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CC BY-NC-ND 4.0 · Thromb Haemost 2022; 122(08): 1361-1368
DOI: 10.1055/a-1772-1069
Cellular Haemostasis and Platelets

Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist

Luis A. Morán 
1   Center 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
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
,
Ying Di
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
,
Marcin A. Sowa
1   Center 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
3   Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences, University of Reading, Reading, United Kingdom
,
Lidia Hermida-Nogueira
1   Center 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
,
María N. Barrachina    †
1   Center 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
,
Eleyna Martin
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
,
Joanne C. Clark 
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
,
Todd H. Mize
4   Advanced Mass Spectrometry Facility, Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, United Kingdom
,
Johannes A. Eble 
5   Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany
,
David Moreira
1   Center 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
,
Alice Y. Pollitt 
3   Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences, University of Reading, Reading, United Kingdom
,
María I. Loza
1   Center 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
,
Eduardo Domínguez
1   Center 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
,
Steve P. Watson 
2   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
,
Ángel García 
1   Center 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
› Author Affiliations Funding L.A.M. and M.S. are supported by the European Union's Horizon 2020 Research and Innovation Program (Marie Sklodowska-Curie grant agreement No. 766118); S.P.W. is a British Heart Foundation Chair (CH03/003); and E.M. is supported by the Wellcome Trust (204951/Z/16/Z). A.G. is supported by the Spanish Ministry of Science and Innovation (Grant No. PID2019–108727RB-I00). J.A.E. is financially supported by the Interdisciplinary Center of Clinical Research (IZKF) of the University of Münster (grant no. Ebl-A/009/21). L.H-.N. receives financial support from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (Centro Singular de investigación de Galicia accreditation 2019–2022, ED431G 2019/02; predoctoral grant 2018 Call).
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Abstract

Background CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2.

Objectives To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2.

Methods ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin–CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand.

Results and Conclusion We developed a CLEC-2–podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)′2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets.

Keywords

platelets - CLEC-2 - high-throughput screening - small-molecule ligands - proanthocyanidin

Informed Consent Statement

Informed consents were obtained from all the individuals involved in the study under the local ethics ERN_11–0175: the regulation of activation of platelets; and also following the approval by the Galician Investigation Ethics Committee (2009/270). The study was developed according to the principles outlined in the Declaration of Helsinki. Mouse experiments were performed in accordance with the UK law (Animal Scientific Procedures Act 1986) with approval of the local ethics committee and UK Home Office approval under PP9677279 to the University of Birmingham.


Author Contributions

L.A.M. performed and designed the experiments, analyzed the data, wrote and edited the manuscript. Y.D., M.A.S., L.H-.N., M.N.B., E.M., J.C.C., T.H.M., and D.M. performed the experiments; M.I.L. provided key reagents and analytical tools; and A.Y.P. provided supervision and edited the manuscript. E.D. designed the study, analyzed the data, and wrote the manuscript; S.P.W and A.G. provided supervision, funding, designed the study, reviewed data, and wrote and edited the manuscript. J.A.E. purified and provided rhodocytin.


† Current affiliation: Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, Boston, United States.


Supplementary Material



Publication History

Received: 17 September 2021

Accepted: 15 January 2022

Accepted Manuscript online:
15 February 2022

Article published online:
28 June 2022

© 2022. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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