oscillations, calcium uptake by mitochondria, opening of the permeability transition pore and collapse of the mitochondrial membrane potential. ADP stimulation of P2Y12 led to cAMP decrease that, in turn, caused changes in phospholipase C phosphorylation by protein kinase A, increase in cytoplasmic calcium level and, consequently, PS+ platelet formation. ADP addition before stimulation of PAR1 produced much greater increase of the PS+ fraction because cAMP concentration had time to go down prior to calcium oscillations; this prediction was also tested and confirmed experimentally. CONCLUSION: These results suggest a mechanism of ADP-dependent PS exposure regulation and show a likely mode of action that could be important for the PS exposure regulation in thrombi, where ADP is released before thrombin formation.

ADP-induced bladder contractility is mediated by P2Y12 receptor and temporally regulated by ectonucleotidases and adenosine signaling.

Purinergic signaling comprises one key pathway in modulating bladder smooth muscle (BSM) contractility, disorders of which become highly prevalent with aging. ADP was first observed to modulate BSM contractility >40 yr ago, yet the underlying molecular mechanism still remains unclear. Here, we demonstrate, using myography, that ADP and ADPßS dose-dependently induce mouse BSM contraction, and ADP-induced BSM contraction is blocked by a selective P2Y12 receptor (P2Y12R) antagonist, PSB 0739 (25 µM), but is unaffected by P2Y1 and P2Y13 receptor antagonists. P2Y12R in BSM exhibits distinct pharmacological properties that are different from P2Y12R in platelets. After an immediate contraction, prolonged exposure to ADP causes BSM to become refractory to further ADP-mediated contraction. However, in mice lacking ectonucleotidases Entpd1 (ATP→ADP→AMP) or Nt5e (AMP→adenosine), or by inhibiting adenosine signaling, the refractory response was altered, resulting in repeated BSM contractions in response to repeated ADP (0.1-1 mM) stimulation. Our data indicate that P2Y12R undergoes slow desensitization; ADP-P2Y12 signaling is tightly regulated by Entpd1/Nt5e activity and adenosine receptors; and ADP-adenosine signaling play an important role in modulating P2X-mediated BSM contraction. The identification of P2Y12R in BSM, and the current clinical availability of P2Y12R inhibitors, such as clopidogrel, offers potentially novel treatment strategies for bladder contractility disorders.

α2A-Adrenergic receptor polymorphism potentiates platelet reactivity in patients with stable coronary artery disease carrying the cytochrome P450 2C19*2 genetic variant.

OBJECTIVE: Platelet α2A-adrenergic receptors (ARs) mediate platelet aggregation in response to sympathetic stimulation. The 6.3-kb variant of α2A-AR gene is associated with increased epinephrine-induced platelet aggregation in healthy volunteers. The cytochrome P450 2C19*2 (CYP2C19*2) loss-of-function allele influences P2Y12-mediated platelet inhibition and hence the rate of major adverse cardiovascular events. We assessed the influence of 6.3-kb α2A-AR gene variant on platelet aggregation and its interaction with CYP2C19*2 loss-of-function allele in patients with stable angina on aspirin and clopidogrel (dual antiplatelet therapy). APPROACH AND RESULTS: Aggregation to 5 increasing doses of epinephrine (from 0.156 to 10 µmol/L) was assessed in aggregation units by Multiplate Analyzer and platelet reactivity in P2Y12 reactivity units and % inhibition by VerifyNow P2Y12 assay before percutaneous revascularization. Gene polymorphisms were analyzed with TaqMan Drug Metabolism assay. Of 141 patients, aggregation was higher in 6.3-kb carriers (n=52) when compared with wild types (n=89) at all epinephrine doses (P<0.05) apart from 10 µmol/L (P=0.077). Percentage inhibition was lower (P=0.048) in 6.3-kb α2A-AR carriers. Percentage inhibition was lower (P=0.005) and P2Y12 reactivity units was higher (P=0.012) in CYP2C19*2 allele carriers. Higher P2Y12 reactivity units (P=0.037) and lower percentage inhibition (P=0.009) were observed in carriers of both 6.3-kb α2A-AR variant and CYP2C19*2 allele when compared with wild-type or with either mutation on its own. CONCLUSIONS: The 6.3-kb α2A-AR variant is associated with increased platelet reactivity to epinephrine and has an additive effect along with CYP2C19*2 loss-of-function allele on P2Y12-mediated platelet responses in patients with stable angina on dual antiplatelet therapy.

