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Background: Platelets contain high levels of amyloid β (Aβ) 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 Aβ peptides Aβ 1-42 and Aβ 1-40 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 Aβ 1-42 and Aβ 1-40 . Notably, LPS preferentially induced the release of Aβ1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective β secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aβ 1-40 or Aβ 1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aβ peptides with platelet alpha granules., Conclusions: Taken together, our data suggest that human platelets release pathogenic Aβ 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 Aβ 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 Aβ 1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of AD patients., Competing Interests: Conflict statement: the authors have no conflict of interest to disclose.

Aims: P-selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N-terminal domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet-leukocyte aggregates., Methods and Results: Using a library of synthetic peptides, we mapped the platelet-binding site to a continuous 20 amino acid stretch. The peptide Efb 68-87 was able to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Dot blots, pull-down assays and P-selectin glycoprotein ligand-1 (PSGL-1) competitive binding experiments identified P-selectin as the cellular docking site mediating Efb 68-87 platelet binding. Accordingly, Efb 68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb 68-87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet-leukocyte aggregates (PLAs). Efb 68-87 also interfered with the platelet-dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro., Conclusions: We have identified Efb 68-87 as a novel selective platelet-binding peptide. Efb 68-87 binds directly to P-selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb 68-87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Secretion of PDI from platelets and endothelial cells is an important step of all thrombotic events. In the absence of extracellular PDI thrombus formation and fibrin generation may be impaired. Thrombin-mediated PDI secretion is regulated by the stimulation of P2Y 12 receptors. This paper provides evidences that P2Y 12 antagonists or AR agonists may modulate release of PDI molecules from platelets and with less efficiency from endothelial cells. Moreover P2Y 12 antagonization or AR agonization modulates platelet-endothelial interaction. We prove that combinations of P2Y 12 antagonists and AR agonists inhibit platelet-dependent adhesion of cancer cells to endothelium and attenuate cancer cell invasiveness, but longer exposition to AR agonists may stimulate migration of invasive breast cancer cells through endothelium thus leading to increased metastasis., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Thrombus formation in vitro in flow conditions and its visualization and quantification with the use of microscopy are widely utilized to evaluate activity of compounds with a potential antithrombotic activity. Visualization and quantification of thrombi can be performed with the use of wide-field or confocal microscopy. Acquiring reliable numerical data from wide-field microscopy images of objects which have a complex three-dimensional structure is strongly influenced by the methods used for image analysis. This can be a possible source of inaccuracy in assessment of antithrombotic activity of a tested substance. We aimed to verify how different approaches to the quantification of wide-field images can affect the evaluation of an antiplatelet effect of a tested substance., Methods: We compared three algorithms of image analysis to evaluate an effect of 2-hexynyl-5'-ethylcarboxamidoadenosine (HE-NECA), a compound of a moderate antiplatelet activity on thrombus formation, and of abciximab - a potent antiplatelet compound. Also, we studied how the results obtained in a wide-field imaging correspond to those obtained by means of confocal imaging., Results: Three algorithms for analysis of wide-field images showed antiplatelet effect of HE-NECA or abciximab. Absolute values of thrombus area and outcomes of the evaluation of inhibition efficacy of HE-NECA were significantly different between the algorithms. Analysis of volumes and heights of thrombi obtained by confocal imaging confirmed inhibitory effect of HE-NECA, but the evaluated levels of inhibition were significantly different from that obtained by wide-field imaging., Discussion: We conclude that wide-field imaging provides reliable qualitative data on an inhibitory effect on thrombus formation, despite differences which can emerge from various approaches to image analysis. However, quantitative evaluation and comparison of the efficacy of inhibitors on the basis of total area occupied by thrombi obtained by wide-field microscopy should be made with caution. To obtain a reliable quantitative assessment of the effect of a tested compound on thrombus structure the use of confocal microscopy is inevitable., (Copyright © 2018 Elsevier Inc. All rights reserved.)