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Tatjana Momic,1 Jehoshua Katzhendler,1 Ela Shai,2 Efrat Noy,3 Hanoch Senderowitz,3 Johannes A Eble,4 Cezary Marcinkiewicz,5 David Varon,2 Philip Lazarovici11School of Pharmacy Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, 2Department of Hematology, Coagulation Unit, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 3Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel; 4Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany; 5Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA, USAAbstract: Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2β1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10-10 M and 1.5×10-10 M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2β1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2β1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy.Keywords: adhesion, collagen I, platelets, integrin antagonist, peptidomimetic

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs. Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Introduction: Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis., Methods and Results: Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors., Discussion: Our findings define integrin α2β1 as an early marker of OC differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Brockhaus, Hemsen, Jauch-Speer, Niland, Vogl and Eble.)

To develop peptide drugs targeting integrin receptors, synthetic peptide ligands endowed with well-defined selective binding motifs are necessary. The snake venom KTS-containing disintegrins, which selectively block collagen α1β1 integrin, were used as lead compounds for the synthesis and structure-activity relationship of a series of linear peptides containing the KTS-pharmacophore and alternating natural amino acids and 3-aminobenzoic acid (MABA). To ensure a better stiffness and metabolic stability, one, two and three MABA residues, were introduced around the KTS pharmacophore motif. Molecular dynamics simulations determined that the solution conformation of MABA peptide 4 is more compact, underwent larger conformational changes until convergence, and spent most of the time in a single cluster. The peptides' binding affinity has been characterized by an enzyme linked immunosorbent assay in which the most potent peptide 4 inhibited with IC 50 of 324 ± 8 µM and 550 ± 45 µM the binding of GST-α1-A domain to collagen IV fragment CB3, and the cell adhesion to collagen IV using α1-overexpressor cells, respectively. Docking studies and MM-GBSA calculations confirmed that peptide 4 binds a smaller region of the integrin near the collagen-binding site and penetrated deeper into the binding site near Trp1. Peptide 4 inhibited tube formation by endothelial cell migration in the Matrigel angiogenesis in vitro assay. Peptide 4 was acutely tolerated by mice, showed stability in human serum, decreased tumor volume and angiogenesis, and significantly increased the survival of mice injected with B16 melanoma cells. These findings propose that MABA-peptide 4 can further serve as an α1β1-integrin antagonist lead compound for further drug optimization in angiogenesis and cancer therapy.

The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, as the lack of characterization and detailed production methods of platelet-based bioproducts persists in the literature and precludes cross-study comparisons. We characterized the molecular composition and in vitro regenerative capacity of platelet-rich plasma (PRP) produced in a closed-system. Furthermore, we performed a parallel characterization on different PRP subfractions (plasma and plasma-free platelet lysate), identifying that the fractions containing platelet-derived cargo exert the most potent regenerative capacity. This observation led us to develop a method to obtain a platelet secretome highly enriched in growth factors, free of plasma and cellular components (PCT/IB2022/057936), with the aim of establishing a superior bioproduct. The molecular characterization of secretomes revealed agonist-dependent differences, which correlates with beneficial grades of regenerative capacity. Importantly, secretomes showed general superiority to PRP in vitro. We discuss the variables influencing the bioproduct quality (inter-donor variation, platelet source and processing methods). Finally, we propose that the characteristics of secretomes circumvents certain limitations of PRP (autologous vs allogeneic), and envision that optimizing post-processing protocols (nanoencapsulation, lyophilization), would allow their clinical application even beyond regenerative medicine. STATEMENT OF SIGNIFICANCE: The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, or to improve/personalize them, as the lack of characterization of the bioproducts and their production methods is a constant in the literature, reason that precludes cross-study comparisons. In the present manuscript, we provide a comprehensive molecular and functional characterization of platelet-based bioproducts and subfractions, including platelet rich plasma, plasma fractions and platelet secretomes produced with a methodology developed by our group. Our results show that the molecular composition of each fraction correlates with its regenerative capacity in vitro. Thus, a rigorous characterization of platelet-derived bioproducts will potentially allow universal use, customizing and new applications., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.A.-H. and L.G. are co-founders of the recently constituted spin off company, Platelet Biotechnologies S.L. (PlaBiTe). The rest of authors have no competing interest to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cancer cells circulating in the blood attach to platelets by direct cell-cell interactions via several receptor-counterreceptor contacts and indirectly by fibrin bridges which connect the two cell types by distinct integrin receptors. In the microenvironment of these tumor cell platelet aggregates (TCPAs), the tumor cells are shielded from the shear stress of the blood flow and from attack by the immune system. This supports hematogenous metastasis and tumor cell induced thrombosis. Platelet activation is triggered by binding of podoplanin on cancer cells to the platelet receptor Clec-2. Therefore, we hypothesize that targeting this initial step will prevent the entire cascade leading to the formation of TCPAs. Rhodocytin, a heterodimeric (αβ) 2 C-type lectin from the Malayan pit viper Calloselasma rhodostoma, binds to Clec-2 and thereby induces TCPA formation. Remarkably, mutations in rhodocytin that disturbed formation of oligomers, blocked the podoplanin-Clec-2 axis and prevented platelet activation. Therefore, we used lysine reactive chemicals to modify rhodocytin isolated from the crude snake venom. Blue native gel electrophoresis and far western blotting showed a change of rhodocytin's suprastructure triggered by acetylation and PEGylation. Mass spectrometry analysis of altered lysines suggested that their modifications interfered with the formation of rhodocytin tetramers. When tested in assays for tumor cell induced platelet aggregation, we found that derivatization turned rhodocytin from an agonist into an antagonist. This observation indicates that Clec-2 is a valid target receptor molecule to curb TCPA formation and to prevent hematogenous metastasis and tumor cell induced thrombosis in cancer patients., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes., (© 2023. Springer Nature Limited.)

