Human platelets express the synaptic markers VGLUT1 and 2 and release glutamate following aggregation

doi:10.1016/j.neulet.2006.06.015

Neuroscience Letters

Volume 404, Issue 3, 1 September 2006, Pages 262-265

https://doi.org/10.1016/j.neulet.2006.06.015 Get rights and content

Abstract

Vesicular glutamate transporters (VGLUTs) are involved in storing glutamate for secretion at the level of glutamatergic axon terminals, and for this reason they have been extensively used as markers to identify glutamate-releasing cells. Platelets have been considered as a suitable model for studying glutamatergic dysfunction because they perform glutamate uptake and express both external transporters, and NMDA-like receptors. Here, we show that platelets express the pre-synaptic markers VGLUT1 and VGLUT2 and release glutamate following aggregation, implying a possible contributory role in the pathophysiology of stroke, migraine, and other excitotoxic disorders.

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Citation Excerpt :
Normal melanocytes do not secrete glutamate but melanoma cells do and glutamate is known to supports melanoma growth not only through the NMDAR but also through metabotropic glutamate receptors [40,41]. Glutamate levels are likely to be highest in advanced melanoma tumors, contributed by seeping plasma, activated platelets [42] and infiltrating white cells [43], which is akin to the wound fluid, where glutamate levels range from 300 to >1000 μM [44]. Half maximal effective concentrations (EC50) for glutamate binding to recombinant NMDARs range from ∼0.5 to 3 μM [6].
GRIN2A mutations are frequent in melanoma tumours but their role in disease is not well understood. GRIN2A encodes a modulatory subunit of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that certain GRIN2A mutations increase NMDAR function and support melanoma growth through oncogenic effects. This hypothesis was tested using 19 low-passage melanoma cell lines, four of which carried novel missense mutations in GRIN2A that we previously reported. We examined NMDAR expression, function of a calcium ion (Ca²⁺) channel and its contribution to cell growth using pharmacological modulators; findings were correlated with the presence or absence of GRIN2A mutations. We found that NMDAR expression was low in all melanoma cell lines, independent of GRIN2A mutations. In keeping with this, NMDAR-mediated Ca²⁺ influx and its contribution to cell proliferation were weak in most cell lines. However, certain GRIN2A mutations and culture media with lower glutamate levels enhanced NMDAR effects on cell growth and invasion. The main finding was that G762E was associated with higher glutamate-mediated Ca²⁺ influx and stronger NMDAR contribution to cell proliferation, compared with wild-type GRIN2A and other GRIN2A mutations. The pro-invasive phenotype of mutated cell lines was increased in culture medium containing less glutamate, implying environmental modulation of mutation effects.
In conclusion, NMDAR ion channel function is low in cultured melanoma cells but supports cell proliferation and invasion. Selected GRIN2A mutations, such as G762E, are associated with oncogenic consequences that can be modulated by extracellular glutamate. Primary cultures may be better suited to determine the role of the NMDAR in melanoma in vivo.
N-methyl-d-aspartate receptors amplify activation and aggregation of human platelets
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Citation Excerpt :
Glutamate is stored in platelet dense granules and released on platelet activation to high serum concentrations in excess of 400 μM [1–4].
Glutamate is stored in platelet dense granules and large amounts (> 400 μM) are released during thrombus formation. N-methyl-d-aspartate glutamate receptors (NMDARs) have been shown in platelets but their roles are unclear.
Platelet activation indices (CD62P expression and PAC-1 binding) and platelet aggregation were tested in the presence of well-characterized agonists (glutamate, NMDA, glycine) and antagonists (MK-801, memantine, AP5) of neuronal NMDARs. Expression of NMDAR subunits in platelets was determined.
NMDAR agonists facilitated and NMDAR antagonists inhibited platelet activation and aggregation. Low concentrations (100 μM) of MK-801 and memantine reduced adrenaline-induced CD62P expression by 47 ± 5 and 42 ± 3%, respectively, and inhibited adrenaline-induced platelet aggregation by 17 ± 6 and 25 ± 5%, respectively (P < 0.05). AP5 caused less inhibition of platelet function, requiring concentrations of at least 250 μM to inhibit aggregation. NMDAR agonists did not aggregate platelets by themselves but enhanced aggregation initiated by low concentrations of ADP. Exogenous glutamate helped reverse inhibition of platelet aggregation by riluzole (inhibitor of glutamate release). Compared with seven possible NMDAR subunits in neurons, human platelets contained four: GluN1, GluN2A, GluN2D and GluN3A, a combination rarely seen in neurons. The presence of NMDAR transcripts in platelets implied platelet ability to regulate NMDAR expression presumably ‘on demand’. Flow cytometry and electron microscopy demonstrated that in non-activated platelets, NMDAR subunits were contained inside platelets but relocated onto platelet blebs, filopodia and microparticles after platelet activation.
Our results support an active role for NMDARs in platelets, in a process that involves activation-dependent receptor relocation towards the platelet surface.
Homocysteine is a novel risk factor for suboptimal response of blood platelets to acetylsalicylic acid in coronary artery disease: A randomized multicenter study
2013, Pharmacological Research
Citation Excerpt :
Glutamate is commonly regarded as the main excitatory neurotransmitter in the central nervous system. However, it can be also detected in blood plasma and platelets, which are the richest cellular sources of this amino acid in a peripheral blood [27,70]. It is believed that glutamate secreted from blood platelets may act autocrinally as platelet co-agonist, as it has been evidenced in early phases after ischaemic stroke [71].
The incomplete inhibition of platelet function by acetylsalicylic acid (ASA), despite the patients are receiving therapeutic doses of the drug (‘aspirin-resistance’), is caused by numbers of risk factors. In this study we verified the idea that plasma homocysteine (Hcy) contributes to ‘aspirin-resistance’ in patients with coronary artery disease (CAD) and with or without type 2 diabetes mellitus (T2DM).
