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Letters to the Editor
tion through activation of integrin GPIIbIIIa at low con- centrations and agglutination at high concentrations. Recombinant CLEC-2 inhibits platelet activation by both mechanisms suggesting that this may represent a novel form of therapeutic intervention to inhibit platelet activa- tion in hemolytic disease.
Ethical approval for collecting blood from healthy vol- unteers was granted by Birmingham University Internal Ethical Review (ERN_11-0175). Mice experiments were performed in accordance with UK laws (Animal [Scientific Procedures] Act 1986) with approval of the local ethical committee and UK Home Office approval (PPL P0e98D513).
Human and mouse washed platelets were prepared as previously described.12,13 Platelet aggregation was assessed using light transmission aggregometry for 6 minutes (ChronoLog, Havertown, PA, USA). Fresh syn- thetic hemin solution was freshly prepared (Frontiers Scientific, USA) for each experiment. PP2 (Tocris/Biotechne Ltd), PRT-060318 (Caltag medsys- tems), indomethacin (Sigma-Aldrich), cangrelor (The Medicines Company), Ibrutinib (Stratech Scientific), N- Acetyl-L-cysteine (Sigma-Aldrich) and TAK-242 (Sigma- Aldrich) were preincubated for 5 minutes (min) at 37oC with platelets prior to addition of hemin. Human (h) and mouse (m) CLEC-2 and GPVI were produced as Fc fusion proteins (hFc-CLEC-2, hFc-GPVI, Fc-mCLEC-2) in human embryonic kidney HEK293 cells and purified using chro- matography affinity. Recombinant human and mouse CLEC-2 were preincubated with hemin for 15 min at 37oC before addition to platelets. Following aggregation, platelets were centrifuged at 1,000g for 10 min and LDH was measured in the supernatant using CyQUANTTM LDH Cytotoxicity Assay (ThermoFisher Scientific). Clec- 2 deficient mice (Clec-1bfl/fl PF4cre) and littermate con- trols were previously described.14
Platelet activation (106 platelets) by different concen- trations of hemin (20 min at 37oC) was assessed by flow cytometry with antibodies against CD62P (PE/Cy5 anti- human CD62P antibody, biolegend) and activated GPIIbIIIa (Alexa Fluor® 647 anti-human CD41/CD61 antibody PAC1, Biolegend) using BD Accuri C6 Plus flow cytometer (BD Bioscience). For western blotting, Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (#2710), Phospho-PLCγ2 (Tyr759) antibody (#3874), Syk antibody (#2712) and PLCγ2 antibody (#3872) were used (Cell sig- nalling). Protein phosphorylation was assessed after 6 min of aggregation as previously described.12
The binding of human and mouse dimeric CLEC-2 to hemin was performed at 25°C and assessed using surface plasma resonance and UV-spectroscopy methods.15
Joshua H. Bourne,1* Martina Colicchia,1* Ying Di,1
Eleyna Martin,1 Alexander Slater,1 Lubka T. Roumenina,2 Jordan D. Dimitrov,2 Steve P. Watson,1,3 and Julie Rayes1,3
1Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK; 2Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, Paris, France and 3Center of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, The Midlands, UK
*JHB and MC contributed equally as co-first authors
Correspondence:
JULIE RAYES - j.rayes@bham.ac.uk
doi:10.3324/haematol.2020.246488
Disclosures: no conflicts of interests to disclose.
Contributions: JHB, MC performed experiments and analyzed data; YD, AS, EM generated reagents; LTR contributed to data inter- pretation; JDD contributed to research design, performed experiments and interpreted data; SPW contributed to research and data interpre- tation and provided reagents; JR designed and performed research, col- lected data, analyzed and interpreted data and wrote the manuscript. All authors reviewed and approved the manuscript.
Acknowledgments: the authors would like to thank Dr B. Grygielska for genotyping of mice.
Funding: this work was supported by a BHF Accelerator Award (AA/18/2/34218), BHF programme grant (RG/13/18/30563), Wellcome Trust 4 year studentship (204951), Wellcome Trust Joint Investigator Award (204951/Z/16/Z) and the Centre of Membrane Proteins and Receptors. JDD hold an H2020 ERC starting grant (CoBABATI 678905). SPW holds a BHF Chair (CH/03/003).
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