Publications by authors named "Andrew C Pearce"

27 Publications

  • Page 1 of 1

GSK137, a potent small molecule BCL6 inhibitor with in vivo activity, suppresses antibody responses in mice.

J Biol Chem 2021 Jul 15:100928. Epub 2021 Jul 15.

Leicester Cancer Research Centre and Ernest and Helen Scott Haematological Research Unit, University of Leicester, Leicester LE1 7HB, UK. Electronic address:

BCL6 is a zinc finger transcriptional repressor possessing a BTB-POZ domain, which is required for homodimerization and association with co-repressors. BCL6 has multiple roles in normal immunity, autoimmunity and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high affinity antibody responses to T-dependent antigens. The co-repressor binding groove in the BTB-POZ domain is a potential target for small compound mediated therapy. Several inhibitors targeting this binding groove have been described but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 = 8 and a cellular pIC50 = 7.3 for blocking binding of a peptide derived from the co-repressor silencing mediator for retinoid or thyroid-hormone receptors (SMRT) to the BCL6 BTB-POZ domain. The compound has good solubility (128 μg/mL) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6 expressing B-cell lymphoma lines, although there was modest dose dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed IgG responses and reduced numbers of germinal centers and germinal center B-cells following immunisation of mice with the hapten trinitro-phenol (TNP). Overall, we report a novel small molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.
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http://dx.doi.org/10.1016/j.jbc.2021.100928DOI Listing
July 2021

The development of highly potent and selective small molecule correctors of Z α-antitrypsin misfolding.

Bioorg Med Chem Lett 2021 06 19;41:127973. Epub 2021 Mar 19.

UCL Respiratory, Rayne Institute, University College London, London WC1E 6JF, United Kingdom.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.
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http://dx.doi.org/10.1016/j.bmcl.2021.127973DOI Listing
June 2021

A Potent and Selective Kallikrein-5 Inhibitor Delivers High Pharmacological Activity in Skin from Patients with Netherton Syndrome.

J Invest Dermatol 2021 Mar 18. Epub 2021 Mar 18.

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1163, Laboratory of Genetic Skin Diseases, Imagine Institute, Paris, France.

Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.
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http://dx.doi.org/10.1016/j.jid.2021.01.029DOI Listing
March 2021

Development of a small molecule that corrects misfolding and increases secretion of Z α -antitrypsin.

EMBO Mol Med 2021 Mar 29;13(3):e13167. Epub 2021 Jan 29.

GlaxoSmithKline, Cambridge, MA, USA.

Severe α -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α -antitrypsin. The lead compound blocks Z α -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α -antitrypsin deficiency.
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http://dx.doi.org/10.15252/emmm.202013167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933930PMC
March 2021

The molecular species responsible for α -antitrypsin deficiency are suppressed by a small molecule chaperone.

FEBS J 2021 04 11;288(7):2222-2237. Epub 2020 Nov 11.

UCL Respiratory, Division of Medicine, University College London, UK.

The formation of ordered Z (Glu342Lys) α -antitrypsin polymers in hepatocytes is central to liver disease in α -antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation-selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse-chase experiments demonstrate that Z α -antitrypsin accumulates as short-chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half-life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by-product thereof) was identified in the cells by a conformation-specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α -antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease.
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http://dx.doi.org/10.1111/febs.15597DOI Listing
April 2021

Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors.

Bioorg Med Chem Lett 2019 10 7;29(20):126675. Epub 2019 Sep 7.

INSERM UMR1163 Laboratory of Genetic Skin Diseases, Imagine Institute and Université Paris Descarte-Sorbonne Paris Cité, Paris, France.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.
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http://dx.doi.org/10.1016/j.bmcl.2019.126675DOI Listing
October 2019

Evaluation of a crystallographic surrogate for kallikrein 5 in the discovery of novel inhibitors for Netherton syndrome.

Acta Crystallogr F Struct Biol Commun 2019 May 26;75(Pt 5):385-391. Epub 2019 Apr 26.

INSERM UMR1163 Laboratory of Genetic Skin Diseases, Imagine Institute and Université Paris Descartes-Sorbonne Paris Cité, Paris, France.

The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.
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http://dx.doi.org/10.1107/S2053230X19003169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497096PMC
May 2019

Optimization of Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors for Duration of Action, as an Inhaled Therapy for Lung Remodeling in Pulmonary Arterial Hypertension.

