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Effects of 2'MOE ASO on human platelets
Table 1. Antisense oligonucleotides included in this study.
Ionis ASO
487660
487660 Sequence (5’3’)
104838
104838
Sequence
(5’3’)
501861
501861
Sequence
(5’3’)
120704
120704
Sequence
(5’3’)
818290 (ODN 2395) ODN 2395
Sequence (5’3’)
Back bone
2’MOE PS
Length PS load*
20 19
Platelet side effects
No reported drops in platelet count
MOEC*MOEC*MOEA*MOEG*MOEC*T*C*A*A*C*C*C*T*T*CMOET*MOET*MOET*MOEA*MOEA*
Phenotype 1-moderate 2’MOE PS 20 19 drops in platelet
count8,10 MOEG*MOEC*MOET*MOEG*MOEA*T*T*A*G*A*G*A*G*A*G*MOEG*MOET*MOEC*MOEC*MOEC*
2’MOE PS 20 19 Unknown MOET*MOEC*MOEA*MOEC*MOEA*G*A*A*T*T*A*T*C*A*G*MOEC*MOEA*MOEG*MOET*MOEA*
CpG PS 24 23 T*C*pG*T*C*pG*T*T*T*T*G*T*C*pG*T*T*T*T*G*T*C*pG*T*T*
CpG PS 22 21 T*C*pG*T*C*pG*T*T*T*T*C*pG*G*C*G*C*G*C*G*C*C*pG*
Platelet activation10
Platelet activation14
All antisense oligonucleotides (ASO) were phosphorothioate (PS)-modified (locations indicated with *). MOE indicates the position of the 2'-O-methoxyethyl (2’MOE)-modified sugar residues with 2ʹ-deoxynucleotides in between.All cytosine residues were methylated at the five position.818290 is ODN 2395 and is referred to as ODN 2395 in the paper, for ease of comparison to previous reports.14ODN 2395 and 120704 are non-MOE ASO with unmethylated CpG dinucleotide-rich motifs.
topenia by affecting proplatelet production from megakaryocytes, human cord blood-derived mature megakaryocytes were incubated with 5 mM ASO for 24 h. Puromycin, an inhibitor of protein synthesis known to impair proplatelet production,21,22 had an inhibitory effect on both human- and murine-derived megakaryocytes (Figure 1A, B). Compared to the vehicle, none of the ASO tested lowered proplatelet counts from the human-derived megakaryocytes (Figure 1A). Similarly, there was no decrease in percent proplatelet producing murine fetal liver- derived megakaryocytes following incubation with any of the ASO (Figure 1B). The CpG ASO 120704 slightly increased proplatelet counts in the human-derived megakaryocytes after 22 h (117±5 vs. vehicle 79±7) (Figure 1A). There was also a small increase in proplatelet-produc- ing murine megakaryocytes after 24 h (Figure 1B); vehicle (17±1%), 487660 (25±1%), 104838 (23±1%), 501861 (25±2%), 120704 (23±1%), and ODN 2395 (26±1%). Representative images of proplatelet production at 0, 8, 16 and 24 h by murine megakaryocytes treated with 2’MOE ASO 104838 or CpG ASO ODN 2395 appeared compara- ble to those of megakaryocytes treated with the vehicle (Figure 1C).
Human platelets internalize both 2’MOE and CpG ASO
Given that the ASO did not appear to inhibit proplatelet production, we focused on examining the direct effects of ASO on human platelets. We used immune-electron microscopy to visualize how ASO (at 5 mM) interact with human washed platelets. ASO with a PS backbone are
hydrophilic, poly-anionic molecules with a high degree of plasma protein binding (typically >90%), and circulate transiently in the blood before interacting with cell-sur- face proteins and typically gaining entry into cells by endocytosis.23 The plasma-free conditions in the experi- ments shown in Figure 2 were intended to maximize visu- alization of ASO binding to platelets. Electron micro- graphs revealed that ASO immunogold staining was either localized to the platelet plasma membrane or, when internalized, to the cytoplasm with sporadic staining of internal membranes and granules (Figure 2B-F). The 2’MOE ASO 487660 appeared to stain less than the other ASO, especially at the plasma membrane (Figure 2B-F).
To exclude any direct platelet cytotoxicity of ASO, we performed a lactate dehydrogenase leakage assay in washed platelets and confirmed that none of the ASO was cytotoxic at doses of 1 or 5 mM (Online Supplementary Figure S1).
2’MOE ASO (104838 and 501861) and CpG ASO increase platelet P-selectin expression in platelet-rich plasma and whole blood
To establish whether the ASO activate human platelets directly, we investigated the effects on platelet surface P- selectin levels using flow cytometry (Figure 3). Platelet- rich plasma treated with the positive control thrombin receptor activating peptide (TRAP) showed a significant increase in P-selectin surface expression, while the 2’MOE ASO 487660 (which does not affect platelet count) did not increase platelet P-selectin compared to vehicle (Figure 3A). Platelet activation nearly doubled after treatment
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