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: With metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy., Methods: Tumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTR asym ) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1 H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry., Results: During tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models., Conclusion: Multiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Invasion of host cells is an important feature of Staphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge between S. aureus Fn binding proteins and α 5 β 1 -integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not only S. aureus, but also of bacteria otherwise poorly taken up by host cells, such as Staphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated β 1 -integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake of S. aureus into non-professional phagocytes. Additionally, Eap supports the binding of S. carnosus to Fn-α 5 β 1 integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap-the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake-and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity. IMPORTANCE Staphylococcus aureus can invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle of S. aureus contributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted by S. aureus promotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such as Staphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course of S. aureus infectious diseases., Competing Interests: The authors declare no conflict of interest.

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca 2+ and Mg 2+ ions, but inhibited by Co 2+ , Zn 2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists., (© 2023. The Author(s).)

The receptor protein CLEC-2 on platelet membranes is the target of the endogenous ligand podoplanin found on cancer cells and of rhodocytin, a snake venom component of the Malayan pit viper Calloselasma rhodostoma. Ligand binding results in platelet activation, increased blood coagulation and thrombosis. In an effort to isolate rhodocytin, we have purified CLEC-2 as bait from E. coli. Affinity captured rhodocytin interacted with mammalian CLEC-2 and stimulated platelet aggregation in a dose dependent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The inflammatory tumor microenvironment (TME) is formed by various immune cells, being closely associated with tumorigenesis. Especially, the interaction between tumor-infiltrating T-cells and macrophages has a crucial impact on tumor progression and metastatic spread. The purpose of this study was to investigate whether oscillating-gradient diffusion-weighted MRI (OGSE-DWI) enables a cell size-based discrimination between different cell populations of the TME., Methods: Sine-shaped OGSE-DWI was combined with the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) approach to measure microscale diffusion distances, here relating to cell sizes. The accuracy of IMPULSED-derived cell radii was evaluated using in vitro spheroid models, consisting of either pure cancer cells, macrophages, or T-cells. Subsequently, in vivo experiments aimed to assess changes within the TME and its specific immune cell composition in syngeneic murine breast cancer models with divergent degrees of malignancy (4T1, 67NR) during tumor progression, clodronate liposome-mediated depletion of macrophages, and immune checkpoint inhibitor (ICI) treatment. Ex vivo analysis of IMPULSED-derived cell radii was conducted by immunohistochemical wheat germ agglutinin staining of cell membranes, while intratumoral immune cell composition was analyzed by CD3 and F4/80 co-staining., Results: OGSE-DWI detected mean cell radii of 8.8±1.3 µm for 4T1, 8.2±1.4 µm for 67NR, 13.0±1.7 for macrophage, and 3.8±1.8 µm for T-cell spheroids. While T-cell infiltration during progression of 4T1 tumors was observed by decreasing mean cell radii from 9.7±1.0 to 5.0±1.5 µm, increasing amount of intratumoral macrophages during progression of 67NR tumors resulted in increasing mean cell radii from 8.9±1.2 to 12.5±1.1 µm. After macrophage depletion, mean cell radii decreased from 6.3±1.7 to 4.4±0.5 µm. T-cell infiltration after ICI treatment was captured by decreasing mean cell radii in both tumor models, with more pronounced effects in the 67NR tumor model., Conclusions: OGSE-DWI provides a versatile tool for non-invasive profiling of the inflammatory TME by assessing the dominating cell type T-cells or macrophages., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: SPW and NSP declare a patent to protect the nanobodies which was filed on 21st December 2021 (PCT/EP2021/087129: ‘Nanobody’). All other authors have declared that they have no conflict of interest., (Copyright: © 2022 Maqsood et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