J Med Chem 2016 09 19;59(17):7901-14. Epub 2016 Aug 19.

Genomics Institute of the Novartis Research Foundation , 10675 John Jay Hopkins Drive, San Diego, California 92121, United States.

A series of potent PDGFR inhibitors has been identified. The series was optimized for duration of action in the lung. A novel kinase occupancy assay was used to directly measure target occupancy after i.t. dosing. Compound 25 shows 24 h occupancy of the PDGFR kinase domain, after a single i.t. dose and has efficacy at 0.03 mg/kg, in the rat moncrotaline model of pulmonary arterial hypertension. Examination of PK/PD data from the optimization effort has revealed in vitro:in vivo correlations which link duration of action in vivo with low permeability and high basicity and demonstrate that nonspecific binding to lung tissue increases with lipophilicity.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00703DOI Listing
September 2016

Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II.

Blood 2011 Oct 7;118(17):4750-8. Epub 2011 Sep 7.

Novartis Institutes for BioMedical Research, West Sussex, UK.

Previous studies from our group have demonstrated that bone morphogenetic protein receptor-II (BMPR-II), expressed on pulmonary artery endothelial cells, imparts profound anti-inflammatory effects by regulating the release of proinflammatory cytokines and promoting barrier function by suppressing the transmigration of leukocytes into the pulmonary vessel wall. Here we demonstrate that, in mice with endothelial-specific loss of BMPR-II expression (L1Cre(+);Bmpr2(f/f)), reduction in barrier function and the resultant pulmonary hypertension observed in vivo are the result of increased leukocyte recruitment through increased CXCR1/2 signaling. Loss of endothelial expressed BMPR-II leads to elevated plasma levels of a wide range of soluble mediators important in regulating leukocyte migration and extravasation, including the CXCR1/2 ligand, KC. Treatment of L1Cre(+);Bmpr2(f/f) mice with the CXCR1/2 antagonist SCH527123 inhibits leukocyte transmigration into lung and subsequently reverses the pulmonary hypertension. Our data have uncovered a previously unrecognized regulatory function of BMPR-II, which acts to regulate the expression of CXCR2 on endothelial cells, suggesting that increased CXCR2 signaling may also be a feature of the human pathology and that CXCR1/2 pathway antagonists may represent a novel therapeutic approach for treating pulmonary hypertension because of defects in BMPR-II expression.
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http://dx.doi.org/10.1182/blood-2011-05-347393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208288PMC
October 2011

SCL-mediated regulation of the cell-cycle regulator p21 is critical for murine megakaryopoiesis.

Blood 2011 Jul 19;118(3):723-35. Epub 2011 May 19.

Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

Megakaryopoiesis is a complex process that involves major cellular and nuclear changes and relies on controlled coordination of cellular proliferation and differentiation. These mechanisms are orchestrated in part by transcriptional regulators. The key hematopoietic transcription factor stem cell leukemia (SCL)/TAL1 is required in early hematopoietic progenitors for specification of the megakaryocytic lineage. These early functions have, so far, prevented full investigation of its role in megakaryocyte development in loss-of-function studies. Here, we report that SCL critically controls terminal megakaryocyte maturation. In vivo deletion of Scl specifically in the megakaryocytic lineage affects all key attributes of megakaryocyte progenitors (MkPs), namely, proliferation, ploidization, cytoplasmic maturation, and platelet release. Genome-wide expression analysis reveals increased expression of the cell-cycle regulator p21 in Scl-deleted MkPs. Importantly, p21 knockdown-mediated rescue of Scl-mutant MkPs shows full restoration of cell-cycle progression and partial rescue of the nuclear and cytoplasmic maturation defects. Therefore, SCL-mediated transcriptional control of p21 is essential for terminal maturation of MkPs. Our study provides a mechanistic link between a major hematopoietic transcriptional regulator, cell-cycle progression, and megakaryocytic differentiation.
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http://dx.doi.org/10.1182/blood-2011-01-328765DOI Listing
July 2011

Differential roles of the PKC novel isoforms, PKCdelta and PKCepsilon, in mouse and human platelets.

PLoS One 2008 24;3(11):e3793. Epub 2008 Nov 24.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

Background: Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.

