Reduced platelet activation and thrombus formation in male transgenic model mice of Alzheimer's disease suggests early sex-specific differences in platelet pathophysiology.

Alzheimer's disease (AD) is the most common form of dementia and characterized by extracellular amyloid-ß (Aß) plaques, intracellular neurofibrillary tau tangles and neurodegeneration. Over 80 % of AD patients also exhibit cerebral amyloid angiopathy (CAA). CAA is a cerebrovascular disease caused by deposition of Aß in the walls of cerebral blood vessels leading to vessel damage and impairment of normal blood flow. To date, different studies suggest that platelet function, including activation, adhesion and aggregation, is altered in AD due to vascular Aß deposition. For example, the transgenic AD model mice APP23 mice that exhibit CAA and parenchymal Aß plaques, show pre-activated platelets in the blood circulation and increased platelet integrin activation leading to a pro-thrombotic phenotype in these mice late stages of AD. However, it is still an open question whether or not platelets exhibit changes in their activation profile before they are exposed to vascular Aß deposits. Therefore, the present study examined platelets from middle-aged transgenic APP23 mice at the age of 8-10 months. At this age, APP23 mice show amyloid plaques in the brain parenchyma but not in the vasculature. Our analyses show that these APP23 mice have unaltered platelet numbers and size, and unaltered surface expression of glycoproteins. However, the number of dense granules in transgenic platelets was increased while the release was unaltered. Male, but not female APP23 mice, exhibited reduced platelet activation after stimulation of the thrombin receptor PAR4 and decreased thrombus stability on collagen under flow conditions ex vivo compared to control mice. In an arterial thrombosis model in vivo, male APP23 mice showed attenuated occlusion of the injured artery compared to controls. These findings provide clear evidence for early changes in platelet activation and thrombus formation in male mice before development of overt CAA. Furthermore, reduced platelet activation and thrombus formation suggest sex-specific differences in platelet physiology in AD that has to be considered in future studies of platelets and their role in AD.

Timing of dense granule biogenesis in asexual malaria parasites.

Malaria is an important infectious disease that continues to claim hundreds of thousands of lives annually. The disease is caused by infection of host erythrocytes by apicomplexan parasites of the genus Plasmodium. The parasite contains three different apical organelles - micronemes, rhoptries and dense granules (DGs) - whose contents are secreted to mediate binding to and invasion of the host cell and the extensive remodelling of the host cell that occurs following invasion. Whereas the roles of micronemes and rhoptries in binding and invasion of the host erythrocyte have been studied in detail, the roles of DGs in Plasmodium parasites are poorly understood. They have been proposed to control host cell remodelling through regulated protein secretion after invasion, but many basic aspects of the biology of DGs remain unknown. Here we describe DG biogenesis timing for the first time, using RESA localization as a proxy for the timing of DG formation. We show that DG formation commences approximately 37 min prior to schizont egress, as measured by the recruitment of the DG marker RESA. Furthermore, using a bioinformatics approach, we aimed to predict additional cargo of the DGs and identified the J-dot protein HSP40 as a DG protein, further supporting the very early role of these organelles in the interaction of the parasite with the host cell.

Evaluation of human platelet granules by structured illumination laser fluorescence microscopy.

Many roles of human platelets in health and disease are linked to their ability to transport and secrete a variety of small molecules and proteins carried in dense (δ-) and α-granules. Determination of granule number and content is important for diagnosis of platelet disorders and for studies of platelet structure, function, and development. We have optimized methods for detection and localization of platelet proteins via antibody and lectin staining, imaging via structured illumination laser fluorescence microscopy (SIM), and three-dimension (3D) image analysis. The methods were validated via comparison with published studies based on electron microscopy and high-resolution fluorescence microscopy. The α-granule cargo proteins thrombospondin-1 (TSP1), osteonectin (SPARC), fibrinogen (FGN), and Von Willebrand factor (VWF) were localized within the granule lumen, as was the proteoglycan serglycin (SRGN). Colocalization analysis indicates that staining with fluorescently labeled wheat germ agglutinin (WGA) allows detection of α-granules as effectively as immunostaining for cargo proteins, with the advantage of not requiring antibodies. RAB27B was observed to be concentrated at dense granules, allowing them to be counted via visual scoring and object analysis. We present a workflow for counting dense and α-granules via object analysis of 3D SIM images of platelets stained for RAB27B and with WGA.Abbreviation: SIM: structured illumination microscopy; WGA: wheat germ agglutinin; FGN: fibrinogen; TSP1: thrombospondin 1; ER: endoplasmic reticulum.