(1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based proteomics experiments. We review the current state-of-the-art and use the identification of an unknown snake venom protein targeting the human tissue factor (TF) as an example to describe the analysis process based on manual spectrum interrogation. (2) Methods: The immobilized TF was incubated with a crude B. moojeni venom solution. The potential binding partners were eluted and further purified by gel electrophoresis. Edman degradation was performed to elucidate the N-terminus of the 31 kDa protein of interest. High-resolution MS with collision-induced dissociation was employed to generate peptide fragmentation spectra. Sequence tags were deduced and used for searches in the NCBI and Uniprot databases. Protein matches from the snake species were further validated by target MS/MS. (3) Results: Sequence tag D [K/Q] D [I/L] VDD [K/Q] led to a snake venom serine protease (SVSP) from lancehead B. jararaca (P81824). With target MS/MS, 24% of the SVSP sequence were confirmed; an additional 41% were tentatively assigned by data-independent MS. Edman sequencing provided information for 10 N-terminal amino acid residues, also confirming the match to SVSP. (4) Conclusions: The identification of unknown proteins continues to be a challenge despite major advances in MS instrumentation and bioinformatic tools. The main requirement is the generation of meaningful, high-quality MS peptide fragmentation spectra. These are used to elucidate sufficiently long sequence tags, which can subsequently be submitted to searches in protein databases. This basic method does not require extensive bioinformatics because peptide MS/MS spectra, especially of doubly-charged ions, can be analysed manually. We demonstrated the procedure with the elucidation of SVSP. While de novo sequencing quickly indicates the correct protein group, the validation of the entire protein sequence of amino acid-by-amino acid will take time. Reasons are the need to properly assign isobaric amino acid residues and modifications. With the ongoing efforts in genomics and transcriptomics and the availability of ever more data in public databases, the need for de novo MS sequencing will decrease. Still, not every animal and plant species will be sequenced, so the combination of MS and Edman sequencing will continue to be of importance for the identification of unknown proteins.

Background:  CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2., Objectives:  To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2., Methods:  ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin-CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand., Results and Conclusion:  We developed a CLEC-2-podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)'2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Integrin-mediated cell contacts with the extracellular matrix (ECM) are essential for cellular adhesion, force transmission, and migration. Several effectors, such as divalent cations and redox-active compounds, regulate ligand binding activities of integrins and influence their cellular functions. To study the role of the Ca 2+ binding site within the hinge region of the integrin α7 subunit, we genetically abrogated it in the α7hiΔCa mutant. This mutant folded correctly, associated with the β1 subunit and was exposed on the cell surface, but showed reduced ligand binding and weaker cell adhesion to laminin-111. Thus, it resembles the α7hiΔSS mutant, in which the redox-regulated pair of cysteines, closeby to the Ca 2+ binding site within the hinge, was abrogated. Comparing both mutants in adhesion strength and cell migration revealed that both Ca 2+ complexation and redox-regulation within the hinge interdepend on each other. Moreover, protein-chemical analyses of soluble integrin ectodomains containing the same α7 hinge mutations suggest that integrin activation via the subunit α hinge is primed by the formation of the cysteine pair-based crosslinkage. Then, this allows Ca 2+ complexation within the hinge, which is another essential step for integrin activation and ligand binding. Thus, the α hinge is an allosteric integrin regulation site, in which both effectors, Ca 2+ and redox-active compounds, synergistically and hierarchically induce far-ranging conformational changes, such as the extension of the integrin ectodomain, resulting in integrin activation of ECM ligand binding and altered integrin-mediated cell functions., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by enhanced platelet clearance and defective platelet production. Diagnosis by exclusion and trial-and-error treatment strategies is common practice, and despite the advancement in treatment options, many patients remain refractory. Although the existence of different pathophysiological entities is acknowledged, we are still far from stratifying and understanding ITP. To investigate, we sought to dissect the platelet proteome dynamics in so-called passive and active preclinical ITP mouse models, with which we propose to phenocopy respectively acute/newly diagnosed and persistent/chronic stages of ITP in humans. We obtained the platelet proteome at the thrombocytopenic stage and after platelet count recovery (reached naturally or by IVIg-treatment, depending on the model). Although most of the proteomic alterations were common to both ITP models, there were model-specific protein dynamics that accompanied and explained alterations in platelet aggregation responses, as measured in the passive ITP model. The expression dynamics observed in Syk may explain, extrapolated to humans and pending validation, the increased bleeding tendency of patients with ITP when treated with fostamatinib as third or later- as opposed to second line of treatment. We propose that the platelet proteome may give diagnostic and prognostic insights into ITP and that such studies should be pursued in humans., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Platelets are the blood cells in charge of maintaining the body hemostasis, recognising the damaged vessel wall, and providing the appropriate cellular surface for the coagulation cascade to act locally. Additionally, platelets are active immunomodulators. At the crossroads of hemostasis and inflammation, platelets may exert either beneficial actions or participate in pathological manifestations, and have been associated with the prothrombotic nature of multi-organ failure in systemic inflammation. Platelet number alterations have been reported in septis, and platelet transfusions are given to thrombocytopenic patients. However, the risk to develop transfusion related acute lung injury (TRALI) is higher in sepsis patients. In this manuscript we show that platelets produced during inflammation in preclinical mouse models of sterile inflammation display lower aggregation capacity when stimulating certain receptors, while responses through other receptors remain intact, and we name them "inflammation-conditioned" platelets. In a cohort of sepsis patients, we observed, as previously reported, alterations in the number of platelets and platelet hyperreactivity. Furthermore, we identified a receptor-wise platelet aggregation response disbalance in these patients, although not similar to platelets from preclinical models of sterile inflammation. Interestingly, we generated evidence supporting the notion that platelet aggregation capacity disbalance was partially triggered by plasma components from sepsis patients. Our findings have implications in the indication of platelet transfusions in sepsis patients: Are fully functional platelets suitable for transfusion in sepsis patients? Current Clinical Trials (RESCUE) will answer whether platelet production stimulation with thrombopoietin receptor agonists (TPO-RAs) could be a substitute of platelet transfusions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α 3 /α 6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background:  Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation., Methods:  Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca 2+ ] i , aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed., Results:  Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca 2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation., Conclusion:  Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding., Competing Interests: J.W.M.H. is a cofounder and shareholder of FlowChamber B.V. The other authors declare no relevant conflicts of interest., (Thieme. All rights reserved.)