Methodology/principal Findings: In this study, we focus on the role of two novel PKC isoforms, PKCdelta and PKCepsilon, in both mouse and human platelets. PKCdelta is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCdelta undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCepsilon, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCepsilon in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRgamma-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.

Conclusions: These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms delta and epsilon in human and mouse platelets and a selective role for PKCepsilon in signalling through GPVI.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583049PMC
June 2009

G6b-B inhibits constitutive and agonist-induced signaling by glycoprotein VI and CLEC-2.

J Biol Chem 2008 Dec 27;283(51):35419-27. Epub 2008 Oct 27.

Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham, B15 2TT, UK.

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.
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http://dx.doi.org/10.1074/jbc.M806895200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602894PMC
December 2008

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

Biochem J 2008 Apr;411(1):133-40

Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.
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http://dx.doi.org/10.1042/BJ20071216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749330PMC
April 2008

The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade.

J Biol Chem 2007 Apr 5;282(17):12397-409. Epub 2007 Mar 5.

Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom.

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.
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http://dx.doi.org/10.1074/jbc.M609558200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1997429PMC
April 2007

Vav family proteins are required for optimal regulation of PLCgamma2 by integrin alphaIIbbeta3.

Biochem J 2007 Feb;401(3):753-61

Centre for Cardiovascular Sciences, Institute of Biomedical Research, Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.
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http://dx.doi.org/10.1042/BJ20061508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1770845PMC
February 2007

DC-SIGN and CLEC-2 mediate human immunodeficiency virus type 1 capture by platelets.

J Virol 2006 Sep;80(18):8951-60

Nikolaus-Fiebiger-Center for Molecular Medicine, University Erlangen-Nürnberg, Glückstrasse 6, 91054 Erlangen, Germany.

Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.
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http://dx.doi.org/10.1128/JVI.00136-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563896PMC
September 2006

Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow.

J Biol Chem 2005 Nov 29;280(47):39474-84. Epub 2005 Sep 29.

Centre for Cardiovascular Sciences, the Institute of Biomedical Research, School of Biosciences, University of Birmingham, United Kingdom.

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo.
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http://dx.doi.org/10.1074/jbc.M504672200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1395485PMC
November 2005

A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2.

Blood 2006 Jan 20;107(2):542-9. Epub 2005 Sep 20.

Department of Clinical and Laboratory Medicine, University of Yamanashi, Shimokato Tamaho Nakakoma, Yamanashi 409-3898, Japan.

The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.
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http://dx.doi.org/10.1182/blood-2005-05-1994DOI Listing
January 2006

Role of the p110delta PI 3-kinase in integrin and ITAM receptor signalling in platelets.

Platelets 2005 May-Jun;16(3-4):191-202

Centre for Cardiovascular Sciences, Division of Medical Sciences, Institute of Biomedical Research, Wolfson Drive, The Medical School, University of Birmingham, Edgbaston, Birmingham, UK.

We have investigated the function of the p110delta catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in platelets using p110delta knock-out (p110delta(-/-)) mice and p110delta knock-in (p110delta(D910A/D910A)) mice, which express a catalytically inactive form of the enzyme. Aggregation to threshold concentrations of the GPVI-specific agonist, CRP, was partially reduced in p110delta(-/-) and p110delta(D910A/D910A) platelets. This inhibition was overcome by higher concentrations of CRP. The degree of inhibition was considerably weaker than that induced by LY294002 and wortmannin, which inhibit all isoforms of PI 3-kinase. p110delta(-/-) platelets showed decreased spreading on fibrinogen- or von Willebrand factor (VWF)-coated surfaces under static conditions, whereas they spread normally on collagen. LY294002 had a more pronounced inhibitory effect on spreading on all three surfaces. Adhesion and aggregate formation of p110delta(-/-) platelets to collagen or fibrinogen/VWF at intermediate/high rates of shear were normal. This study demonstrates a minor role for the p110delta catalytic subunit in mediating platelet activation by the collagen receptor GPVI and integrin alphaIIbeta3. The more pronounced inhibitory effect of LY294002 and wortmannin indicates that other isoforms of PI 3-kinase play a more significant role in signalling by the two platelet glycoprotein receptors.
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http://dx.doi.org/10.1080/09537100400016711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868960PMC
October 2005

Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen.

J Biol Chem 2004 Dec 27;279(52):53955-62. Epub 2004 Sep 27.

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK.