Clopidogrel preserves whole kidney autoregulatory behavior in ANG II-induced hypertension.

This study tested the hypothesis that P2Y12 receptor blockade with clopidogrel preserves renal autoregulatory ability during ANG II-induced hypertension. Clopidogrel was administered orally to male Sprague-Dawley rats chronically infused with ANG II. After 14 days of treatment, whole kidney autoregulation of renal blood flow was assessed in vivo in pentobarbital-anesthetized rats using an ultrasonic flow probe placed around the left renal artery. In ANG II-vehicle-treated rats, decreasing arterial pressure over a range from 160 to 100 mmHg resulted in a 25 ± 5% decrease in renal blood flow, demonstrating a significant loss of autoregulation with an autoregulatory index of 0.66 ± 0.15. However, clopidogrel treatment preserved autoregulatory behavior in ANG II-treated rats to levels indistinguishable from normotensive sham-operated (sham) rats (autoregulatory index: 0.04 ± 0.14). Compared with normotensive sham-vehicle-treated rats, ANG II infusion increased renal CD3-positive T cell infiltration by 66 ± 6%, induced significant thickening of the preglomerular vessels and glomerular basement membrane and increased glomerular collagen I deposition, tubulointerstitial fibrosis, damage to the proximal tubular brush border, and protein excretion. Clopidogrel significantly reduced renal infiltration of T cells by 39 ± 9% and prevented interstitial artery thickening, ANG II-induced damage to the glomerular basement membrane, deposition of collagen type I, and tubulointerstitial fibrosis, despite the maintenance of hypertension. These data demonstrate that systemic P2Y12 receptor blockade with clopidogrel protects against impairment of autoregulatory behavior and renal vascular injury in ANG II-induced hypertension, possibly by reducing renal T cell infiltration.

Molecular mechanisms of platelet P2Y(12) receptor regulation.

Platelets are critical for haemostasis, however inappropriate activation can lead to the development of arterial thrombosis, which can result in heart attack and stroke. ADP is a key platelet agonist that exerts its actions via stimulation of two surface GPCRs (G-protein-coupled receptors), P2Y(1) and P2Y(12). Similar to most GPCRs, P2Y receptor activity is tightly regulated by a number of complex mechanisms including receptor desensitization, internalization and recycling. In the present article, we review the molecular mechanisms that underlie P2Y(1) and P2Y(12) receptor regulation, with particular emphasis on the structural motifs within the P2Y(12) receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.

Roles of purinergic receptor P2Y, G protein-coupled 12 in the development of atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: The aim of the study was to evaluate the role of purinergic receptor P2Y, G protein-coupled 12 (P2Y12), an ADP receptor, in the development of atherosclerotic lesions. METHODS AND RESULTS: Apolipoprotein E-null mice were crossed with P2y12(-/-) mice to generate double knockout mice. The double knockout mice and the control apolipoprotein E-null mice were fed a high-fat diet for 20 weeks. Assessment of the atherosclerotic lesions in the control and double knockout mice demonstrated that P2Y12 deficiency caused a diminished lesion area, an increased fibrous content at the plaque site, and decreased monocyte/macrophage infiltration of the lesions. Polymerase chain reaction studies revealed that white blood cells do not express significant levels of P2Y12. Bone marrow transplantation experiments confirmed that P2Y12 expressed on platelets is a key factor responsible for atherosclerosis, but do not exclude a role of smooth muscle cell P2Y12. Supernatant fluid from activated P2y12(+/+) but not P2y12(-/-) platelets was capable of causing monocyte migration. In vitro studies showed that platelet P2Y12 deficiency suppressed platelet factor 4 secretion and P-selectin expression. Further work demonstrated that platelet P2Y12, through inhibition of the cAMP/protein kinase A pathway, critically regulates the release of platelet factor 4, and thereby affects monocyte recruitment and infiltration. CONCLUSIONS: These results demonstrate that P2Y12 modulates atherogenesis, at least in part by augmenting inflammatory cell recruitment via regulation of platelet α-granule release.