Cancer progression with uncontrolled tumor growth, local invasion, and metastasis depends largely on the proteolytic activity of numerous matrix metalloproteinases (MMPs), which affect tissue integrity, immune cell recruitment, and tissue turnover by degrading extracellular matrix (ECM) components and by releasing matrikines, cell surface-bound cytokines, growth factors, or their receptors. Among the MMPs, MMP-14 is the driving force behind extracellular matrix and tissue destruction during cancer invasion and metastasis. MMP-14 also influences both intercellular as well as cell-matrix communication by regulating the activity of many plasma membrane-anchored and extracellular proteins. Cancer cells and other cells of the tumor stroma, embedded in a common extracellular matrix, interact with their matrix by means of various adhesive structures, of which particularly invadopodia are capable to remodel the matrix through spatially and temporally finely tuned proteolysis. As a deeper understanding of the underlying functional mechanisms is beneficial for the development of new prognostic and predictive markers and for targeted therapies, this review examined the current knowledge of the interplay of the various MMPs in the cancer context on the protein, subcellular, and cellular level with a focus on MMP14.

While Staphylococcus aureus has classically been considered an extracellular pathogen, these bacteria are also capable of being taken up by host cells, including nonprofessional phagocytes such as endothelial cells, epithelial cells, or osteoblasts. The intracellular S. aureus lifestyle contributes to infection development. The predominant recognition and internalization pathway appears to be the binding of the bacteria via a fibronectin bridge to the α5β1-integrin on the host cell membrane, followed by phagocytosis. Although osteoblasts showed high expression of α5β1-integrin and fibronectin, and bacteria adhered to osteoblasts to a high proportion, here we demonstrate by internalization assays and immunofluorescence microscopy that S. aureus was less engulfed in osteoblasts than in epithelial cells. The addition of exogenous fibronectin during the infection of cells with S. aureus resulted in an increased uptake by epithelial cells but not by osteoblasts. This contrasts with the previous conception of the uptake mechanism, where high expression of integrin and fibronectin would promote the bacterial uptake into host cells. Extracellular fibronectin surrounding osteoblasts, but not epithelial cells, is organized in a fibrillary network. The inhibition of fibril formation, the short interfering RNA-mediated reduction of fibronectin expression, and the disruption of the fibronectin-fibril meshwork all resulted in a significant increase in S. aureus uptake by osteoblasts. Thus, the network of fibronectin fibrils appears to strongly reduce the uptake of S. aureus into a given host cell, indicating that the supramolecular structure of fibronectin determines the capacity of particular host cells to internalize the pathogen. IMPORTANCE Traditionally, Staphylococcus aureus has been considered an extracellular pathogen. However, among other factors, the frequent failure of antimicrobial therapy and the ability of the pathogen to cause recurrent disease have established the concept of eukaryotic invasion of the pathogen, thereby evading the host's immune system. In the current model of host cell invasion, bacteria initially bind to α5β1 integrin on the host cell side via a fibronectin bridge, which eventually leads to phagocytosis of S. aureus by host cells. However, in this study, we demonstrate that not the crude amount but the supramolecular structure of fibronectin molecules deposited on the eukaryotic cell surface plays an essential role in bacterial uptake by host cells. Our findings explain the large differences of S. aureus uptake efficacy in different host cell types as well as in vivo differences between courses of bacterial infections and the localization of bacteria in different clinical settings.

