BPA Exposure Affects Mouse Gastruloids Axial Elongation by Perturbing the Wnt/ß-Catenin Pathway.

Mammalian embryos are very vulnerable to environmental toxicants (ETs) exposure. Bisphenol A (BPA), one of the most diffused ETs, exerts endocrine-disrupting effects through estro-gen-mimicking and hormone-like properties, with detrimental health effects, including on reproduction. However, its impact during the peri-implantation stages is still unclear. This study, using gastruloids as a 3D stem cell-based in vitro model of embryonic development, showed that BPA exposure arrests their axial elongation when present during the Wnt/ß-catenin pathway activation period by ß-catenin protein reduction. Gastruloid reshaping might have been impeded by the downregulation of Snail, Slug and Twist, known to suppress E-cadherin expression and to activate the N-cadherin gene, and by the low expression of the N-cadherin protein. Also, the lack of gastruloids elongation might be related to altered exit of BPA-exposed cells from the pluripotency condition and their following differentiation. In conclusion, here we show that the inhibition of gastruloids' axial elongation by BPA might be the result of the concomitant Wnt/ß-catenin perturbation, reduced N-cadherin expression and Oct4, T/Bra and Cdx2 altered patter expression, which all together concur in the impaired development of mouse gastruloids.

Platelet P2Y12 signalling pathway in the dysregulated immune response during sepsis.

Sepsis is a complicated pathological condition in response to severe infection. It is characterized by a strong systemic inflammatory response, where multiple components of the immune system are involved. Currently, there is no treatment for sepsis. Blood platelets are known for their role in haemostasis, but they also participate in inflammation through cell-cell interaction and the secretion of inflammatory mediators. Interestingly, an increase in platelet activation, secretion, and aggregation with other immune cells (such as monocytes, T-lymphocytes and neutrophils) has been detected in septic patients. Therefore, antiplatelet therapy in terms of P2Y12 antagonists has been evaluated as a possible treatment for sepis. It was found that blocking P2Y12 receptors decreased platelet marker expression and limited attachment to immune cells in some studies, but not in others. This review addresses the role of platelets in sepsis and discusses whether antagonizing P2Y12 signalling pathways can alter the disease outcome. Challenges in studying P2Y12 antagonists in sepsis also are discussed. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

The C-Type Lectin Receptor CD93 Regulates Platelet Activation and Surface Expression of the Protease Activated Receptor 4.

BACKGROUND: The C-type lectin receptor CD93 is a single pass type I transmembrane glycoprotein involved in inflammation, immunity, and angiogenesis. This study investigates the role of CD93 in platelet function. CD93 knockout (KO) mice and wild-type (WT) controls were compared in this study. METHODS: Platelet activation and aggregation were investigated by flow cytometry and light transmission aggregometry, respectively. Protein expression and phosphorylation were analyzed by immunoblotting. Subcellular localization of membrane receptors was investigated by wide-field and confocal microscopy. RESULTS: The lack of CD93 in mice was not associated to any evident bleeding defect and no alterations of platelet activation were observed upon stimulation with thromboxane A2 analogue and convulxin. Conversely, platelet aggregation induced by stimulation of the thrombin receptor PAR4 was significantly reduced in the absence of CD93. This defect was associated with a significant reduction of α-granule secretion, integrin αIIbß3 activation, and protein kinase C (PKC) stimulation. Resting WT and CD93-deficient platelets expressed comparable amounts of PAR4. However, upon stimulation with a PAR4 activating peptide, a more pronounced clearance of PAR4 from the platelet surface was observed in CD93-deficient platelets compared with WT controls. Confocal microscopy analysis revealed a massive movement of PAR4 in cytosolic compartments of activated platelets lacking CD93. Accordingly, platelet desensitization following PAR4 stimulation was more pronounced in CD93 KO platelets compared with WT controls. CONCLUSION: These results demonstrate that CD93 supports platelet activation triggered by PAR4 stimulation and is required to stabilize the expression of the thrombin receptor on the cell surface.