Platelets support blood clotting, wound healing, and other essential processes. These functions rely on the ability of platelets to transport and release small molecules like serotonin carried in dense granules and a wide range of proteins carried in alpha granules. Several conditions have been linked to abnormalities in one or more of platelet granule number, content, structure, and function. These conditions can be difficult to diagnose because platelet granules are so small they cannot be consistently resolved by conventional light microscopy, while higher power electron microscopy is not widely accessible.The goal of this study was to develop a method for counting and examining platelet dense and alpha granules without the need of electron microscopy. Key to this was the discovery that alpha granules can be reliably stained with the plant lectin wheat germ agglutinin, which has the advantages of being a smaller and less expensive molecule than the antibodies commonly used to detect alpha granule cargo proteins. We also establish that dense granules can be detected with high specificity via antibody staining of the membrane-associated protein RAB27B. We used structured illumination laser fluorescence microscopy to obtain high-resolution images of stained platelets. These were assembled into 3D renders using image analysis software, which was used to validate a protocol for rapidly counting granules within individual platelets.Our method supports the relatively rapid, accurate, and cost-effective assessment of platelet granules. We have already shown that it can confirm dense granule deficiency, and we anticipate that this approach will also prove useful in diagnosing and studying alpha granule abnormalities.

Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation.

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

Identification of Ca-rich dense granules in human platelets using scanning transmission X-ray microscopy.

Whole-mount (WM) platelet preparation followed by transmission electron microscopy (TEM) observation is the standard method currently used to assess dense granule (DG) deficiency (DGD). However, due to the electron-density-based contrast mechanism in TEM, other granules such as α-granules might cause false DG detection. Here, scanning transmission X-ray microscopy (STXM) was used to identify DGs and minimize false DG detection of human platelets. STXM image stacks of human platelets were collected at the calcium (Ca) L2,3 absorption edge and then converted to optical density maps. Ca distribution maps, obtained by subtracting the optical density maps at the pre-edge region from those at the post-edge region, were used to identify DGs based on the Ca richness. DGs were successfully detected using this STXM method without false detection, based on Ca maps for four human platelets. Spectral analysis of granules in human platelets confirmed that DGs contain a richer Ca content than other granules. The Ca distribution maps facilitated more effective DG identification than TEM which might falsely detect DGs. Correct identification of DGs would be important to assess the status of platelets and DG-related diseases. Therefore, this STXM method is proposed as a promising approach for better DG identification and diagnosis, as a complementary tool to the current WM TEM approach.

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii.

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+ , and we show that this Ca2+ -mediated response is contingent on the function of calcium-dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+ signalling plays in the invasion biology of apicomplexan parasites.

Platelet Electron Microscopy: Utilizing LEAN Methodology to Optimize Laboratory Workflow.