Fractalkine activates a signal transduction pathway similar to P2Y12 and is associated with impaired clopidogrel responsiveness.

OBJECTIVE: Fractalkine (FKN) activates a G(αi) protein-coupled signaling pathway similar to the one activated by ADP via P2Y(12), which is the drug target of clopidogrel. FKN levels are increased under several disease conditions associated with impaired clopidogrel responsiveness. METHODS AND RESULTS: Blood samples were obtained from healthy volunteers and from 40 patients under chronic clopidogrel treatment. FKN reduced prostaglandin E1-induced vasodilator-stimulated phosphoprotein phosphorylation by ≈ 25% (P<0.01) at least partially mimicking the effect of ADP via P2Y(12). In vitro, FKN increased platelet reactivity index in clopidogrel-treated patients indicating potential activation of downstream targets of P2Y(12). When stratifying patients by their FKN levels, patients within the highest quartile of FKN (2042 ± 25 pg/mL) had the weakest response to clopidogrel (platelet reactivity index, 68 ± 4%), and patients within the lowest quartile (479 ± 50 pg/mL) had the strongest response (platelet reactivity index, 48 ± 7%; P=0.0106). FKN by itself induced phosphoinositide 3-kinase activation leading to Akt phosphorylation at Ser(473) (P<0.01 versus basal). CONCLUSIONS: In addition to desensitizing platelets to prostaglandin E1 via G(αi), FKN induces phosphoinositide 3-kinase-dependent Akt phosphorylation via a G(ßγ) protein similar to ADP signaling through P2Y(12). FKN increased the platelet ADP response in clopidogrel-treated patients. Once released from an atherosclerotic lesion, this mechanism could contribute locally to impaired clopidogrel responsiveness at the vulnerable plaque.

[Molecular mechanisms of platelet activation]. / Les mécanismes moléculaires de l'activation plaquettaire.

The main role of blood platelets is to ensure vascular integrity and hemostasis in case of vascular damage. The platelet functions involved in these physiological processes are also at work in arterial thrombosis, which is a dramatic complication of atherosclerosis that may lead to vascular occlusion. These functions of platelets include their ability to adhere to the injured vessel wall, to be activated by contact with various substrates and soluble activators, and to form aggregates stabilized by a fibrin network. Platelets are also involved in metastasis, various inflammatory processes, innate and adaptive immune defenses, and embryonic development. These roles are supported by multiple molecular mechanisms, some of which are common to several functions while others are distinct. Defects in one or other of these mechanisms do not necessarily disrupt all platelet functions.

P2Y(12) receptors in platelets and other hematopoietic and non-hematopoietic cells.

The P2Y(12) receptor is a Gi-coupled ADP receptor first described in blood platelets where it plays a central role in the complex processes of activation and aggregation. Platelet granules store important amounts of ADP which are released upon stimulation by interaction of platelets with the damaged vessel wall. Therefore, the P2Y(12) receptor is a key player in primary hemostasis and in arterial thrombosis and is an established target of antithrombotic drugs like the thienopyridine compounds ticlopidine, clopidogrel, and prasugrel or the direct, reversible antagonists ticagrelor and cangrelor. Beyond the platelet physiology and pharmacology, recent studies have revealed the expression of the P2Y(12) receptor in other hematopoietic cells including leukocyte subtypes and microglia in the central nervous system as well as in vascular smooth muscle cells. These studies indicate putative roles of the P2Y(12) receptor in inflammatory states and diseases of the brain, lung, and blood vessels. The selective role of P2Y(12) among other P2 receptors as well as the possible impact of P2Y(12) targeting drugs in these processes remain to be evaluated.

P2Y11 purinoceptor mediates the ATP-enhanced chemotactic response of rat neutrophils.