Human platelets release amyloid peptides ß1-40 and ß1-42 in response to haemostatic, immune, and hypoxic stimuli.

Background: Platelets contain high levels of amyloid ß (Aß) peptides and have been suggested to participate in the deposition of amyloid plaques in Alzheimer's Disease (AD). Objectives: This study aimed to determine whether human platelets release pathogenic Aß peptides Aß1-42 and Aß1-40 and to characterise the mechanisms regulating this phenomenon. Methods and Results: Enzyme-linked immunosorbent assays (ELISAs) revealed that the haemostatic stimulus thrombin and the pro-inflammatory molecule lipopolysaccharide (LPS) induce platelet release of both Aß1-42 and Aß1-40. Notably, LPS preferentially induced the release of Aß1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective ß secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aß1-40 or Aß1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aß peptides with platelet alpha granules. Conclusions: Taken together, our data suggest that human platelets release pathogenic Aß peptides as a result of a store-and-release mechanism rather than a de novo proteolytic event. Although further studies are required to fully characterise this phenomenon, we suggest the possibility of a role for platelets in the deposition of Aß peptides and the formation of amyloid plaques. Interestingly, the combination of hypoxia and inflammation that we simulated in vitro with reduced oxygen tension and LPS may increase the release of fibrillogenic Aß1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of AD patients.

Proteomic and functional profiling of platelet-derived extracellular vesicles released under physiological or tumor-associated conditions.

During hemostasis, thrombosis, and inflammation, activated blood platelets release extracellular vesicles (PEVs) that represent biological mediators of physiological and pathological processes. We have recently demonstrated that the activation of platelets by breast cancer cells is accompanied by a massive release of PEVs, evidence that matches with the observation that breast cancer patients display increased levels of circulating PEVs. A core concept in PEVs biology is that their nature, composition and biological function are strongly influenced by the conditions that induced their release. In this study we have performed a comparative characterization of PEVs released by platelets upon activation with thrombin, a potent thrombotic stimulus, and upon exposure to the breast cancer cell line MDA-MB-231. By nanoparticle tracking analysis and tandem mass spectrometry we have characterized the two populations of PEVs, showing that the thrombotic and tumoral stimuli produced vesicles that largely differ in protein composition. The bioinformatic analysis of the proteomic data led to the identification of signaling pathways that can be differently affected by the two PEVs population in target cells. Specifically, we have demonstrated that both thrombin- and cancer-cell-induced PEVs reduce the migration and potentiate Ca2+-induced apoptosis of Jurkat cells, but only thrombin-derived PEVs also potentiate cell necrosis. Our results demonstrate that stimulation of platelets by thrombotic or tumoral stimuli induces the release of PEVs with different protein composition that, in turn, may elicit selective biological responses in target cells.

Platelet-Derived Extracellular Vesicles Stimulate Migration through Partial Remodelling of the Ca2+ Handling Machinery in MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Platelets can support cancer progression via the release of microparticles and microvesicles that enhance the migratory behaviour of recipient cancer cells. We recently showed that platelet-derived extracellular vesicles (PEVs) stimulate migration and invasiveness in highly metastatic MDA-MB-231 cells by stimulating the phosphorylation of p38 MAPK and the myosin light chain 2 (MLC2). Herein, we assessed whether the pro-migratory effect of PEVs involves the remodelling of the Ca2+ handling machinery, which drives MDA-MB-231 cell motility. METHODS: PEVs were isolated from human blood platelets, and Fura-2/AM Ca2+ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca2+ dynamics and Ca2+-dependent migratory processes in MDA-MB-231 cells. RESULTS: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca2+ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP3R1/InsP3R2 mRNAs and proteins. The consequent enhancement of ER Ca2+ depletion led to a significant increase in store-operated Ca2+ entry. The larger Ca2+ mobilization from the ER was required to potentiate serum-induced migration by recruiting p38 MAPK and MLC2. CONCLUSIONS: PEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca2+ handling machinery.