BACKGROUND: Platelet electron microscopy (PEM) is the gold standard methodology for diagnosing storage pool disorder, defined as a paucity of delta granules, alpha granules, or both. PEM literature is limited with few published resources and without well-developed interlaboratory standardization for the preparation and examination of platelet samples. METHODS: Whole mount (WM) dense body (DB) counts for 300 pediatric cases were reviewed to determine whether counting fewer platelets could yield the same results. For 6 cases, DB average was determined on the day of WM preparation and on 2 consecutive days. Both WM and thin section (TS) preparations were examined for all cases. RESULTS: Employing LEAN methodology, an algorithm was developed to markedly decrease the number of platelets counted and still ensure accurate results. WMs decay with time; a statistically significant difference in DB counts was noted between day 0 and day 1 (p < .1). Twelve of 300 cases required both WM and TS preparations for a complete diagnosis. CONCLUSION: It is possible to maintain accuracy and decrease 100 platelet DB counts by >75%. WMs must be counted on the day they are prepared to avoid false paucity of DB secondary to sample decay. An accurate evaluation of platelet morphology requires both the WM and TS techniques.

The dense granule protein 8 (GRA8) is a component of the sub-pellicular cytoskeleton in Toxoplasma gondii.

After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8I may interact with both Toxoplasma ß-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.

Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395.

The release of ADP from platelet dense granules and its binding to platelet P2Y12 receptors is key to amplifying the initial hemostatic response and propagating thrombus formation. P2Y12 has thus emerged as a therapeutic target to safely and effectively prevent secondary thrombotic events in patients with acute coronary syndrome or a history of myocardial infarction. Pharmacological inhibition of P2Y12 receptors represents a useful approach to better understand the signaling mediated by these receptors and to elucidate the role of these receptors in a multitude of platelet hemostatic and thrombotic responses. The present work examined and compared the effects of four different P2Y12 inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function in a series of in vitro studies of platelet dense granule secretion and trafficking, calcium generation, and protein phosphorylation. Our results show that in platelets activated with the PAR-1 agonist TRAP-6 (thrombin receptor-activating peptide), inhibition of P2Y12 with the antagonist MRS2395, but not ticagrelor, PSB 0739 or AR-C 66096, potentiated human platelet dense granule trafficking to the plasma membrane and release into the extracellular space, cytosolic Ca2+ influx, and phosphorylation of GSK3ß-Ser9 through a PKC-dependent pathway. These results suggest that inhibition of P2Y12 with MRS2395 may act in concert with PAR-1 signaling and result in the aberrant release of ADP by platelet dense granules, thus reducing or counteracting the anticipated anti-platelet efficacy of this inhibitor.

Metastatic TFE3-overexpressing renal clear cell carcinoma with dense granules: a histological, immunohistochemical, and ultrastructural study.

This is a case report of a 46-year-old white male who presented with dyspnea. Thoracic and abdominal examinations showed a heterogeneously enhancing mass in the right kidney, multiple pulmonary nodules, and left pleural thickening with large pleural effusion. Pleura biopsy revealed a malignant neoplasm composed of cells with predominantly clear cytoplasm. Considering the large mass in the right kidney, clear cell renal cell carcinoma (RCC) was the main differential diagnosis. The diagnosis in this case was not definitive by histology alone since clear cell RCC markers such as RCC and AE1/AE3 were negative, and CD10 was only focally positive. Transcription factor E3 (TFE3) immunohistochemistry was positive, while the XP11.2 translocation testing was negative. Electron microscopy demonstrated that the tumor cells had abundant cytoplasmic glycogen and lipid, focal long microvilli lining rare lumina, and adjacent interdigitating cell membranes joining the neoplastic cells, indicating a diagnosis of renal clear cell carcinoma. In addition, numerous crystalline-like dense granules were identified in the cytoplasm of the neoplastic cells, which are reminiscent of those typically seen in alveolar soft part sarcoma and rarely described in XP11.2 translocation RCC. Overall, this renal tumor likely represents a variant of XP11.2 translocation RCC, overexpressing TFE3 with dense granules.

