A case of acquired transient bleeding diathesis associated with acquired platelet storage pool deficiency and defective thromboxane A2 production.

Acquired disorders of platelet function are an underdiagnosed cause of bleeding tendency. A 14-year-old girl developed moderate mucocutaneous bleeding two weeks after a Mycoplasma pneumoniae infection successfully treated with clarithromycin. The patient was referred to us 7 months later for laboratory investigation of the persisting bleeding diathesis. The patient's personal and family histories were negative for bleeding disorders. Complete blood count, von Willebrand Factor levels and coagulation tests were normal; platelet aggregation, ATP secretion, δ-granules content and serum thromboxane B2 levels were defective. At follow-up visits, laboratory parameters and the bleeding diathesis progressively normalized within 2 years. The patient's condition is compatible with a diagnosis of acquired Storage Pool Deficiency (SPD), associated with defective thromboxane A2 production. To our knowledge, this is the first case of acquired, transient SPD with spontaneous remission. The pathogenic role of Mycoplasma pneumoniae infection or clarithromycin is possible, albeit uncertain.

Bioisosteric analogs of MDMA with improved pharmacological profile.

3,4-Methylenedioxymethamphetamine (MDMA, ' ecstasy' ) is re-emerging in clinical settings as a candidate for the treatment of specific psychiatric disorders (e.g. post-traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter-mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose-, individual-, and context-dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off-target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubation with isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin and dopamine transporters (hSERT and hDAT, respectively) but decreased activity at 5-HT 2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N -demethylation being the only metabolic route shared, and without forming phase II metabolites. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA analogs might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT and hDAT, but displaying a reduced activity at 5-HT 2A/2B/2C receptors and reduced hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

The teleost fish PepT1-type peptide transporters and their relationships with neutral and charged substrates.

In teleosts, two PepT1-type (Slc15a1) transporters, i.e., PepT1a and PepT1b, are expressed at the intestinal level. They translocate charged di/tripeptides with different efficiency, which depends on the position of the charged amino acid in the peptide and the external pH. The relation between the position of the charged amino acid and the capability of transporting the dipeptide was investigated in the zebrafish and Atlantic salmon PepT1-type transporters. Using selected charged (at physiological pH) dipeptides: i.e., the negatively charged Asp-Gly and Gly-Asp, and the positively charged Lys-Gly and Gly-Lys and Lys-Met and Met-Lys, transport currents and kinetic parameters were collected. The neutral dipeptide Gly-Gln was used as a reference substrate. Atlantic salmon PepT1a and PepT1b transport currents were similar in the presence of Asp-Gly and Gly-Asp, while zebrafish PepT1a elicited currents strongly dependent on the position of Asp in the dipeptide and zebrafish PepT1b elicited small transport currents. For Lys- and Met-containing dipeptides smaller currents compared to Gly-Gln were observed in PepT1a-type transporters. In general, for zebrafish PepT1a the currents elicited by all tested substrates slightly increased with membrane potential and pH. For Atlantic salmon PepT1a, the transport current increased with negative potential but only in the presence of Met-containing dipeptides and in a pH-dependent way. Conversely, large currents were shown for PepT1b for all tested substrates but Gly-Lys in Atlantic salmon. This shows that in Atlantic salmon PepT1b for Lys-containing substrates the position of the charged dipeptides carrying the Lys residue defines the current amplitudes, with larger currents observed for Lys in the N-terminal position. Our results add information on the ability of PepT1 to transport charged amino acids and show species-specificity in the kinetic behavior of PepT1-type proteins. They also suggest the importance of the proximity of the substrate binding site of residues such as LysPepT1a/GlnPepT1b for recognition and specificity of the charged dipeptide and point out the role of the comparative approach that exploits the natural protein variants to understand the structure and functions of membrane transporters.

Impairment of platelet function in both mild and severe COVID-19 patients.

Abnormalities of platelet function were reported in patients with severe COVID-19 (severe-C), but few data are available in patients with mild COVID-19 (mild-C) and after COVID-19 recovery. The aim of this study was to investigate platelet parameters in mild-C patients (n = 51), with no evidence of pneumonia, and severe-C patients (n = 49), during the acute phase and after recovery, compared to 43 healthy controls. Both mild-C and severe-C patients displayed increased circulating activated platelets, low δ-granule content (ADP, serotonin), impaired platelet activation by collagen (light transmission aggregometry) and impaired platelet thrombus formation on collagen-coated surfaces under controlled flow conditions (300/s shear rate). The observed abnormalities were more marked in severe-C patients than in mild-C patients. Overall, 61% (30/49) of mild-C and 73% (33/45) of severe-C patients displayed at least one abnormal platelet parameter. In a subgroup of just 13 patients who showed no persisting signs/symptoms of COVID-19 and were re-evaluated at least 1 month after recovery, 11 of the 13 subjects exhibited normalization of platelet parameters. In conclusion, mild abnormalities of platelet parameters were present not only in severe-C but also, albeit to a lesser extent, in mild-C patients during the acute phase of COVID-19 and normalized in most tested patients after clinical recovery.