The Controversial Role of LPS in Platelet Activation In Vitro.

Circulating platelets are responsible for hemostasis and thrombosis but are also primary sensors of pathogens and are involved in innate immunity, inflammation, and sepsis. Sepsis is commonly caused by an exaggerated immune response to bacterial, viral, and fungal infections, and leads to severe thrombotic complications. Among others, the endotoxin lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria is the most common trigger of sepsis. Since the discovery of the expression of the LPS receptor TLR4 in platelets, several studies have investigated the ability of LPS to induce platelet activation and to contribute to a prothrombotic phenotype, per se or in combination with plasma proteins and platelet agonists. This issue, however, is still controversial, as different sources, purity, and concentrations of LPS, different platelet-purification protocols, and different methods of analysis have been used in the past two decades, giving contradictory results. This review summarizes and critically analyzes past and recent publications about LPS-induced platelet activation in vitro. A methodological section illustrates the principal platelet preparation protocols and significant differences. The ability of various sources of LPS to elicit platelet activation in terms of aggregation, granule secretion, cytokine release, ROS production, and interaction with leukocytes and NET formation is discussed.

Proline-rich tyrosine kinase Pyk2 regulates deep vein thrombosis.

Deep vein thrombosis results from the cooperative action of leukocytes, platelets, and endothelial cells. The proline-rich tyrosine kinase Pyk2 regulates platelet activation and supports arterial thrombosis. In this study, we combined pharmacological and genetic approaches to unravel the role of Pyk2 in venous thrombosis. We found that mice lacking Pyk2 almost completely failed to develop deep venous thrombi upon partial ligation of the inferior vena cava. Pyk2-deficient platelets displayed impaired exposure of phosphatidylserine and tissue factor expression by endothelial cells and monocytes was completely prevented by inhibition of Pyk2. In human umbilical vein endothelial cells (HUVEC), inhibition of Pyk2 hampered IL-1b-induced expression of VCAM and P-selectin, and von Willebrand factor release. Pyk2-deficient platelets showed defective adhesion on von Willebrand factor and reduced ability to bind activated HUVEC under flow. Moreover, inhibition of Pyk2 in HUVEC strongly reduced platelet adhesion. Similarly, Pyk2-deficient neutrophils were unable to efficiently roll and adhere to immobilized endothelial cells under venous flow conditions. Moreover, platelets and neutrophils from Pyk2- knockout mice showed defective ability to form heterogeneous aggregates upon stimulation, while platelet monocyte interaction occurred normally. Consequently, platelet neutrophil aggregates, abundant in blood of wild-type mice upon inferior vena cava ligation, were virtually undetectable in Pyk2-knockout mice. Finally, we found that expression of Pyk2 was required for NETosis induced by activated platelets. Altogether our results demonstrate a critical role of Pyk2 in the regulation of the coordinated thromboinflammatory responses of endothelial cells, leukocytes and platelets leading to venous thrombosis. Pyk2 may represent a novel promising target in the treatment of deep vein thrombosis.

Pin1 as Molecular Switch in Vascular Endothelium: Notes on Its Putative Role in Age-Associated Vascular Diseases.