Current antiplatelet drugs for the treatment of arterial thrombosis often coincide with increased bleeding risk. Several tyrosine kinase inhibitors (TKIs) for cancer treatment inhibit platelet function, with minor reported bleeding symptoms. The aim of this study was to compare the antiplatelet properties of eight TKIs to explore their possible repurposing as antiplatelet drugs. Samples of whole blood, platelet-rich plasma (PRP), or isolated platelets from healthy donors were treated with TKI or the vehicle. Measurements of platelet aggregation, activation, intracellular calcium mobilization, and whole-blood thrombus formation under flow were performed. Dasatinib and sunitinib dose-dependently reduced collagen-induced aggregation in PRP and washed platelets; pazopanib, cabozantinib, and vatalanib inhibited this response in washed platelets only; and fostamatinib, axitinib, and lapatinib showed no/limited effects. Fostamatinib reduced thrombus formation by approximately 50% on collagen and other substrates. Pazopanib, sunitinib, dasatinib, axitinib, and vatalanib mildly reduced thrombus formation on collagen by 10-50%. Intracellular calcium responses in isolated platelets were inhibited by dasatinib (>90%), fostamatinib (57%), sunitinib (77%), and pazopanib (82%). Upon glycoprotein-VI receptor stimulation, fostamatinib, cabozantinib, and vatalanib decreased highly activated platelet populations by approximately 15%, while increasing resting populations by 39%. In conclusion, the TKIs with the highest affinities for platelet-expressed molecular targets most strongly inhibited platelet functions. Dasatinib, fostamatinib, sunitinib, and pazopanib interfered in early collagen receptor-induced molecular-signaling compared with cabozantinib and vatalanib. Fostamatinib, sunitinib, pazopanib, and vatalanib may be promising for future evaluation as antiplatelet drugs.

All irreversible Bruton tyrosine kinase (Btk) inhibitors including ibrutinib and acalabrutinib induce platelet dysfunction and increased bleeding risk. New reversible Btk inhibitors were developed, like MK-1026. The mechanism underlying increased bleeding tendency with Btk inhibitors remains unclear. We investigated the effects of ibrutinib, acalabrutinib and MK-1026 on platelet function in healthy volunteers, patients and Btk-deficient mice, together with off-target effects on tyrosine kinase phosphorylation. All inhibitors suppressed GPVI- and CLEC-2-mediated platelet aggregation, activation and secretion in a dose-dependent manner. Only ibrutinib inhibited thrombus formation on vWF-co-coated surfaces, while on collagen this was not affected. In blood from Btk-deficient mice, collagen-induced thrombus formation under flow was reduced, but preincubation with either inhibitor was without additional effects. MK-1026 showed less off-target effects upon GPVI-induced TK phosphorylation as compared to ibrutinib and acalabrutinib. In ibrutinib-treated patients, GPVI-stimulated platelet activation, and adhesion on vWF-co-coated surfaces were inhibited, while CLEC-2 stimulation induced variable responses. The dual inhibition of GPVI and CLEC-2 signalling by Btk inhibitors might account for the increased bleeding tendency, with ibrutinib causing more high-grade bleedings due to additional inhibition of platelet-vWF interaction. As MK-1026 showed less off-target effects and only affected activation of isolated platelets, it might be promising for future treatment., (© 2021 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Glycoprotein (GP)Ib that binds von Willebrand factor (vWF) and glycoprotein (GP)VI, that binds collagen play a significant role in platelet activation and aggregation, and are potential targets for antithrombotic treatment. They are targeted by snake venom proteinases. The effect of a such proteinase, mutalysin-II, on platelet aggregation was examined using washed human platelets and platelet-rich plasma. Its proteolytic activity on vWF, on its binding partner GPIbα, and on GPVI was analyzed by SDS-PAGE, and immunodetection with the corresponding antibodies after blotting. Dose- and time-dependently, mutalysin-II inhibits aggregation of washed platelets induced by vWF plus ristocetin and by convulxin, but with no significant effect on platelet-rich-plasma. Furthermore, mutalysin-II cleaves vWF into low molecular mass multimers of vWF and a rvWF-A1 domain to realease a ∼27-kDa fragment detectable by SDS-PAGE and blotting with mouse anti-rvWF-A1-domain IgG. Moreover, GPVI was cut by mutalysin-II into a soluble ∼55-kDa ectodomain and a fragment of ∼35-kDa. Thus, mutalysin-II inhibits vWF-induced platelet aggregation via cleavage of bound vWF-A1, and its receptor GPIbα. The additional cleavage of, GPVI, blocks collagen-induced platelets. Our data highlight mutalysin-II as an interesting platelet-directed tool targeting vWF-GPIbα binding and particularly GPVI. Thus, it might be suited for antithrombotic therapy as its combined inactivation of two receptors does not significantly compromise hemostasis, but shows high efficacy and safety. Studies are needed to further develop and demonstrate its potential benefits., Competing Interests: Declaration of competing interest The authors state no conflicts of interest., (Copyright © 2021. Published by Elsevier B.V.)

