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Effects of 2'MOE ASO on human platelets
with 5 mM of the 2’MOE ASO 104838 and 501861 (19±3% and 20±3%, respectively), compared to vehicle (10±2%); the CpG ASO 120704 and ODN 2395 activated the platelets more potently (38±3% and 38±4%, respec- tively) (Figure 3A).
We also studied platelet activation in whole blood, in which platelet P-selectin was elevated by the platelet ago- nists TRAP and collagen (Figure 3B). Pre-treatment with a Spleen tyrosine kinase (SYK) inhibitor (PRT-060318) decreased collagen, but not TRAP activation since SYK is downstream of the collagen receptor GPVI signaling in platelets (Figure 3B). The selective inhibitory effect on col- lagen signaling is demonstrated more clearly in Figure 6A. The 2’MOE ASO 104838 and 501861 had mild platelet- activating effects in whole blood (similar to that of colla- gen) whereas the CpG ASO 120704 and ODN 2395 had
stronger effects (comparable to the effect of TRAP) (Figure 3B). Pre-treatment with the SYK inhibitor blocked the ASO-induced P-selectin expression (Figure 3B).
Responsiveness to ASO treatment is strongly correlated to individual GPVI levels
Since we noticed donor-to-donor variability in the responsiveness to ASO treatment (Figure 3B), we investi- gated whether this could be related to differential platelet surface expression of GPVI receptors, which we measured by flow cytometry. Basal platelet GPVI levels varied between donors and activating the platelet-rich plasma with TRAP reduced the platelet GPVI levels (Online Supplementary Figure S2), consistent with reports of GPVI shedding upon platelet activation.24 Pearson correlation analysis showed a strong positive correlation (correlation
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Figure 2. Uptake and localization of antisense oligonucleotides in human washed platelets. Washed platelets were incubated with a therapeutically relevant con- centration of the antisense oligonucleotides (ASO) (5 mM), then fixed and stained with an anti-ASO antibody and labeled with protein A-gold. Representative electron microscopy images at 15000x of ASO localization, using anti-ASO immunogold labeling (shown as black dots) of human washed platelets treated for 30 min with (A) vehicle (HEPES, 10 mM), (B) 2’MOE ASO 487660, (C) 2’MOE ASO 104838, (D) 2’MOE ASO 501861, (E) CpG ASO 120704, or (F) CpG ASO ODN 2395 (all at 5 mM). Black arrows indicate ASO localizing on the platelet membrane and white arrows indicate internalized ASO.
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