By controlling the change of the backbones of several cellular substrates, the peptidyl-prolyl cis-trans isomerase Pin1 acts as key fine-tuner and amplifier of multiple signaling pathways, thereby inducing several biological consequences, both in physiological and pathological conditions. Data from the literature indicate a prominent role of Pin1 in the regulating of vascular homeostasis. In this review, we will critically dissect Pin1's role as conformational switch regulating the homeostasis of vascular endothelium, by specifically modulating nitric oxide (NO) bioavailability. In this regard, Pin1 has been reported to directly control NO production by interacting with bovine endothelial nitric oxide synthase (eNOS) at Ser116-Pro117 (human equivalent is Ser114-Pro115) in a phosphorylation-dependent manner, regulating its catalytic activity, as well as by regulating other intracellular players, such as VEGF and TGF-ß, thereby impinging upon NO release. Furthermore, since Pin1 has been found to act as a critical driver of vascular cell proliferation, apoptosis, and inflammation, with implication in many vascular diseases (e.g., diabetes, atherosclerosis, hypertension, and cardiac hypertrophy), evidence indicating that Pin1 may serve a pivotal role in vascular endothelium will be discussed. Understanding the role of Pin1 in vascular homeostasis is crucial in terms of finding a new possible therapeutic player and target in vascular pathologies, including those affecting the elderly (such as small and large vessel diseases and vascular dementia) or those promoting the full expression of neurodegenerative dementing diseases.

HA and HS Changes in Endothelial Inflammatory Activation.

Cardiovascular diseases are a group of disorders caused by the presence of a combination of risk factors, such as tobacco use, unhealthy diet and obesity, physical inactivity, etc., which cause the modification of the composition of the vessel's matrix and lead to the alteration of blood flow, matched with an inflammation condition. Nevertheless, it is not clear if the inflammation is a permissive condition or a consequent one. In order to investigate the effect of inflammation on the onset of vascular disease, we treated endothelial cells with the cytokine TNF-α that is increased in obese patients and is reported to induce cardiometabolic diseases. The inflammation induced a large change in the extracellular matrix, increasing the pericellular hyaluronan and altering the heparan sulfate Syndecans sets, which seems to be related to layer permeability but does not influence cell proliferation or migration nor induce blood cell recruitment or activation.

Platelet-derived extracellular vesicles regulate cell cycle progression and cell migration in breast cancer cells.

Platelets have been extensively implicated in the progression of cancer and platelet-derived extracellular vesicles (PEVs) are gaining growing attention as potential mediators of the platelet-cancer interplay. PEVs are shed from platelet membrane in response to extracellular stimuli and carry important biological signals for intercellular communication. In this study we demonstrate that PEVs specifically bind to different breast cancer cells and elicit cell-specific functional responses. PEVs were massively internalized by the metastatic cell lines MDA-MB-231 and SKBR3 and the ductal carcinoma cell line BT474, but not by the MCF-7 cell line. In SKBR3 cells, PEVs decreased mitochondrial dehydrogenase activities and altered cell cycle progression without affecting cell viability. Conversely, PEVs potently stimulated migration and invasion of MDA-MB-231, without affecting the distribution in the different phases of the cell cycle. In all the analyzed breast cancer cells, PEVs triggered a sustained increase of intracellular Ca2+, but only in MDA-MB-231 cells, this was associated to the stimulation of selected signaling proteins implicated in migration, including p38MAPK and myosin light chain. Importantly, inhibition of myosin light chain phosphorylation by a Rho kinase inhibitor prevented PEVs-stimulated migration of MDA-MB-231 cells. Our results demonstrate that PEVs are versatile regulators of cancer cell behavior and elicit a variety of different responses depending on the specific breast cancer cell subtype.

Fibrillar amyloid peptides promote platelet aggregation through the coordinated action of ITAM- and ROS-dependent pathways.