Although platelets and the coagulation factors are components of the blood system, they become part of and contribute to the tumor microenvironment (TME) not only within a solid tumor mass, but also within a hematogenous micrometastasis on its way through the blood stream to the metastatic niche. The latter basically consists of blood-borne cancer cells which are in close association with platelets. At the site of the primary tumor, the blood components reach the TME via leaky blood vessels, whose permeability is increased by tumor-secreted growth factors, by incomplete angiogenic sprouts or by vasculogenic mimicry (VM) vessels. As a consequence, platelets reach the primary tumor via several cell adhesion molecules (CAMs). Moreover, clotting factor VII from the blood associates with tissue factor (TF) that is abundantly expressed on cancer cells. This extrinsic tenase complex turns on the coagulation cascade, which encompasses the activation of thrombin and conversion of soluble fibrinogen into insoluble fibrin. The presence of platelets and their release of growth factors, as well as fibrin deposition changes the TME of a solid tumor mass substantially, thereby promoting tumor progression. Disseminating cancer cells that circulate in the blood stream also recruit platelets, primarily by direct cell-cell interactions via different receptor-counterreceptor pairs and indirectly by fibrin, which bridges the two cell types via different integrin receptors. These tumor cell-platelet aggregates are hematogenous micrometastases, in which platelets and fibrin constitute a particular TME in favor of the cancer cells. Even at the distant site of settlement, the accompanying platelets help the tumor cell to attach and to grow into metastases. Understanding the close liaison of cancer cells with platelets and coagulation factors that change the TME during tumor progression and spreading will help to curb different steps of the metastatic cascade and may help to reduce tumor-induced thrombosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Obermann, Brockhaus and Eble.)

Background: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms., Methods: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time., Results: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug., Conclusion: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans.

Proteomics studies have revealed that adhesomes are assembled from a plethora of proteins at integrin-mediated cellular contact sites with the extracellular matrix. By combining dimedone-trapping of sulfenylated proteins with the purification of the adhesome complex, we extended previous proteomics approaches on adhesomes to a redox proteomic analysis. This added a new aspect of adhesome complexity as individual adhesome proteins change their redox state in response to environmental signals. As model system, rat pheochromocytoma PC12 cells were studied in contact with type IV collagen and in response to nerve growth factor (NGF). NGF stimulates the endogenous production of reactive oxygen species (ROS) and the formation of neurite-like cell protrusions, which are anchored to the substratum via adhesomes. Dimedone detects the reversible oxidation of cysteine thiol groups into sulfenic acid groups which was used in proteomic analysis of adhesome proteins revealing that sulfenylation and location of proteins mutually influence each other. For some proteins, identified by the redox proteomics approach, among them Nck-associated protein-1 (Nap-1), proximity ligation analysis and co-immunoprecipitation assays proved that protein sulfenylation sites colocalize with adhesomes of protrusions. In conclusion, the suprastructural composition and function of adhesomes is redox-regulated by ROS. Of interest in this respect, isoform-selective pharmacological inhibition of NADPH-oxidases (Noxs) reduced the adhesomal location of the collagen-binding α1β1 integrin and the length of the outgrowing neurites, indicative of a role of Nox isoforms in the redox-regulation of adhesomes. Thus, our novel redox proteomics approach not only revealed redox-modifications and the potential redox-regulation of adhesomes and their constituents but it may also provide a tool to analyze the ROS-stimulated neurite repair of peripheral neurons., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.

The tumor microenvironment (TME) has become the focus of interest in cancer research and treatment. It includes the extracellular matrix (ECM) and ECM-modifying enzymes that are secreted by cancer and neighboring cells. The ECM serves both to anchor the tumor cells embedded in it and as a means of communication between the various cellular and non-cellular components of the TME. The cells of the TME modify their surrounding cancer-characteristic ECM. This in turn provides feedback to them via cellular receptors, thereby regulating, together with cytokines and exosomes, differentiation processes as well as tumor progression and spread. Matrix remodeling is accomplished by altering the repertoire of ECM components and by biophysical changes in stiffness and tension caused by ECM-crosslinking and ECM-degrading enzymes, in particular matrix metalloproteinases (MMPs). These can degrade ECM barriers or, by partial proteolysis, release soluble ECM fragments called matrikines, which influence cells inside and outside the TME. This review examines the changes in the ECM of the TME and the interaction between cells and the ECM, with a particular focus on MMPs.