ATP and hydrolysis products of ATP like adenosine regulate the chemotaxis of neutrophils by activating purinoceptors and adenosine receptors. The present study was designed to examine exogenous ATP, activation of purinoceptors, and activation of A(3) adenosine receptor as key steps in the signal cascades that control cell orientation and migration of rat neutrophils. One or more of those steps might be potential therapeutic targets for treatment of inflammatory diseases. The chemotaxis of rat neutrophils was stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured with an EZ-TAXIScan apparatus. The effects of apyrase, exogenous ATP, suramin (P2X and P2Y blocker), PPADS (a P2X blocker), TNP-ATP (P2X(1) and P2X(3) antagonist), and Reactive Blue 2 (a P2Y blocker) on the chemotactic response were also investigated. Rat neutrophil chemotaxis was significantly suppressed by apyrase. fMLP induced rat neutrophil chemotaxis was potentiated by ATP, blocked by suramin, not affected by PPADS or TNP-ATP, and significantly inhibited by RB-2. Western blotting showed that A(3), P2Y(2), and P2Y(11) were expressed in rat neutrophils. The chemotactic response of rat neutrophils to fMLP stimulation is potentiated by ATP via P2Y(11) purinoceptors but not P2X purinoceptors or A(3) adenosine receptor, and that the response plays a critical role in host defense and pathogenicity.

P2Y12 platelet inhibition in clinical practice.

Platelet adhesion, activation and aggregation play a pivotal role in atherothrombosis. Intracoronary atherothrombosis is the most common cause of the development of acute coronary syndrome (ACS), and plays a central role in complications occurring around percutaneous coronary intervention (PCI) including recurrent ACS, procedure-related myocardial infarction or stent thrombosis. Inhibition of platelet aggregation by medical treatment impairs formation and progression of thrombotic processes and is therefore of great importance in the prevention of complications after an ACS or around PCI. An essential part in the platelet activation process is the interaction of adenosine diphosphate (ADP) with the platelet P2Y12 receptor. The P2Y12 receptor is the predominant receptor involved in the ADP-stimulated activation of the glycoprotein IIb/IIIa receptor. Activation of the glycoprotein IIb/IIIa receptor results in enhanced platelet degranulation and thromboxane production, and prolonged platelet aggregation. The objectives of this review are to discuss the pharmacological limitations of the P2Y12 inhibitor clopidogrel, and describe the novel alternative P2Y12 inhibitors prasugrel and ticagrelor and the clinical implications of the introduction of these new medicines.

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions.

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases.

Extracellular Ca(2+) modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation.

ADP is considered a weak platelet agonist due to the limited aggregation responses it induces in vitro at physiological concentrations of extracellular Ca(2+) [(Ca(2+) )(o) ]. Lowering [Ca(2+) ](o) paradoxically enhances ADP-evoked aggregation, an effect that has been attributed to enhanced thromboxane A(2) production. This study examined the role of ectonucleotidases in the [Ca(2+) ](o) -dependence of platelet activation. Reducing [Ca(2+) ](o) from millimolar to micromolar levels converted ADP (10 µmol/l)-evoked platelet aggregation from a transient to a sustained response in both platelet-rich plasma and washed suspensions. Blocking thromboxane A(2) production with aspirin had no effect on this [Ca(2+) ](o) -dependence. Prevention of ADP degradation abolished the differences between low and physiological [Ca(2+) ](o) resulting in a robust and sustained aggregation in both conditions. Measurements of extracellular ADP revealed reduced degradation in both plasma and apyrase-containing saline at micromolar compared to millimolar [Ca(2+) ](o) . As reported previously, thromboxane A(2) generation was enhanced at low [Ca(2+) ](o) , however this was independent of ectonucleotidase activity(.) P2Y receptor antagonists cangrelor and MRS2179 demonstrated the necessity of P2Y(12) receptors for sustained ADP-evoked aggregation, with a minor role for P2Y(1) . In conclusion, Ca(2+) -dependent ectonucleotidase activity is a major factor determining the extent of platelet aggregation to ADP and must be controlled for in studies of P2Y receptor activation.