BACKGROUND: Amyloid peptides Aß40 and Aß42, whose deposition in brain correlates with Alzheimer disease, are also present in platelets and have prothrombotic activities. OBJECTIVE: In this study, we analyze the ability of Aß peptides to form fibrils and to induce platelet activation and aggregation. METHODS: Aß40, Aß42, and their scrambled peptides were diluted in phosphate buffered saline and fibrillogenesis was investigated by ThioflavinT and Congo Red. Aggregation, protein phosphorylation, and reactive oxygen species (ROS) production were analyzed. RESULTS: Aß40 and Aß42, but not scrambled peptides, were able to form fibrils when diluted in phosphate buffered saline. Fibrillogenesis of Aß42 was very rapid, whereas fibril formation by Aß40 was completed only after 48 hours of incubation. Fibrillar Aß40 and Aß42 promoted dose-dependent aggregation of washed platelets in the presence of extracellular CaCl2 . Cleavage of GPIbα by mocarhagin or blockade of the ITAM-containing FcγRIIA prevented platelet aggregation induced by fibrillary Aß40 and Aß42. Fibrillar Aß peptides stimulated the phosphorylation of FcγRIIA, resulting in the downstream stimulation of PLC, protein kinase C, and phosphoinositide 3-kinases, whose activity was necessary for full aggregation of platelets. Fibrillar Aß peptides also induced ROS generation, and NOX inhibitors, as well as ROS scavengers, prevented platelet aggregation. However, Aß peptide-induced ROS production did not require binding to GPIbα or activation of FcγRIIA, but was initiated by CD36, which provided an important contribution to full platelet aggregation. CONCLUSION: These results suggest that fibrillar amyloid Aß40 and Aß42 induce platelet aggregation through the recruitment of GPIb-IX-V and CD36, which requires the convergence of ITAM- and ROS-dependent pathways.

The proline-rich tyrosine kinase Pyk2 modulates integrin-mediated neutrophil adhesion and reactive oxygen species generation.

Neutrophils are first responders in infection and inflammation. They are able to roll, adhere and transmigrate through the endothelium to reach the site of infection, where they fight pathogens through secretion of granule contents, production of reactive oxygen species, extrusion of neutrophil extracellular traps, and phagocytosis. In this study we explored the role of the non-receptor focal adhesion kinase Pyk2 in neutrophil adhesion and activation. Using a specific Pyk2 pharmacological inhibitor, PF-4594755, as well as Pyk2-deficient murine neutrophils, we found that Pyk2 is activated upon integrin αMß2-mediated neutrophil adhesion to fibrinogen. This process is triggered by Src family kinases-mediated phosphorylation and supported by Pyk2 autophosphorylation on Y402. In neutrophil adherent to fibrinogen, Pyk2 activates PI3K-dependent pathways promoting the phosphorylation of Akt and of its downstream effector GSK3. Pyk2 also dynamically regulates MAP kinases in fibrinogen-adherent neutrophils, as it stimulates p38MAPK but negatively regulates ERK1/2. Pharmacological inhibition of Pyk2 significantly prevented adhesion of human neutrophils to fibrinogen, and neutrophils from Pyk2-knockout mice showed a reduced ability to adhere compared to wildtype cells. Accordingly, neutrophil adhesion to fibrinogen was reduced upon inhibition of p38MAPK but potentiated by ERK1/2 inhibition. Neutrophil adherent to fibrinogen, but not to polylysine, were able to produce ROS upon lipopolysaccharide challenge and ROS production was completely suppressed upon inhibition of Pyk2. By contrast PMA-induced ROS production by neutrophil adherent to either fibrinogen or polylysine was independent from Pyk2. Altogether these results demonstrate that Pyk2 is an important effector in the coordinated puzzle regulating neutrophil adhesion and activation.

Stimulation of mTORC2 by integrin αIIbß3 is required for PI3Kß-dependent activation of Akt but is dispensable for platelet spreading on fibrinogen.

Phosphatidylinositol 3 kinase (PI3K) is a major player in platelet activation and regulates thrombus formation and stabilization. The ß isoform of PI3K is implicated in integrin αIIbß3 outside-in signaling, is required for the phosphorylation of Akt, and controls efficient platelet spreading upon adhesion to fibrinogen. In this study we found that during integrin αIIbß3 outside-in signaling PI3Kß-dependent phosphorylation of Akt on Serine473 is mediated by the mammalian target of rapamycin complex 2 (mTORC2). The activity of mTORC2 is stimulated upon platelet adhesion to fibrinogen, as documented by increased autophosphorylation. However, mTORC2 activation downstream of integrin αIIbß3 is PI3Kß-independent. Inhibition of mTORC2, but not mTORC1, also prevents Akt phosphorylation of Threonine308 and affects Akt activity, resulting in the inhibition of GSK3α/ß phosphorylation. Nevertheless, mTORC2 or Akt inhibition does not alter PI3Kß-dependent platelet spreading on fibrinogen. The activation of the small GTPase Rap1b downstream of integrin αIIbß3 is regulated by PI3Kß but is not affected upon inhibition of either mTORC2 or Akt. Altogether, these results demonstrate for the first time the activation of mTORC2 and its involvement in Akt phosphorylation and stimulation during integrin αIIbß3 outside-in signaling. Moreover, the results demonstrate that the mTORC2/Akt pathway is dispensable for PI3Kß-regulated platelet spreading on fibrinogen.