Hermansky-Pudlak syndrome: Report of two patients with updated genetic classification and management recommendations.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder caused by mutations in one of nine genes involved in the packaging and formation of specialized lysosomes, including melanosomes and platelet-dense granules. The cardinal features are pigmentary dilution, bleeding diathesis, and accumulation of ceroid-like material in reticuloendothelial cells. Pulmonary fibrosis induced by tissue damage is seen in the most severe forms, and one subtype is characterized by immunodeficiency. We describe two patients with HPS type 1 and review the updated gene-based classification, clinical features, and recommendations for evaluation and follow-up.

[The role of polyphosphates in the blood coagulation process and the inflammation progress].

Polyphosphates (polyP) are commonly found in prokaryotic and eukaryotic cells linear, highly anionic polymers, composed of a few to many hundreds of orthophosphate residues, linked by high-energy phosphoanhydride bonds. These polymers are stored in dense granules of platelets and secreted after their activation. The recently studies indicate that polyP are a potent procoagulant agent accelerating blood coagulation by activating the contact pathway and by promoting FV and FXI activation by thrombin, which in turn abrogates the anticoagulant function of tissue factor pathway inhibitor (TFPI). Furthermore polyP enhance fibrin clot structure, which increase its resistance to fibrinolysis. PolyP using different signaling pathways have pro-inflammatory effects by promoting bradykinin release, enhancing the pro-inflammatory activity of histones, and activating NF-κB.

Human platelets contain a pool of free zinc in dense granules.

Background: Activated platelets release procoagulant factors that include Ca2+ and Zn2+. Releasable Ca2+ stores have been identified in platelet dense granules and the dense tubular system, but similar stores of free Zn2+ have not been identified. Objectives: Guided by studies of platelet Ca2+, we employed minimally disruptive methods to identify and localize concentrated free Zn2+ in human platelets. Methods: Resting platelets from normal donors (NDs), patients with gray platelet syndrome (GPS) lacking α-granules, and patients with Hermansky-Pudlak syndrome (HPS) deficient in dense granules were loaded with cell-permeant fluorescent probes specific to free Ca2+ or Zn2+. Ion concentrations were detected in fixed cells as bright puncta via high-resolution confocal microscopy. Ions were also directly detected via transmission electron microscopy and energy dispersive X-ray analysis. Levels of total platelet Ca, Zn, and Mg were measured via inductively coupled plasma optical emission spectroscopy. Results: Fluorescent Zn2+ puncta counts were similar in ND and GPS platelets and markedly lower in HPS platelets, pointing to dense granules as likely reservoirs of free Zn2+. This localization was supported by direct detection of Ca2+, Zn2+, and Na+ in platelet dense granules via transmission electron microscopy and energy dispersive X-ray analysis. Measurements of total platelet Ca, Zn, and Mg via inductively coupled plasma optical emission spectroscopy indicated that free Zn2+ represents a small proportion of total platelet zinc, consistent with the strong affinity of Zn2+ for binding proteins, including several abundant in platelet α-granules. Conclusion: We conclude that normal human platelets contain a pool of free Zn2+ concentrated in dense granules that is available for secretion upon platelet activation and potentially contributes to hemostasis.

Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes.

SUMMARYAcidocalcisomes are organelles conserved during evolution and closely related to the so-called volutin granules of bacteria and archaea, to the acidocalcisome-like vacuoles of yeasts, and to the lysosome-related organelles of animal species. All these organelles have in common their acidity and high content of polyphosphate and calcium. They are characterized by a variety of functions from storage of phosphorus and calcium to roles in Ca2+ signaling, osmoregulation, blood coagulation, and inflammation. They interact with other organelles through membrane contact sites or by fusion, and have several enzymes, pumps, transporters, and channels.

Protists: Eukaryotic single-celled organisms and the functioning of their organelles.

Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes. These diverse organelles have lost the major, at one time, definitive components of the mitochondrion (tricarboxylic cycle enzymes and cytochromes), however they all contain the machinery for the assembly of Fe-S clusters, which is the single unifying feature they share. The plasticity of organelles, like the mitochondrion, is therefore evident from its ability to lose its identity as an aerobic energy generating powerhouse while retaining key ancestral functions common to both aerobes and anaerobes. It is interesting to note that the apicoplast, a non-photosynthetic plastid that is present in all apicomplexan protozoa, apart from Cryptosporidium and possibly the gregarines, is also the site of Fe-S cluster assembly proteins. It turns out that in Cryptosporidium proteins involved in Fe-S cluster biosynthesis are localized in the mitochondrial remnant organelle termed the mitosome. Hence, different organisms have solved the same problem of packaging a life-requiring set of reactions in different ways, using different ancestral organelles, discarding what is not needed and keeping what is essential. Don't judge an organelle by its cover, more by the things it does, and always be prepared for surprises.

Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma.

Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) - an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

Recombinant Dense Granule Protein (NcGRA4) Is a Novel Serological Marker for Neospora caninum Infection in Goats.

Neospora caninum is widely recognised as one of the most significant causes of abortion in cattle, with infections also occurring in sheep and goats. To prevent and control animal neosporosis, it is crucial to develop sensitive and specific methods for detecting N. caninum infection. Recently, several recombinant proteins have been utilised in serological assays for the diagnosis of neosporosis. In this study, we used commercial gene synthesis to produce dense granular antigen 4 (NcGRA4) recombinant protein. NcGRA4 plasmids were expressed in the Escherichia coli system and then purified. The purified recombinant protein was analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To evaluate the diagnostic potential of recombinant NcGRA4 protein, we tested 214 serum samples from goat farms via indirect enzyme-linked immunosorbent assay (iELISA) and compared the results to those from the indirect fluorescent antibody test (IFAT). Western blotting analysis revealed a single NcGRA4 band with an expected molecular weight of 32 kDa. The specific IgG against N. caninum was detected in 34.1% and 35% of samples evaluated by NcGRA4 iELISA and IFAT, respectively. The sensitivity and specificity of the NcGRA4 iELISA were 71.6% and 86.3%, respectively, when compared with the results from IFAT. Our results demonstrate that a recombinant protein that can be used to detect animal neosporosis can be produced using a synthetic NcGRA4 gene. Overall, recombinant NcGRA4 shows promise as a sensitive and specific serological marker for identifying target IgG in goat samples.

Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell.

Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively remodels intestinal epithelial cells, building an elaborate interface structure. How this occurs at the molecular level and the contributing parasite factors are largely unknown. Here, we generated a whole-cell spatial proteome of the Cryptosporidium sporozoite and used genetic and cell biological experimentation to discover the Cryptosporidium-secreted effector proteome. These findings reveal multiple organelles, including an original secretory organelle, and generate numerous compartment markers by tagging native gene loci. We show that secreted proteins are delivered to the parasite-host interface, where they assemble into different structures including a ring that anchors the parasite into its unique epicellular niche. Cryptosporidium thus uses a complex set of secretion systems during and following invasion that act in concert to subjugate its host cell.

ß-N-methylamino-l-alanine is a non-competitive inhibitor of vesicular monoamine transporter 2.

The neurotoxic, non-proteinogenic amino acid ß-N-methylamino-l-alanine (BMAA) has been implicated in the development of neurodegenerative diseases; however, the mechanism(s) and mode(s) of toxicity remain unclear. Similarities in the neuropathology and behavioural deficits of neonatal rats exposed to either BMAA or reserpine, a known vesicular monoamine transporter 2 (VMAT2) inhibitor, suggest a similar mode of action. The aims of this study were therefore to determine if BMAA could prevent the uptake of serotonin into dense granules via inhibition of VMAT2, and, if so, the type of inhibition caused by BMAA. Exposing platelet dense granules to BMAA resulted in a concentration-dependent reduction in serotonin uptake. The inhibition of VMAT2 was non-competitive. The findings from this study support previous reports that BMAA-associated neuropathologies in a neonatal rat model may be due to VMAT2 inhibition during critical periods of neurogenesis.