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet β -cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic β -cells via the α v β 3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions., Competing Interests: A.Z., S.L.L., G.P.D., and K.S.‐L. are inventors on patent application EP19154849.4 associated with this work and owned by the University Tübingen. S.L.L., M.Z., and K.S.‐L. are inventors on patents (EP3027201B1 and CN105517564A), and patent applications (US20160158314A1, CA2916614A1, JP2016530532A, and KR20160037170A) associated with this work and owned by the NMI, Reutlingen., (© 2020 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Redox-mediated signal transduction depends on the enzymatic production of second messengers such as hydrogen peroxide, nitric oxide and hydrogen sulfite, as well as specific, reversible redox modifications of cysteine-residues in proteins. So-called thiol switches induce for instance conformational changes in specific proteins that regulate cellular pathways e.g . , cell metabolism, proliferation, migration, gene expression and inflammation. Reduction, oxidation and disulfide isomerization are controlled by oxidoreductases of the thioredoxin family, including thioredoxins, glutaredoxins, peroxiredoxins and protein dsisulfide isomerases. These proteins are located in different cellular compartments, interact with substrates and catalyze specific reactions. Interestingly, some of these proteins are released by cells. Their extracellular functions and generally extracellular redox control have been widely underestimated. Here, we give an insight into extracellular redox signaling, extracellular thiol switches and their regulation by secreted oxidoreductases and thiol-isomerases, a topic whose importance has been scarcely studied so far, likely due to methodological limitations. We focus on the secreted redox proteins and characterized thiol switches in the ectodomains of membrane proteins, such as integrins and the metalloprotease ADAM17, which are among the best-characterized proteins and discuss their underlying mechanisms and biological implications., (© 2020 Inken Lorenzen et al., published by De Gruyter, Berlin/Boston.)

Cadherins mediate cohesive contacts between isotypic cells by homophilic interaction and prevent contact between heterotypic cells. Breast cancer cells neighboring endothelial cells (ECs) atypically express vascular endothelial (VE)-cadherin. To understand this EC-induced VE-cadherin expression in breast cancer cells, MCF7 and MDA-MB-231 cells expressing different endogenous cadherins were co-cultured with ECs and analyzed for VE-cadherin at the transcriptional level and by confocal microscopy, flow cytometry, and immunoblotting. After losing their endogenous cadherins and neo-expression of VE-cadherin, these cells integrated into an EC monolayer without compromising the barrier function instantly. However, they induced the death of nearby ECs. EC-derived extracellular vesicles (EVs) contained soluble and membrane-anchored forms of VE-cadherin. Only the latter was re-utilized by the cancer cells. In a reporter gene assay, EC-adjacent cancer cells also showed a juxtacrine but no paracrine activation of the endogenous VE-cadherin gene. This cadherin switch enabled intimate contact between cancer and endothelial cells in a chicken chorioallantoic membrane tumor model showing vasculogenic mimicry (VM). This EV-mediated, EC-induced cadherin switch in breast cancer cells and the neo-expression of VE-cadherin mechanistically explain the mutual communication in the tumor microenvironment. Hence, it may be a target to tackle VM, which is often found in breast cancers of poor prognosis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by fast tumor progression and diagnosis at advanced, inoperable stages. Previous studies could demonstrate an involvement of miR-192-5p in epigenetic regulation of visceral carcinomas. Due to contradictory results, however, the clinical utility of miR-192-5p in PDAC has yet to be determined. MiR-192-5p expression was analyzed by RT-qRT-PCR in human PDAC and benign tissue ( n = 78), blood serum ( n = 81) and serum exosomes ( n = 74), as well as in PDAC cell lines ( n = 5), chemoresistant cell clones ( n = 2), and pancreatic duct cell line H6c7. Analysis of EMT-associated (epithelial-to-mesenchymal transition) proteins was performed by immunohistochemistry and Western blot. MiR-192-5p was deregulated in PDAC as compared to healthy controls (HCs), with downregulation in macrodissected tissue ( p < 0.001) and upregulation in blood serum of PDAC UICC (Union for International Cancer Control) stage IV ( p = 0.016) and serum exosomes of PDAC UICC stages II to IV ( p < 0.001). MiR-192-5p expression in tumor tissue was significantly lower as compared to corresponding peritumoral tissue (PDAC UICC stage II: p < 0.001; PDAC UICC stage III: p = 0.024), while EMT markers ZEB1 and ZEB2 were more frequently expressed in tumor tissue as compared to peritumoral tissue, HCs, and chronic pancreatitis. Tissue-derived (AUC of 0.86; p < 0.0001) and exosomal (AUC of 0.83; p = 0.0004) miR-192-5p could differentiate between PDAC and HCs with good accuracy. Furthermore, high expression of miR-192-5p in PDAC tissue of curatively resected PDAC patients correlated with prolonged overall and recurrence-free survival in multivariate analysis. In vitro, miR-192-5p was downregulated in gemcitabine-resistant cell clones of AsPC-1 ( p = 0.029). Transient transfection of MIA PaCa-2 cells with miR-192-5p mimic resulted in downregulation of ZEB2. MiR-192-5p seems to possess a tumor-suppressive role and high potential as a diagnostic and prognostic marker in PDAC.