Biology and Role of Extracellular Vesicles (EVs) in the Pathogenesis of Thrombosis.

Extracellular vesicles (EVs) are well-established mediators of cell-to-cell communication. EVs can be released by every cell type and they can be classified into three major groups according to their biogenesis, dimension, density, and predominant protein markers: exosomes, microvesicles, and apoptotic bodies. During their formation, EVs associate with specific cargo from their parental cell that can include RNAs, free fatty acids, surface receptors, and proteins. The biological function of EVs is to maintain cellular and tissue homeostasis by transferring critical biological cargos to distal or neighboring recipient cells. On the other hand, their role in intercellular communication may also contribute to the pathogenesis of several diseases, including thrombosis. More recently, their physiological and biochemical properties have suggested their use as a therapeutic tool in tissue regeneration as well as a novel option for drug delivery. In this review, we will summarize the impact of EVs released from blood and vascular cells in arterial and venous thrombosis, describing the mechanisms by which EVs affect thrombosis and their potential clinical applications.

Amyloid Peptide ß1-42 Induces Integrin αIIbß3 Activation, Platelet Adhesion, and Thrombus Formation in a NADPH Oxidase-Dependent Manner.

The progression of Alzheimer's dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of ß amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide ß1-42 (Aß1-42), Aß1-40, and Aß25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aß1-42 compared to other Aß peptides. In addition, significant platelet spreading was observed over Aß1-42, while Aß1-40, Aß25-35, and the scAß1-42 control did not seem to induce any platelet spreading, which suggested that only Aß1-42 activates platelet signalling in our experimental conditions. Aß1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aß peptides did not. The molecular mechanism of Aß1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbß3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aß1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aß1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbß3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide ß1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer's patients.

Focal Adhesion Kinases in Platelet Function and Thrombosis.

The focal adhesion kinase family includes 2 homolog members, FAK and Pyk2 (proline-rich tyrosine kinase 2), primarily known for their roles in nucleated cells as regulators of cytoskeletal dynamics and cell adhesion. FAK and Pyk2 are also expressed in megakaryocytes and platelets and are activated by soluble agonists and on adhesion to the subendothelial matrix. Despite high sequence homology and similar molecular organization, FAK and Pyk2 play different roles in platelet function. Whereas FAK serves mostly as a traditional focal adhesion kinase activated downstream of integrins, Pyk2 coordinates multiple signals from different receptors. FAK, but not Pyk2, is involved in megakaryocyte maturation and platelet production. In circulating platelets, FAK is recruited by integrin αIIbß3 to regulate hemostasis, whereas it plays minimal roles in thrombosis. By contrast, Pyk2 is implicated in platelet activation and is an important regulator of thrombosis. The direct activation of Pyk2 by calcium ions provides a connection between GPCRs (G-protein coupled receptors) and Src family kinases. In this review, we provide the comprehensive overview of >20 years of investigations on the role and regulation of focal adhesion kinases in blood platelets, highlighting common and distinctive features of FAK and Pyk2 in hemostasis and thrombosis.

A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide ß.