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain.

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Its homeostasis is maintained by neuronal and glial GABA transporters (GATs). The four GATs identified in humans are GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and betaine/GABA transporter-1 BGT-1 (SLC6A12) which are all members of the solute carrier 6 (SLC6) family of sodium-dependent transporters. While GAT1 has been investigated extensively, the other GABA transporters are less studied and their role in CNS is not clearly defined. Altered GABAergic neurotransmission is involved in different diseases, but the importance of the different transporters remained understudied and limits drug targeting. In this review, the well-studied GABA transporter GAT1 is compared with the less-studied BGT-1 with the aim to leverage the knowledge on GAT1 to shed new light on the open questions concerning BGT-1. The most recent knowledge on transporter structure, functions, expression, and localization is discussed along with their specific role as drug targets for neurological and neurodegenerative disorders. We review and discuss data on the binding sites for Na+, Cl-, substrates, and inhibitors by building on the recent cryo-EM structure of GAT1 to highlight specific molecular determinants of transporter functions. The role of the two proteins in GABA homeostasis is investigated by looking at the transport coupling mechanism, as well as structural and kinetic transport models. Furthermore, we review information on selective inhibitors together with the pharmacophore hypothesis of transporter substrates.

Bile acid interactions with neurotransmitter transporters.

Synthesized in the liver from cholesterol, the bile acids (BAs) primary role is emulsifying fats to facilitate their absorption. BAs can cross the blood-brain barrier (BBB) and be synthesized in the brain. Recent evidence suggests a role for BAs in the gut-brain signaling by modulating the activity of various neuronal receptors and transporters, including the dopamine transporter (DAT). In this study, we investigated the effects of BAs and their relationship with substrates in three transporters of the solute carrier 6 family. The exposure to obeticholic acid (OCA), a semi-synthetic BA, elicits an inward current (IBA) in the DAT, the GABA transporter 1 (GAT1), and the glycine transporter 1 (GlyT1b); this current is proportional to the current generated by the substrate, respective to the transporter. Interestingly, a second consecutive OCA application to the transporter fails to elicit a response. The full displacement of BAs from the transporter occurs only after exposure to a saturating concentration of a substrate. In DAT, perfusion of secondary substrates norepinephrine (NE) and serotonin (5-HT) results in a second OCA current, decreased in amplitude and proportional to their affinity. Moreover, co-application of 5-HT or NE with OCA in DAT, and GABA with OCA in GAT1, did not alter the apparent affinity or the Imax, similar to what was previously reported in DAT in the presence of DA and OCA. The findings support the previous molecular model that suggested the ability of BAs to lock the transporter in an occluded conformation. The physiological significance is that it could possibly avoid the accumulation of small depolarizations in the cells expressing the neurotransmitter transporter. This achieves better transport efficiency in the presence of a saturating concentration of the neurotransmitter and enhances the action of the neurotransmitter on their receptors when they are present at reduced concentrations due to decreased availability of transporters.

Betaine-The dark knight of the brain.

The role of betaine in the liver and kidney has been well documented, even from the cellular and molecular point of view. Despite literature reporting positive effects of betaine supplementation in Alzheimer's, Parkinson's and schizophrenia, the role and function of betaine in the brain are little studied and reviewed. Beneficial effects of betaine in neurodegeneration, excitatory and inhibitory imbalance and against oxidative stress in the central nervous system (CNS) have been collected and analysed to understand the main role of betaine in the brain. There are many 'dark' aspects needed to complete the picture. The understanding of how this osmolyte is transported across neuron and glial cells is also controversial, as the expression levels and functioning of the known protein capable to transport betaine expressed in the brain, betaine-GABA transporter 1 (BGT-1), is itself not well clarified. The reported actions of betaine beyond BGT-1 related to neuronal degeneration and memory impairment are the focus of this work. With this review, we underline the scarcity of detailed molecular and cellular information about betaine action. Consequently, the requirement of detailed focus on and study of the interaction of this molecule with CNS components to sustain the therapeutic use of betaine.

The "www" of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research.