A cross-designed randomized controlled intervention study has been performed (126 CAD pts incl. 26 with T2DM) to determine whether increasing ASA dose from 75 mg to 150 mg daily may result in the increased antiplatelet effect, in the course of four-week treatment. Platelet response to collagen (coll) or arachidonic acid (AA) was monitored with whole blood aggregometry, plasma thromboxane (Tx), and Hcy levels were determined immunochemically.
The ASA-mediated reductions in platelet response to coll (by 12 ± 3%) or AA (by 10 ± 3%) and in plasma Tx (by 20 ± 9%; p < 0.02 or less) were significantly greater for higher ASA dose and significantly correlated with plasma Hcy, which was significantly lower in “good” ASA responders compared to “poor” responders (p < 0.001). Higher plasma Hcy appeared a significant risk factor for blood platelet refractoriness to low ASA dose (OR = 1.11; ±95%CI: 1.02–1.20, p < 0.02, adjusted to age, sex and CAD risk factors). Hcy diminished in vitro antiplatelet effect of low ASA concentration and augmented platelet aggregation (by up to 62% (p < 0.005) for coll and up to 15% (p < 0.005) for AA), whereas its acetyl derivative acted oppositely. Otherwise, Hcy intensified antiplatelet action of high ASA.
Hyperhomocysteinaemia may be a novel risk factor for the suppressed blood platelet response to ASA, and homocysteine may act as a specific sensitizer of blood platelets to some agonists. While homocysteine per se acts as a proaggregatory agent to blood platelets, its acetylated form is able to reverse this effect. Thus, these findings reveal a possibly new challenging potential of the acetylating properties of ASA therapy.
Glutamate release from platelets: Exocytosis versus glutamate transporter reversal
2013, International Journal of Biochemistry and Cell Biology
Platelets express neuronal and glial glutamate transporters EAAT 1–3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-β-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K⁺] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[¹⁴C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H⁺-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K⁺], dl-THA, FCCP and bafilomycin A1.
In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is rather ambiguous.
The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin
2011, Neurochemistry International
Citation Excerpt :
Recently, it has been found that platelets are able to accomplish glutamate uptake and express neuronal high-affinity Na+-dependent glutamate transporters EAAT 1-3 in the plasma membrane, which use Na+/K+ gradient as a driving force (Begni et al., 2005; Hoogland et al., 2005; Kasatkina and Borisova, 2010; Mangano and Schwarcz, 1981; Rainesalo et al., 2003). Subsequently, cytosolic glutamate is accumulated by dense secretory granules by special vesicular glutamate transporters VGLUT 1 and 2, which utilize the proton gradient for glutamate transport (Tremolizzo et al., 2006). Vesicular glutamate may be released by exocytosis during platelet activation.
Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na⁺-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15 mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD–cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H⁺-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.
Impaired Na<sup>+</sup>-dependent glutamate uptake in platelets during depolarization of their plasma membrane
2010, Neurochemistry International
Blood platelets contain neuronal Na⁺-dependent glutamate transporters and are able to accomplish glutamate uptake. Applying high-KCl, we have demonstrated dose-dependent depolarization of the plasma membrane of rabbit platelets that was registered as an increase in the fluorescence of the potential-sensitive fluorescent dye rhodamine 6G. The initial velocity of L-[¹⁴C]glutamate uptake (10 μM) in platelets was decreased by 20% during 35 mM KCl-evoked depolarization and consisted of 1.2 ± 0.09 pmol × min⁻¹ × mg⁻¹ protein in control and 0.96 ± 0.08 pmol × min⁻¹ × mg⁻¹ protein during depolarization. Confocal laser scanning microscopy and flow cytometry revealed that these changes in glutamate uptake were not a result of platelet aggregation/activation. Also, addition of high-KCl did not change acidification of platelet secretory granules that was found with pH-sensitive fluorescent dye acridine orange, thereby showing that changes in their proton gradient could not cause glutamate uptake alteration. This malfunction of glutamate transporters has to take place under: (i) the conditions of pseudohyperkalemia or hyperkalemia, i.e. activation and clotting of platelets, haemolysis, leucocytosis, acute renal failure, hypofunction of adrenal cortex, lack of aldosterone, stroke, trauma and (ii) depolarization of the plasma membrane of platelets during their activation by ADP, thrombin, platelet-activating factor. Weak glutamate uptake might have considerable consequences for platelets per se (and thus for haemostatic system) and glutamate homeostasis in the CNS.

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¹: These authors contributed equally to the work.

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Human platelets express the synaptic markers VGLUT1 and 2 and release glutamate following aggregation

Abstract

Neurosci. Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Brain Res.

J. Neuroimmunol.

Blood

Neurobiol. Aging

Increased plasma glutamate in stroke patients may be linked to altered platelet release and uptake

J. Cereb. Blood Flow. Metab.

Substrate modulation of glutamate uptake in human platelets

Br. J. Pharmacol.

Progression of ischaemic stroke and excitotoxic aminoacids

Lancet

Amino acid transmitters in patients with headache during the acute phase of cerebrovascular ischemic disease

Stroke

Platelet glycine, glutamate and aspartate in primary headache

Cephalalgia

Treatment of aura: solving the puzzle

Neurol. Sci.

Inhibition of glutamate release via recovery of ATP levels accounts for a neuroprotective effect of aspirin in rat cortical neurons exposed to oxygen-glucose deprivation

Stroke

Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats

J. Neurochem.