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Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists
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 a...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10082178/ https://www.ncbi.nlm.nih.gov/pubmed/37029319 http://dx.doi.org/10.1038/s42003-023-04766-6 |
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author | 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. |
author_facet | 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. |
author_sort | Clark, Joanne C. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-10082178 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-100821782023-04-09 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. Commun Biol Article 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. Nature Publishing Group UK 2023-04-07 /pmc/articles/PMC10082178/ /pubmed/37029319 http://dx.doi.org/10.1038/s42003-023-04766-6 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article 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. Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists |
title | Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists |
title_full | Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists |
title_fullStr | Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists |
title_full_unstemmed | Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists |
title_short | Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists |
title_sort | divalent nanobodies to platelet clec-2 can serve as agonists or antagonists |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10082178/ https://www.ncbi.nlm.nih.gov/pubmed/37029319 http://dx.doi.org/10.1038/s42003-023-04766-6 |
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