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M.H. Lundberg Slingsby et al.
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Figure 5. Immunostimulatory effects after 30 minutes and 6 hours in antisense oligonucleotide-treated whole blood. Whole blood (WB) from four human donors was incubated with vehicle (HEPES, 10 mM), thrombin receptor activating peptide (TRAP, 25 mM), lipopolysaccharide (LPS 0.01 mg/mL) or 1, 5 or 10 mM of the anti- sense oligonucleotides (ASO). Interleukin 8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) release was measured in plasma (pg/mL), using an MSD U-plex assay, after incubation for 30 min and 6 h at 37°C in 5% CO2. (A) IL-8 after 30 min. (B) MCP-1 after 30 min. (C) IL-8 after 6 h. (D) MCP-1 after 6 h. *P<0.05 compared to vehicle by one-way analysis of variance, Dunnett post-test. Concentration-response curves for (E) IL-8 release after 6 h and (F) MCP-1 release after 6 h.
not at a dose of 1 mM (Figure 3E, F). The 2’MOE ASO 104838 and 501861 triggered a small release at 5 mM in whole blood only (Figure 3F). The 2’MOE ASO 487660 did not trigger SDF1a release at any concentration tested, in either platelet-rich plasma or whole blood (Figure 3E, F).
2’MOE ASO 104838 increases platelet reactivity to collagen
To investigate whether the platelet activation docu- mented in Figure 3 would translate into an effect on platelet aggregation, we performed 96-well aggregometry and traditional light transmission aggregometry under both unstimulated and stimulated conditions (Figure 4A- D). The 2’MOE ASO 104838 triggered a small potentiat- ing effect on collagen-induced platelet aggregation, but this effect was not seen after treatment with the other two 2’MOE ASO (Figure 4A). The CpG ASO 120704 and ODN 2395 also increased platelet reactivity to collagen (Figure 4B), including ~40% spontaneous aggregation in two
donors with high platelet GPVI levels (Online Supplementary Figure S2). However, no significant sponta- neous aggregation was detected by light transmission aggregometry in any of the ASO-treated platelet-rich plas- ma samples and stimulating the 5 mM ASO-treated sam- ples with TRAP resulted in a similar light transmission aggregometry-measured aggregation response to that fol- lowing stimulation with the vehicle (Figure 4C). Differences in the mechanical shear stress environment in 96-well aggregometry versus light transmission aggregom- etry25 may explain the discrepancy in results between these assays.
2’MOE ASO (104838 and 501861) and CpG ASO poten- tiate platelet reactivity to TRAP in whole
blood impedance aggregometry
When whole blood was incubated with 5 mM of the CpG ASO ODN 2395 in an impedance aggregometer, spontaneous aggregation was detected over the course of
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haematologica | 2022; 107(2)