Vav family proteins are guanine nucleotide exchange factors for the Rho/Rac family of small GTP-binding proteins. In addition, they have domains that mediate protein-protein interactions, including one Src homology 2 (SH2) and two Src homology 3 (SH3) domains. Vav1, Vav2, and Vav3 play a crucial role in the regulation of phospholipase C gamma (PLC gamma) isoforms by immuno-tyrosine-based activation motif (ITAM)-coupled receptors, including the T- and B-cell antigen receptors. We have reported in platelets, however, that Vav1 and Vav2 are not required for activation of PLC gamma 2 in response to stimulation of the ITAM-coupled collagen receptor glycoprotein VI (GPVI). Here we report that Vav3 is tyrosinephosphorylated upon activation of GPVI but that Vav3-deficient platelets also exhibit a normal response upon activation of the ITAM receptor. In sharp contrast, platelets deficient in both Vav1 and Vav3 show a marked inhibition of aggregation and spreading upon activation of GPVI, which is associated with a reduction in tyrosine phosphorylation of PLC gamma 2. The phenotype of Vav1/2/3 triple-deficient platelets is similar to that of Vav1/3 double-deficient cells. These results demonstrate that Vav3 and Vav1 play crucial but redundant roles in the activation of PLC gamma 2 by GPVI. This is the first time that absolute redundancy between two protein isoforms has been observed with respect to the regulation of PLC gamma 2 in platelets.
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http://dx.doi.org/10.1074/jbc.M410355200DOI Listing
December 2004

Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry.

Proteomics 2004 Mar;4(3):656-68

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, UK.

Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.
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http://dx.doi.org/10.1002/pmic.200300665DOI Listing
March 2004

Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins.

Blood 2004 Mar 26;103(6):2088-95. Epub 2003 Nov 26.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Rd, Oxford, OX1 3QU, United Kingdom.

We have applied a proteomics approach to analyze signaling cascades in human platelets stimulated by thrombin receptor activating peptide (TRAP). By analyzing basal and TRAP-activated platelets using 2-dimensional gel electrophoresis (2-DE), we detected 62 differentially regulated protein features. From these, 41 could be identified by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) and were found to derive from 31 different genes, 8 of which had not previously been reported in platelets, including the adapter downstream of tyrosine kinase 2 (Dok-2). Further studies revealed that the change in mobility of Dok-2 was brought about by tyrosine phosphorylation. Dok-2 tyrosine phosphorylation was also found to be involved in collagen receptor, glycoprotein VI (GPVI), signaling as well as in outside-in signaling through the major platelet integrin, alpha IIIb beta 3. These studies also provided the first demonstration of posttranslational modification of 2 regulator of G protein signaling (RGS) proteins, RGS10 and 18. Phosphorylation of RGS18 was mapped to Ser49 by MS/MS analysis. This study provides a new approach for the identification of novel signaling molecules in activated platelets, providing new insights into the mechanisms of platelet activation and building the basis for the development of therapeutic agents for thrombotic diseases.
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http://dx.doi.org/10.1182/blood-2003-07-2392DOI Listing
March 2004

The heptapeptide LSARLAF mediates platelet activation through phospholipase Cgamma2 independently of glycoprotein IIb-IIIa.

Biochem J 2004 Feb;378(Pt 1):193-9

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.

The seven-amino-acid peptide LSARLAF has been reported to activate platelets via the integrin GPIIb-IIIa (glycoprotein IIb-IIIa). Activation by LSARLAF is reinforced by release of ADP and thromboxanes, but the initiating event in the signalling cascade is not known. In the present study, we demonstrate that LSARLAF stimulates Src kinase-dependent tyrosine phosphorylation of many of the proteins in the GPIIb-IIIa cascade, including the tyrosine kinase Syk, the adapter SLP-76 (SH2-containing leucocyte phosphoprotein of 76 kDa) and PLCgamma2 (phospholipase Cgamma2). A critical role for PLCgamma2 in signalling by LSARLAF was demonstrated by abolition of aggregation in PLCgamma2-/- murine platelets to low concentrations of the peptide, although a partial recovery was seen with higher concentrations. In sharp contrast with the GPIIb-IIIa-regulated signalling cascade, aggregation was inhibited in murine platelets deficient in the adapter LAT (linker for activation of T-cells) and the Fc receptor gamma-chain. Aggregation was also partially inhibited by the cholesterol-lowering reagent, beta-methyl-cyclodextrin, at concentrations that disrupt membrane rafts, but do not interfere with signalling by GPIIb-IIIa. Furthermore, LSARLAF also stimulated tyrosine phosphorylation in GPIIb-deficient murine platelets, confirming that the integrin is not critical for activation of intracellular signalling pathways. LSARLAF also stimulated Ca2+ elevation in RBL-2H3 cells, which lack the platelet glycoproteins GPIIb, GPVI and GPIb. These results demonstrate that LSARLAF activates platelets through a PLCgamma2-dependent pathway that lies downstream of Src kinases and which is partially dependent on the Fc receptor gamma-chain, LAT and lipid rafts. The mechanism of cell activation by LSARLAF remains to be established, although the present results indicate that more than one surface glycoprotein may mediate this response.
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http://dx.doi.org/10.1042/BJ20031298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223919PMC
February 2004

