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Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists.
Clark, Joanne C; Martin, Eleyna M; Morán, Luis A; Di, Ying; Wang, Xueqing; Zuidscherwoude, Malou; Brown, Helena C; Kavanagh, Deirdre M; Hummert, Johan; Eble, Johannes A; Nieswandt, Bernhard; Stegner, David; Pollitt, Alice Y; Herten, Dirk-Peter; Tomlinson, Michael G; García, Angel; Watson, Steve P.
Afiliación
  • Clark JC; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. j.clark.5@bham.ac.uk.
  • Martin EM; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, UK. j.clark.5@bham.ac.uk.
  • Morán LA; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • Di Y; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • Wang X; Centre for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de Santiago de Compostela, and Instituto de Investigación Sanitaria de Santiago (IDIS), Santiago de Compostela, Spain.
  • Zuidscherwoude M; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • Brown HC; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • Kavanagh DM; Centre for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de Santiago de Compostela, and Instituto de Investigación Sanitaria de Santiago (IDIS), Santiago de Compostela, Spain.
  • Hummert J; School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • Eble JA; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • Nieswandt B; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, UK.
  • Stegner D; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • Pollitt AY; Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany.
  • Herten DP; Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 QU3, UK.
  • Tomlinson MG; Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
  • García A; Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, UK.
  • Watson SP; Institute for Physiological Chemistry & Pathobiochemistry, University of Münster, Waldeyerstraße 15, 48149, Münster, Germany.
Commun Biol ; 6(1): 376, 2023 04 07.
Article en En | MEDLINE | ID: mdl-37029319
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Lectinas Tipo C / Anticuerpos de Dominio Único Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: Commun Biol Año: 2023 Tipo del documento: Article Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Lectinas Tipo C / Anticuerpos de Dominio Único Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: Commun Biol Año: 2023 Tipo del documento: Article Pais de publicación: Reino Unido