Reactive oxygen species (ROS) generation is critical in the regulation of platelets, which has important implications in the modulation of hemostasis and thrombosis. Nonetheless, despite several assays have been described and successfully utilized in the past, the analysis of ROS generation in human platelets remains challenging. Here we show that dihydroethidium (DHE) allows the characterization of redox responses upon platelet activation by physiological and pathological stimuli. In particular, the flow cytometry assay that we describe here allowed us to confirm that thrombin, collagen-related peptide (CRP) and arachidonic acid but not adenosine diphosphate (ADP) stimulate superoxide anion formation in a concentration-dependent manner. 0.1unit/ml thrombin, 3 µg/ml CRP and 30 µM arachidonic acid are commonly used to stimulate platelets in vitro and here were shown to stimulate a significant increase in superoxide anion formation. The ROS scavenger N-acetylcysteine (NAC) abolished superoxide anion generation in response to all tested stimuli, but the pan-NADPH oxidase (NOX) inhibitor VAS2870 only inhibited superoxide anion formation in response to thrombin and CRP. The involvement of NOXs in thrombin and CRP-dependent responses was confirmed by the inhibition of platelet aggregation induced by these stimuli by VAS2870, while platelet aggregation in response to arachidonic acid was insensitive to this inhibitor. In addition, the pathological platelet stimulus amyloid ß (Aß) 1-42 peptide induced superoxide anion formation in a concentration-dependent manner. Aß peptide stimulated superoxide anion formation in a NOX-dependent manner, as proved by the use of VAS2870. Aß 1-42 peptide displayed only moderate activity as an aggregation stimulus, but was able to significantly potentiate platelet aggregation in response to submaximal agonists concentrations, such as 0.03 unit/ml thrombin and 10 µM arachidonic acid. The inhibition of NOXs by 10 µM VAS2870 abolished Aß-dependent potentiation of platelet aggregation in response to 10 µM arachidonic acid, suggesting that the pro-thrombotic activity of Aß peptides depends on NOX activity. Similar experiments could not be performed with thrombin or collagen, as NOXs are required for the signaling induced by these stimuli. These findings shed some new light on the pro-thrombotic activity of Aß peptides. In summary, here we describe a novel and reliable assay for the detection of superoxide anion in human platelets. This is particularly important for the investigation of the pathophysiological role of redox stress in platelets, a field of research of increasing importance, but hindered by the absence of a reliable and easily accessible ROS detection methodology applicable to platelets.

Amyloid precursor protein is required for in vitro platelet adhesion to amyloid peptides and potentiation of thrombus formation.

Amyloid precursor protein (APP) is the precursor of amyloid ß (Aß) peptides, whose accumulation in the brain is associated with Alzheimer's disease. APP is also expressed on the platelet surface and Aß peptides are platelet agonists. The physiological role of APP is largely unknown. In neurons, APP acts as an adhesive receptor, facilitating integrin-mediated cell adhesion, while in platelets it regulates coagulation and venous thrombosis. In this work, we analyzed platelets from APP KO mice to investigate whether membrane APP supports platelet adhesion to physiological and pathological substrates. We found that APP-null platelets adhered and spread normally on collagen, von Willebrand Factor or fibrinogen. However, adhesion on immobilized Aß peptides Aß1-40, Aß1-42 and Aß25-35 was completely abolished in platelets lacking APP. By contrast, platelet activation and aggregation induced by Aß peptides occurred normally in the absence of APP. Adhesion of APP-transfected HEK293 to Aß peptides was significantly higher than that of control cells expressing low levels of APP. Co-coating of Aß1-42 and Aß25-35 with collagen strongly potentiated platelet adhesion when whole blood from wild type mice was perfused at arterial shear rate, but had no effects with blood from APP KO mice. These results demonstrate that APP selectively mediates platelet adhesion to Aß under static condition but not platelet aggregation, and is responsible for Aß-promoted potentiation of thrombus formation under flow. Therefore, APP may facilitate an early step in thrombus formation when Aß peptides accumulate in cerebral vessel walls or atherosclerotic plaques.