A critical role for phospholipase Cgamma2 in alphaIIbbeta3-mediated platelet spreading.

J Biol Chem 2003 Sep 27;278(39):37520-9. Epub 2003 Jun 27.

Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.

The interaction of fibrinogen with the integrin alphaIIbbeta3 plays a crucial role in platelet adhesion and platelet activation leading to the generation of intracellular signals that nucleate the reorganization of the cytoskeleton. Presently, we have only a limited understanding of the signaling cascades and effector proteins through which changes in the cytoskeletal architecture are mediated. The present study identifies phospholipase Cgamma2 (PLCgamma2) as an important target of the Src-dependent signaling cascade regulated by alphaIIbbeta3. Real time phasecontrast microscopy is used to show that formation of filopodia and lamellapodia in murine platelets on a fibrinogen surface is dramatically inhibited in the absence of PLCgamma2. Significantly, the formation of these structures is mediated by Ca2+ elevation and activation of protein kinase C, both directly regulated by PLC activity. With the involvement of Syk, SLP-76, and Btk, alphaIIbbeta3-induced PLCgamma2 activation partly overlaps with the pathway used by the collagen receptor glycoprotein VI. Important differences, however, exist between the two signaling cascades in that activation of PLCgamma2 by alphaIIbbeta3 is unaltered in murine platelets, which lack the FcR gamma-chain or the adaptor LAT, but is abolished in the presence of cytochalasin D. Therefore, PLCgamma2 plays not only a crucial role in activation of alphaIIbbeta3 by collagen receptors but also in alphaIIbbeta3-mediated responses.
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http://dx.doi.org/10.1074/jbc.M305077200DOI Listing
September 2003

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C.

Blood 2002 Nov;100(10):3561-9

Department of Pharmacology, University of Oxford, United Kingdom.

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.
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http://dx.doi.org/10.1182/blood.V100.10.3561DOI Listing
November 2002

Towards complete analysis of the platelet proteome.

Proteomics 2002 Mar;2(3):288-305

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford Oxford, UK.

Platelets exert a crucial function in haemostasis, wound repair, and the formation of vascular plugs, underlying thrombotic diseases such as stroke and myocardial infarction. Analysis of platelet biochemistry is largely dependent on protein analysis as platelets are anucleated cells providing little analytical target for DNA or RNA based strategies. Here we present data from our analysis of the human platelet proteome, the entire set of proteins building a platelet at a given point in time. Proteins were separated by two-dimensional electrophoresis (2-DE) using broad and narrow range pH gradients in the isoelectric focusing step. Consequently, a high-resolution 2-DE proteome map has been generated that comprises approximately 2300 different protein features. From the 536 protein features detected in the 4-5 pI range 284 features were identified by electrospray ionisation time of flight tandem mass spectrometry. These 284 proteins originate from 123 different open reading frames. This includes the five human proteins KIAA0193, KIAA0573, KIAA0830, WUGSC:H_DJ0777O23 protein, and cytokine receptor related protein 4, all isolated for the first time. The data are discussed with regard to proteome characteristics, protein function, and the high prevalence of signalling molecules. This study contributes to a more thorough and holistic understanding of platelet biology, helping to build the basis for future identification of new drug targets and therapeutic strategies.
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http://dx.doi.org/10.1002/1615-9861(200203)2:3<288::aid-prot288>3.0.co;2-0DOI Listing
March 2002
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