Page 182 - 2022_02-Haematologica-web

M.H. Lundberg Slingsby et al.
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Figure 3. Effect of antisense oligonucleotides on platelet activation marker P-selectin and stromal cell derived factor 1a release in human platelet-rich plasma and whole blood and correlation with individual platelet glycoprotein VI receptor levels. Platelet activation was identified through increases in P-selectin expression on the platelet surface after 30 min treatment with vehicle (HEPES, 10 mM) or thrombin receptor activating peptide (TRAP, 25 mM, to activate the platelets) or 5 mM of the antisense oligonucleotides (ASO): 104838 (2’MOE ASO), 501861 (2’MOE ASO), 120704 (CpG ASO) and ODN 2395 (CpG ASO), assessed by flow cytometry in (A) platelet-rich plasma (PRP) (from 9 human donors, the 2’MOE ASO 487660 was included in 3 of the experiments) and (B) whole blood (WB) (from 8 human donors) pretreated or not with the spleen tyrosine kinase (SYK) inhibitor PRT-060318 (10 mM) before addition of vehicle, TRAP (25 μM) or ASO (5 μM) that increased P-selectin in PRP (i.e., 104838, 501861, 120704 and ODN 2395). *P<0.05 compared to vehicle by one-way analysis of variance (ANOVA), with the Dunnett post-test. #P<0.05 paired Student t-test for the effect of the SYK inhibitor. (C, D) Individual donor platelet glycoprotein (GP)VI receptor levels (median fluorescence intensity, MFI) were correlated to the same individual platelet P-selectin levels after treatment with: (C) 2’MOE ASO 104838 or 501861, and (D) CpG ASO 120704 or ODN 2395, (7 human donors). P<0.05 by Pearson correlation analysis. (E, F) Stromal cell derived factor 1α (SDF1a), released from platelets upon activation, was measured by Mesoscale U-plex multiplex assay in (E) PRP and (F) WB blood treated for 30 min with vehicle (HEPES), TRAP (25 mM) or 1, 5 or 10 mM of the ASO in blood from four human donors. *P<0.05 compared to vehicle by one-way ANOVA, with Dunnett post-test.
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haematologica | 2022; 107(2)