After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old methods for investigating the activity and the expression of Solute Carriers (SLC) are reviewed, and the kinds of experiment that are still useful to perform with this kind of cell are reported. Xenopus laevis oocytes at the full-grown stage have a highly efficient biosynthetic apparatus that correctly targets functional proteins at the defined compartment. This small protein factory can produce, fold, and localize almost any kind of wild-type or recombinant protein; some tricks are required to obtain high expression and to verify the functionality. The methodologies examined here are mainly related to research in the field of membrane transporters. This work is certainly not exhaustive; it has been carried out to be helpful to researchers who want to quickly find suggestions and detailed indications when investigating the functionality and expression of the different members of the solute carrier families.

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease.

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson's disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knock-in mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular re-localization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters.

The high-affinity/low-capacity system Slc15a2 (PepT2) is responsible for the reuptake of di/tripeptides from the renal proximal tubule, but it also operates in many other tissues and organs. Information regarding PepT2 in teleost fish is limited and, to date, functional data are available from the zebrafish (Danio rerio) only. Here, we report the identification of two slc15a2 genes in the Atlantic salmon (Salmo salar) genome, namely slc15a2a and slc15a2b. The two encoded PepT2 proteins share 87% identity and resemble both structurally and functionally the canonical vertebrate PepT2 system. The mRNA tissue distribution analyses reveal a widespread distribution of slc15a2a transcripts, being more abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and the distal part of the gastrointestinal tract. The function of the two transporters was investigated by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp recordings of transport and presteady-state currents. Both PepT2a and PepT2b in the presence of Gly-Gln elicit pH-dependent and Na+ independent inward currents. The biophysical and kinetic analysis of the recorded currents defined the transport properties, confirming that the two Atlantic salmon PepT2 proteins behave as high-affinity/low-capacity transporters. The recent structures and the previous kinetic schemes of rat and human PepT2 qualitatively account for the characteristics of the two Atlantic salmon proteins. This study is the first to report on the functional expression of two PepT2-type transporters that operate in the same vertebrate organism as a result of (a) gene duplication process(es). KEY POINTS: Two slc15a2-type genes, slc15a2a and slc15a2b coding for PepT2-type peptide transporters were found in the Atlantic salmon. slc15a2a transcripts, widely distributed in the fish tissues, are abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and distal gastrointestinal tract. Amino acids involved in vertebrate Slc15 transport function are conserved in PepT2a and PepT2b proteins. Detailed kinetic analysis indicates that both PepT2a and PepT2b operate as high-affinity transporters. The kinetic schemes and structures proposed for the mammalian models of PepT2 are suitable to explain the function of the two Atlantic salmon transporters.

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure-Function Relationships in Mammalian SLC6 Transporters.

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional-structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts.

Bile Acids Gate Dopamine Transporter Mediated Currents.

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764-25773; Cheng and Bahar, Structure, 2015, 23, 2171-2181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method.

Low-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these "non-responders" patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC-MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37-63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8-1222) for EC-ASA, and 823.1(624-1196) ng h/mL (median, 25-75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching.

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities.Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein.This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48-72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals.The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

TRPV4 and TRPM8 as putative targets for chronic low back pain alleviation.

The purpose of this study is to investigate the presence of nervous fibers and expression of TRP channels in samples harvested during decompressive/fusion spine surgeries from patients affected by chronic low back pain (CLBP). The aim was to understand if members of this family of receptors played a role in detection and processing of painful stimuli, to eventually define them as potential targets for CLBP alleviation. Expression of transient receptor potential (TRP) channels (A1, V1, V2, V4, and M8) was evaluated in samples from different periarticular sites of 6 patients affected by CLBP, at both protein and transcript levels. The capsular connective pathological tissue appeared infiltrated by sensitive unmyelinated nervous fibers. An increase in TRP channel mRNAs and proteins was observed in the pathological capsule compared with tissues collected from the non-symptomatic area in five of the six analyzed patients, independently by the location and number of affected sites. In particular, TRPV4 and TRPM8 were consistently upregulated in pathological tissues. Interestingly, the only patient showing a different pattern of expression also had a different clinical history. TRPV4 and TRPM8 channels may play a role in CLBP and warrant further investigations as possible therapeutic targets.

Patients with Essential Thrombocythemia may be Poor Responders to Enteric-Coated Aspirin, but not to Plain Aspirin.

Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10-1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

Correction to: The peptide transporter 1a of the zebrafish Danio rerio, an emerging model in nutrigenomics and nutrition research: molecular characterization, functional properties, and expression analysis.

[This corrects the article DOI: 10.1186/s12263-019-0657-3.].