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold β/β hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease., Competing Interests: Declaration of competing interest The authors declared there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

While adhering to extracellular matrix (ECM) proteins, such as laminin-111, cells temporarily produce hydrogen peroxide at adhesion sites. To study the redox regulation of α7β1 integrin-mediated cell adhesion to laminin-111, a conserved cysteine pair within the α-subunit hinge region was replaced for alanines. The molecular and cellular effects were analyzed by electron and atomic force microscopy, impedance-based migration assays, flow cytometry and live cell imaging. This cysteine pair constitutes a thiol-switch, which redox-dependently governs the equilibrium between an extended and a bent integrin conformation with high and low ligand binding activity, respectively. Hydrogen peroxide oxidizes the cysteines to a disulfide bond, increases ligand binding and promotes cell migration toward laminin-111. Inversely, extracellular thioredoxin-1 reduces the disulfide, thereby decreasing laminin binding. Mutation of this cysteine pair into the non-oxidizable hinge-mutant shows molecular and cellular effects similar to the reduced wild-type integrin, but lacks redox regulation. This proves the existence of a dominant thiol-switch within the α subunit hinge of α7β1 integrin, which is sufficient to implement activity regulation by extracellular redox agents in a redox-regulatory circuit. Our data reveal a novel and physiologically relevant thiol-based regulatory mechanism of integrin-mediated cell-ECM interactions, which employs short-lived hydrogen peroxide and extracellular thioredoxin-1 as signaling mediators.

C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A 2 (TXA 2 ). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA 2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr 525+526 ) and Src(p-Tyr 419 ), and for PLCγ2 activity (p-Tyr 759 ). We demonstrated that Syk phosphorylation at Tyr 525+526 also happens in the presence of ADP and TXA 2 inhibitors, which is not the case for Src-pTyr 419 and PLCγ2-pTyr 759 . Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca 2+ mobilization assays confirmed the relevance of ADP and TXA 2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Neuropilin-1 and neuropilin-2 form a small family of transmembrane receptors, which, due to the lack of a cytosolic protein kinase domain, act primarily as co-receptors for various ligands. Performing at the molecular level both the executive and organizing functions of a handyman as well as of a power broker, they are instrumental in controlling the signaling of various receptor tyrosine kinases, integrins, and other molecules involved in the regulation of physiological and pathological angiogenic processes. In this setting, the various neuropilin ligands and interaction partners on various cells of the tumor microenvironment, such as cancer cells, endothelial cells, cancer-associated fibroblasts, and immune cells, are surveyed. The suitability of various neuropilin-targeting substances and the intervention in neuropilin-mediated interactions is considered as a possible building block of tumor therapy.

Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.

Environmental DNA (eDNA) is increasingly being used to document species distributions and habitat use in marine systems, with much of the recent effort focused on leveraging advances in next-generation DNA sequencing to assess and track biodiversity across taxonomic groups. Environmental DNA offers a number of important advantages over traditional survey techniques, including non-invasive sampling, sampling where traditional approaches are impractical or inefficient (e.g. deep oceans), reduced cost, and increased detection sensitivity. However, eDNA applications are currently limited because of an insufficient understanding of the influence of sample source, analytical approach, and marker type on eDNA detections. Because approaches vary considerably among eDNA studies, we present a summary of the current state of the field and emerging best practices. The impact of observed variation in rates of eDNA production, persistence, and transport are also discussed and future research needs are highlighted with the goal of expanding eDNA applications, including the development of statistical models to improve the predictability of eDNA detection and quantification., (© 2020 Elsevier Ltd All rights reserved.)

Despite significant advances in the treatment of thrombogenic diseases, antiplatelet therapies are still associated with a high bleeding risk. Consequently, potential benefits of preventing thromboembolic events by pharmacological agents need to be balanced with the potential harm of inducing hemorrhage. Glycoprotein VI (GPVI) is a platelet-specific receptor, which plays a crucial role in thrombus formation. GPVI deficiency has been identified in patients who suffer from significant reduction of collagen-induced thrombus formation, with a slight tendency for mild bleeding. However, an isolated GPVI deficiency can reduce thrombus formation while not resulting in severe bleeding. Together, these observations strongly suggest that physiological hemostasis does not require GPVI, but pharmacological GPVI modulation may provide novel "bleeding-free" antithrombotic therapies. In this review, we discuss recent findings regarding the biological role of GPVI in platelet-related disorders and highlight the efforts to develop potential therapeutic strategies based on its structure, signaling pathways, and biological effects., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox . This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg 2+ and Ca 2+ , enhance its activity, whereas excess Zn 2+ inhibits it. Chemical modification of the Zn 2+ -complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2β1 integrin. Neither the α2β1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox .