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BackgroundBruton's tyrosine kinase (Btk) is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-gamma2 (PLCgamma2) and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCgamma2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCgamma2-deficient DT40 B cell line.ResultsActivation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCgamma2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCgamma2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, and apoptosis. In DT40 B cells deficient for PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis.ConclusionsThese data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCgamma2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure., (Copyright © 2024 The Authors. Published by Elsevier Inc. 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).)

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy., Competing Interests: Conflict of interests The authors have no competing interests. A patent “INHIBITORS OF SARS-COV-2 INFECTION AND OTHER CORONA VIRUS INFECTIONS” SF-2021-201-2-PCT-0 has been filed., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Receptor diffusion plays an essential role in cellular signalling via the plasma membrane microenvironment and receptor interactions, but the regulation is not well understood. To aid in understanding of the key determinants of receptor diffusion and signalling, we developed agent-based models (ABMs) to explore the extent of dimerisation of the platelet- and megakaryocyte-specific receptor for collagen glycoprotein VI (GPVI). This approach assessed the importance of glycolipid enriched raft-like domains within the plasma membrane that lower receptor diffusivity. Our model simulations demonstrated that GPVI dimers preferentially concentrate in confined domains and, if diffusivity within domains is decreased relative to outside of domains, dimerisation rates are increased. While an increased amount of confined domains resulted in further dimerisation, merging of domains, which may occur upon membrane rearrangements, was without effect. Modelling of the proportion of the cell membrane which constitutes lipid rafts indicated that dimerisation levels could not be explained by these alone. Crowding of receptors by other membrane proteins was also an important determinant of GPVI dimerisation. Together, these results demonstrate the value of ABM approaches in exploring the interactions on a cell surface, guiding the experimentation for new therapeutic avenues., (© 2023. The Author(s).)

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.)

The platelet-activating collagen receptor GPVI represents the focus of clinical trials as an antiplatelet target for arterial thrombosis, and soluble GPVI is a plasma biomarker for several human diseases. A disintegrin and metalloproteinase 10 (ADAM10) acts as a 'molecular scissor' that cleaves the extracellular region from GPVI and many other substrates. ADAM10 interacts with six regulatory tetraspanin membrane proteins, Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33, which are collectively termed the TspanC8s. These are emerging as regulators of ADAM10 substrate specificity. Human platelets express Tspan14, Tspan15 and Tspan33, but which of these regulates GPVI cleavage remains unknown. To address this, CRISPR/Cas9 knockout human cell lines were generated to show that Tspan15 and Tspan33 enact compensatory roles in GPVI cleavage, with Tspan15 bearing the more important role. To investigate this mechanism, a series of Tspan15 and GPVI mutant expression constructs were designed. The Tspan15 extracellular region was found to be critical in promoting GPVI cleavage, and appeared to achieve this by enabling ADAM10 to access the cleavage site at a particular distance above the membrane. These findings bear implications for the regulation of cleavage of other ADAM10 substrates, and provide new insights into post-translational regulation of the clinically relevant GPVI protein.

Tetraspanins are four-pass transmembrane proteins that function by regulating trafficking of partner proteins and organizing signaling complexes in the membrane. Tspan15, one of a six-member TspanC8 subfamily, forms a complex that regulates the trafficking, maturation, and substrate selectivity of the transmembrane protease ADAM10, an essential enzyme in mammalian physiology that cleaves a wide variety of membrane-anchored substrates, including Notch receptors, amyloid precursor protein, cadherins, and growth factors. We present here crystal structures of the Tspan15 large extracellular loop (LEL) required for functional association with ADAM10 both in isolation and in complex with the Fab fragment of an anti-Tspan15 antibody. Comparison of the Tspan15 LEL with other tetraspanin LEL structures shows that a core helical framework buttresses a variable region that structurally diverges among LELs. Using co-immunoprecipitation and a cellular N-cadherin cleavage assay, we identify a site on Tspan15 required for both ADAM10 binding and promoting substrate cleavage., Competing Interests: Declaration of interests S.C.B. receives funding for an unrelated project from Novartis, is on the scientific advisory board for Erasca, Inc., is an advisor to MPM Capital, and is a consultant for IFM, Scorpion Therapeutics, Odyssey Therapeutics, and Ayala Pharmaceuticals for unrelated projects., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity., Competing Interests: None declared., (Thieme. All rights reserved.)

The ubiquitously expressed transmembrane protein a disintegrin and metalloproteinase 10 (ADAM10) functions as a "molecular scissor", by cleaving the extracellular regions from its membrane protein substrates in a process termed ectodomain shedding. ADAM10 is known to have over 100 substrates including Notch, amyloid precursor protein, cadherins, and growth factors, and is important in health and implicated in diseases such as cancer and Alzheimer's. The tetraspanins are a superfamily of membrane proteins that interact with specific partner proteins to regulate their intracellular trafficking, lateral mobility, and clustering at the cell surface. We and others have shown that ADAM10 interacts with a subgroup of six tetraspanins, termed the TspanC8 subgroup, which are closely related by protein sequence and comprise Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. Recent evidence suggests that different TspanC8/ADAM10 complexes have distinct substrates and that ADAM10 should not be regarded as a single scissor, but as six different TspanC8/ADAM10 scissor complexes. This review discusses the published evidence for this "six scissor" hypothesis and the therapeutic potential this offers.

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss.

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex., (© 2020 Koo et al.)

The interplay between thrombosis and inflammation, termed thrombo-inflammation, causes acute organ damage in diseases such as ischaemic stroke and venous thrombosis. We have recently identified tetraspanin Tspan18 as a novel regulator of thrombo-inflammation. The tetraspanins are a family of 33 membrane proteins in humans that regulate the trafficking, clustering, and membrane diffusion of specific partner proteins. Tspan18 partners with the store-operated Ca 2+ entry channel Orai1 on endothelial cells. Orai1 appears to be expressed in all cells and is critical in health and disease. Orai1 mutations cause human immunodeficiency, resulting in chronic and often lethal infections, while Orai1-knockout mice die at around the time of birth. Orai1 is a promising drug target in autoimmune and inflammatory diseases, and Orai1 inhibitors are in clinical trials. The focus of this review is our work on Tspan18 and Orai1 in Tspan18-knockout mice and Tspan18-knockdown primary human endothelial cells. Orai1 trafficking to the cell surface is partially impaired in the absence of Tspan18, resulting in impaired Ca 2+ signaling and impaired release of the thrombo-inflammatory mediator von Willebrand factor following endothelial stimulation. As a consequence, Tspan18-knockout mice are protected in ischemia-reperfusion and deep vein thrombosis models. We provide new evidence that Tspan18 is relatively highly expressed in endothelial cells, through the analysis of publicly available single-cell transcriptomic data. We also present new data, showing that Tspan18 is required for normal Ca 2+ signaling in platelets, but the functional consequences are subtle and restricted to mildly defective platelet aggregation and spreading induced by the platelet collagen receptor GPVI. Finally, we generate structural models of human Tspan18 and Orai1 and hypothesize that Tspan18 regulates Orai1 Ca 2+ channel function at the cell surface by promoting its clustering.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

ADAM10 is a transmembrane metalloprotease that is essential for development and tissue homeostasis. It cleaves the ectodomain of many proteins, including amyloid precursor protein, and plays an essential role in Notch signaling. ADAM10 associates with six members of the tetraspanin superfamily referred to as TspanC8 (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33), which regulate its exit from the endoplasmic reticulum and its substrate selectivity. We now show that ADAM10, Tspan5, and Tspan15 influence each other's expression level. Notably, ADAM10 undergoes faster endocytosis in the presence of Tspan5 than in the presence of Tspan15, and Tspan15 stabilizes ADAM10 at the cell surface yielding high expression levels. Reciprocally, ADAM10 stabilizes Tspan15 at the cell surface, indicating that it is the Tspan15/ADAM10 complex that is retained at the plasma membrane. Chimeric molecules indicate that the cytoplasmic domains of these tetraspanins contribute to their opposite action on ADAM10 trafficking and Notch signaling. In contrast, an unusual palmitoylation site at the end of Tspan15 C-terminus is dispensable. Together, these findings uncover a new level of ADAM10 regulation by TspanC8 tetraspanins., (© 2019 Eschenbrenner et al.)

Ca 2+ entry via Orai1 store-operated Ca 2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca 2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca 2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca 2+ signaling and von Willebrand factor release in response to inflammatory stimuli., (Copyright© 2019 Ferrata Storti Foundation.)

Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton tyrosine kinase used in the treatment of B-cell malignancies. They bind irreversibly to cysteine 481 of Bruton tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of downstream substrates including phospholipase C-γ2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton tyrosine kinase activity, as shown by loss of phosphorylation at tyrosine 223 and phospholipase C-γ2, delay but do not block aggregation in response to a maximally-effective concentration of collagen-related peptide or collagen. In contrast, 10- to 20-fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation in response to glycoprotein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, showed a reduction of platelet aggregation in response to collagen-related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton tyrosine kinase blockade. Unexpectedly, low concentrations of ibrutinib inhibited aggregation in response to collagen-related peptide in patients deficient in Bruton tyrosine kinase. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavorable pharmacodynamics., (Copyright© 2018 Ferrata Storti Foundation.)

Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b ), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 ( Cd37 -/- ) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37 -/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37 -/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37 -/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane protein that functions as a "molecular scissor" to cleave the extracellular regions from its transmembrane target proteins. ADAM10 is well characterized as the ligand-dependent activator of Notch proteins, which control cell fate decisions. Indeed, conditional knockouts of ADAM10 in mice reveal impaired B-, T-, and myeloid cell development and/or function. ADAM10 cleaves many other leukocyte-expressed substrates. On B-cells, ADAM10 cleavage of the low-affinity IgE receptor CD23 promotes allergy and asthma, cleavage of ICOS ligand impairs antibody responses, and cleavage of the BAFF-APRIL receptor transmembrane activator and CAML interactor, and BAFF receptor, reduce B-cell survival. On microglia, increased ADAM10 cleavage of a rare variant of the scavenger receptor triggering receptor expressed on myeloid cells 2 may increase susceptibility to Alzheimer's disease. We and others recently showed that ADAM10 interacts with one of six different regulatory tetraspanin membrane proteins, which we termed the TspanC8 subgroup, comprising Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. The TspanC8s are required for ADAM10 exit from the endoplasmic reticulum, and emerging evidence suggests that they dictate ADAM10 subcellular localization and substrate specificity. Therefore, we propose that ADAM10 should not be regarded as a single scissor, but as six different scissors with distinct substrate specificities, depending on the associated TspanC8. In this review, we collate recent transcriptomic data to present the TspanC8 repertoires of leukocytes, and we discuss the potential role of the six TspanC8/ADAM10 scissors in leukocyte development and function.

The transmembrane protein, ADAM10 (a disintegrin and metalloprotease 10), has key physiologic functions-for example, during embryonic development and in the brain. During transit through the secretory pathway, immature ADAM10 (proADAM10) is converted into its proteolytically active, mature form (mADAM10). Increasing or decreasing the abundance and/or activity of mADAM10 is considered to be a therapeutic approach for the treatment of such diseases as Alzheimer's disease and cancer. Yet biochemical detection and characterization of mADAM10 has been difficult. In contrast, proADAM10 is readily detected-for example, in immunoblots-which suggests that mADAM10 is only a fraction of total cellular ADAM10. Here, we demonstrate that mADAM10, but not proADAM10, unexpectedly undergoes rapid, time-dependent degradation upon biochemical cell lysis in different cell lines and in primary neurons, which prevents the detection of the majority of mADAM10 in immunoblots. This degradation required the catalytic activity of ADAM10, was efficiently prevented by adding active site inhibitors to the lysis buffer, and did not affect proADAM10, which suggests that ADAM10 degradation occurred in an intramolecular and autoproteolytic manner. Inhibition of postlysis autoproteolysis demonstrated efficient cellular ADAM10 maturation with higher levels of mADAM10 than proADAM10. Moreover, a cycloheximide chase experiment revealed that mADAM10 is a long-lived protein with a half-life of approximately 12 h. In summary, our study demonstrates that mADAM10 autoproteolysis must be blocked to allow for the proper detection of mADAM10, which is essential for the correct interpretation of biochemical and cellular studies of ADAM10.-Brummer, T., Pigoni, M., Rossello, A., Wang, H., Noy, P. J., Tomlinson, M. G., Blobel, C. P., Lichtenthaler, S. F. The metalloprotease ADAM10 (a disintegrin and metalloprotease 10) undergoes rapid, postlysis autocatalytic degradation.

Objective: TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg 2+ and Ca 2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown., Approach and Results: Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice ( Trpm7 R/R ) causes a marked signaling defect in platelets. Trpm7 R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca 2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and β3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca 2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo., Conclusions: Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca 2+ entry in platelets. The protection of Trpm7 R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target., (© 2017 American Heart Association, Inc.)

The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.

The recruitment of blood leukocytes across the endothelium to sites of tissue infection is central to inflammation, but also promotes chronic inflammatory diseases. A disintegrin and metalloproteinase 10 (ADAM10) is a ubiquitous transmembrane molecular scissor that is implicated in leukocyte transmigration by proteolytically cleaving its endothelial substrates. These include VE-cadherin, a homotypic adhesion molecule that regulates endothelial barrier function, and transmembrane chemokines CX3CL1 and CXCL16, which have receptors on leukocytes. However, a definitive role for endothelial ADAM10 in transmigration of freshly isolated primary leukocytes under flow has not been demonstrated, and the relative importance of distinct ADAM10 substrates is unknown. Emerging evidence suggests that ADAM10 can be regarded as six different molecular scissors with different substrate specificities, depending on which of six TspanC8 tetraspanins it is associated with, but TspanC8s remain unstudied in leukocyte transmigration. In the current study, ADAM10 knockdown on primary HUVECs was found to impair transmigration of freshly isolated human peripheral blood T lymphocytes, but not neutrophils or B lymphocytes, in an in vitro flow assay. This impairment was due to delayed transmigration rather than a complete block, and was overcome in the presence of neutrophils. Transmigration of purified lymphocytes was dependent on ADAM10 regulation of VE-cadherin, but not CX3CL1 and CXCL16. Tspan5 and Tspan17, the two most closely related TspanC8s by sequence, were the only TspanC8s that regulated VE-cadherin expression and were required for lymphocyte transmigration. Therefore endothelial Tspan5- and Tspan17-ADAM10 complexes may regulate inflammation by maintaining normal VE-cadherin expression and promoting T lymphocyte transmigration., (Copyright © 2017 The Authors.)

The Platelet Derived Growth Factor (PDGF) family of ligands have well established functions in the induction of cell proliferation and migration during development, tissue homeostasis and interactions between tumours and stroma. However, the mechanisms by which these actions are executed are incompletely understood. Here we report a differential phosphoproteomics study, using a SILAC approach, of PDGF-stimulated mouse embryonic fibroblasts (MEFs). 116 phospho-sites were identified as up-regulated and 45 down-regulated in response to PDGF stimulation. These encompass proteins involved in cell adhesion, cytoskeleton regulation and vesicle-mediated transport, significantly expanding the range of proteins implicated in PDGF signalling pathways. Included in the down-regulated class was the microtubule bundling protein Collapsin Response Mediator Protein 2 (CRMP2). In response to stimulation with PDGF, CRMP2 was dephosphorylated on Thr514, an event known to increase CRMP2 activity. This was reversed in the presence of micromolar concentrations of the protein phosphatase inhibitor okadaic acid, implicating PDGF-induced activation of protein phosphatase 1 (PP1) in CRMP2 regulation. Depletion of CRMP2 resulted in impairment of PDGF-mediated cell migration in an in vitro wound healing assay. These results show that CRMP2 is required for PDGF-directed cell migration in vitro.

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane protein which is essential for embryonic development through activation of Notch proteins. ADAM10 regulates over 40 other transmembrane proteins and acts as a 'molecular scissor' by removing their extracellular regions. ADAM10 is also a receptor for α-toxin, a major virulence factor of Staphylococcus aureus Owing to the importance of its substrates, ADAM10 is a potential therapeutic target for cancer, neurodegenerative diseases such as Alzheimer's and prion diseases, bacterial infection and inflammatory diseases such as heart attack, stroke and asthma. However, targetting ADAM10 is likely to result in toxic side effects. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals which interact with and regulate specific partner proteins within membrane nanodomains. Tetraspanins appear to have a cone-shaped structure with a cholesterol-binding cavity, which may enable tetraspanins to undergo cholesterol-regulated conformational change. An emerging paradigm for tetraspanin function is the regulation of ADAM10 by the TspanC8 subgroup of tetraspanins, namely Tspan5, 10, 14, 15, 17 and 33. This review will describe how TspanC8s are required for ADAM10 trafficking from the endoplasmic reticulum and its enzymatic maturation. Moreover, different TspanC8s localise ADAM10 to different subcellular localisations and may cause ADAM10 to adopt distinct conformations and cleavage of distinct substrates. We propose that ADAM10 should now be regarded as six different scissor proteins depending on the interacting TspanC8. Therapeutic targetting of specific TspanC8/ADAM10 complexes could allow ADAM10 targetting in a cell type- or substrate-specific manner, to treat certain diseases while minimising toxicity., (© 2017 The Author(s).)

A disintegrin and metalloprotease (ADAM) 10 and ADAM17 are ubiquitous transmembrane "molecular scissors" which proteolytically cleave, or shed, the extracellular regions of other transmembrane proteins. ADAM10 is essential for development because it cleaves Notch proteins to induce Notch signaling and regulate cell fate decisions. ADAM17 is regarded as a first line of defense against injury and infection, by releasing tumor necrosis factor α (TNFα) to promote inflammation and epidermal growth factor (EGF) receptor ligands to maintain epidermal barrier function. However, the regulation of ADAM10 and ADAM17 trafficking and activation are not fully understood. This review will describe how the TspanC8 subgroup of tetraspanins (Tspan5, 10, 14, 15, 17, and 33) and the iRhom subgroup of protease-inactive rhomboids (iRhom1 and 2) have emerged as important regulators of ADAM10 and ADAM17, respectively. In particular, they are required for the enzymatic maturation and trafficking to the cell surface of the ADAMs, and there is evidence that different TspanC8s and iRhoms target the ADAMs to distinct substrates. The TspanC8s and iRhoms have not been studied functionally on platelets. On these cells, ADAM10 is the principal sheddase for the platelet collagen receptor GPVI, and the regulatory TspanC8s are Tspan14, 15, and 33, as determined from proteomic data. Platelet ADAM17 is the sheddase for the von Willebrand factor (vWF) receptor GPIb, and iRhom2 is the only iRhom that is expressed. Induced shedding of either GPVI or GPIb has therapeutic potential, since inhibition of either receptor is regarded as a promising anti-thrombotic therapy. Targeting of Tspan14, 15, or 33 to activate platelet ADAM10, or iRhom2 to activate ADAM17, may enable such an approach to be realized, without the toxic side effects of activating the ADAMs on every cell in the body.

A major impediment when studying primary human endothelial cell function is the resistance of these cells to gene transfer. Here we describe methods for transferring genes into primary endothelial cells prior to incorporation into a static adhesion assay to analyze the adhesion and migration of isolated lymphocytes. Human embryonic kidney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) cells are initially transfected with plasmids containing the lentiviral packaging and envelope genes and the target sequence, such as a gene of interest or short hairpin loop RNA (shRNA). These cells produce lentivirus packaged with this target sequence and are used to transduce primary human umbilical vein endothelial cells (HUVECs). Human peripheral blood lymphocytes (PBLs) isolated from venous blood are co-incubated with lentivirally transduced cytokine-stimulated endothelial cells to assess lymphocyte adhesion in a static adhesion assay. Direct observations of lymphocyte adhesion and migration over a time course can also be made. In general, lentiviral transduction of primary endothelial cells provides an invaluable system to manipulate gene expression levels when studying the cellular adhesion dynamics that regulate leukocyte adhesion and extravasation.

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix., (© 2016. Published by The Company of Biologists Ltd.)

Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke., (© 2015 by The American Society of Hematology.)

Vitamin D stimulates transcription of antiangiogenic and apoptotic factors that may suppress tumours, while vitamin D binding protein (DBP) may be a biomarker in murine lung cancer models. We sought to ascertain whether the vitamin D axis is altered in lung cancer or influences prognosis. 148 lung cancer patients, 68 other intrathoracic cancer patients and 33 noncancer controls were studied for up to 5 yrs. Circulating DBP and vitamin D levels were compared between groups and their effect on survival assessed by Cox regression analysis. Expression of DBP and vitamin D receptor (VDR) was examined in lung cancer cell lines and in normal and tumour lung tissue by Western blot and immunohistochemistry. Low serum DBP levels predicted lung cancer-specific death (p=0.04), and DBP was poorly expressed in lung cancer cells on Western blot and immunohistochemistry. Vitamin D did not predict cancer survival and VDR expression was variable in tumours. Preservation of serum DBP is a significant independent factor associated with better cancer outcome in operated lung cancer patients. Given the established role of DBP in macrophage activation and clearance of abnormal cells, further study on its involvement in lung cancer is merited.

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.

Tetraspanins function as organizers of the cell surface by recruiting specific partner proteins into tetraspanin-enriched microdomains, which regulate processes such as cell adhesion, signalling and intracellular trafficking. Endothelial cells appear to express at least 23 of the 33 human tetraspanins, and a number of recent studies have demonstrated their importance in endothelial cell biology. Tetraspanin CD151 is essential for pathological angiogenesis, which may in part be due to regulation of its main partner proteins, the laminin-binding integrins α3β1, α6β1 and α6β4. CD9 and CD151 are essential for leucocyte recruitment during an inflammatory response, through the formation of pre-assembled nano-platforms containing the adhesion molecules ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1), which ultimately coalesce to form docking structures around captured leucocytes. Tetraspanin CD63 also facilitates leucocyte capture by promoting clustering of the adhesion molecule P-selectin. Finally, Tspan12 is required for blood vessel development in the eye, through regulation of Norrin-induced Frizzled-4 signalling, such that Tspan12 mutations can lead to human disease. Future studies on these and other endothelial tetraspanins are likely to provide further novel insights into angiogenesis and inflammation.

Platelets are crucial for preventing excessive blood loss at sites of injury by plugging holes in damaged blood vessels through thrombus formation. Platelet thrombi can, however, cause heart attack or stroke by blocking diseased vessels upon rupture of atherosclerotic plaques. Current anti-platelet therapy is not effective in all patients and carries a risk of bleeding. As such, a major goal in platelet research is to identify new drug targets to specifically inhibit platelets in disease processes. Tetraspanins are potential candidates because of their capacity to regulate other proteins in microdomains, and their defined roles in cell adhesion and signalling. In the last 6 years, analyses of tetraspanin-deficient mice have suggested that tetraspanins are indeed important for fine-tuning platelet responses. The future characterization of novel regulatory mechanisms in tetraspanin microdomains may lead to new drug targets for the prevention and treatment of heart attack and stroke.

The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor.

Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase-linked and G protein-coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein-coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.

Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet glycoprotein receptors that signal via an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex glycoprotein VI (GPVI)-FcR gamma-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cgamma (PLCgamma). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory activity of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5'-phosphatase, SHIP. Constitutive signaling via Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR gamma-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation.

Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcRgamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM)., Objective: To determine why GPVI-FcRgamma signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin., Methods and Results: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI-FcRgamma signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin., Conclusion: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcRgamma has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.

Background: Glycoprotein VI (GPVI) is a physiologic receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerization of GPVI has been proposed as being necessary for the interaction with collagen, although direct evidence of dimerization has not been reported in cell lines or platelets., Objectives: To investigate oligomerization of GPVI in transfected cell lines and in platelets under non-stimulated conditions., Methods and Results: By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extracellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerization of GPVI under these conditions. A chemical crosslinker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the Fc receptor (FcR) gamma-chain., Conclusions: The present results directly demonstrate GPVI-FcR gamma-chain oligomerization at the surface of the platelet, and thereby add to the growing evidence that oligomerization of GPVI may be a prerequisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

The two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase Cgamma. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters.

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.

Objective: The differentiation of megakaryocytes is characterized by polyploidization and cytoplasmic maturation leading to platelet production. Studying these processes is hindered by the paucity of bone marrow megakaryocytes and their precursors. We describe a method for the expansion and purification of committed megakaryocyte progenitors and demonstrate their usefulness by studying changes in the expression of Ets and GATA family transcription factors throughout megakaryocytopoiesis., Methods: A two-step serum-free method was developed. Cells isolated using this method were analyzed for surface marker expression by flow cytometry, and for their ability to differentiate using single-cell culture. Purified progenitors were induced to differentiate and analyzed with respect to their ploidy by flow cytometry and expression of specific genes by RT-PCR., Results: A population of Lin- c-kit+ CD45+ CD41+ CD31+ CD34low CD9low FcgammaRII/IIIlow Sca-1med/low committed megakaryocyte progenitors was purified. These cells could be differentiated efficiently, achieving ploidy of up to 128N. Analysis of RNA demonstrated the expected increases in expression of key megakaryocyte-associated genes. RT-PCR analysis also revealed that a range of Ets and GATA factors are expressed, their individual levels and patterns of expression varying widely. Surprisingly, we find that GATA-6 is specifically expressed in late differentiated megakaryocytes and has the potential to regulate megakaryocyte-expressed genes in cooperation with Ets factors., Conclusion: Purified primary megakaryocytic progenitors are able to differentiate as a cohort into fully mature megakaryocytes. The number of cells obtainable, and the synchrony of the differentiation process, facilitates analysis of the dynamics of molecular processes involved in megakaryocytopoiesis. The expression pattern of Ets and GATA family transcription factors reveals the complexity of the involvement of these key megakaryocytic regulators. The finding of GATA-6 expression and demonstration of its functional activity suggests a novel mechanism for the regulation of certain genes late in megakaryocytopoiesis.

The Tec family of protein-tyrosine kinases (PTKs), that includes Tec, Itk, Btk, Bmx, and Txk, plays an essential role in phospholipase Cgamma (PLCgamma) activation following antigen receptor stimulation. This function requires activation of phosphatidylinositol 3-kinase (PI 3-kinase), which promotes Tec membrane localization through phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P(3)) generation. The mechanism of negative regulation of Tec family PTKs is poorly understood. In this study, we show that the inositol 5' phosphatases SHIP1 and SHIP2 interact preferentially with Tec, compared with other Tec family members. Four lines of evidence suggest that SHIP phosphatases are negative regulators of Tec. First, SHIP1 and SHIP2 are potent inhibitors of Tec activity. Second, inactivation of the Tec SH3 domain, which is necessary and sufficient for SHIP binding, generates a hyperactive form of Tec. Third, SHIP1 inhibits Tec membrane localization. Finally, constitutively targeting Tec to the membrane relieves SHIP1-mediated inhibition. These data suggest that SHIP phosphatases can interact with and functionally inactivate Tec by de-phosphorylation of local PtdIns 3,4,5-P(3) and inhibition of Tec membrane localization.

The Tec protein tyrosine kinase is the founding member of a family that includes Btk, Itk, Bmx, and Txk. Btk is essential for B-cell receptor signaling, because mutations in Btk are responsible for X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice, whereas Itk is involved in T-cell receptor signaling. Tec is expressed in both T and B cells, but its role in antigen receptor signaling is not clear. In this study, we show that Tec protein is expressed at substantially lower levels in primary T and B cells relative to Itk and Btk, respectively. However, Tec is up-regulated upon T-cell activation and in Th1 and Th2 cells. In functional experiments that mimic Tec up-regulation, we find that Tec overexpression in lymphocyte cell lines is sufficient to induce phospholipase Cgamma (PLC-gamma) phosphorylation and NFAT (nuclear factor of activated T cells) activation. In contrast, overexpression of Btk, Itk, or Bmx does not induce NFAT activation. Tec-induced NFAT activation requires PLC-gamma, but not the adapters LAT, SLP-76, and BLNK, which are required for Btk and Itk to couple to PLC-gamma. Finally, we show that the unique effector function for Tec correlates with a unique subcellular localization. We hypothesize that Tec functions in activated and effector T lymphocytes to induce the expression of genes regulated by NFAT transcription factors.