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M.H. Lundberg Slingsby et al.
vivo conditions or be due to species differences. Further investigation is warranted to see if in vivo treatment, or indeed clinical use of certain 2’MOE ASO sequences leads to increased platelet activation and formation of platelet- leukocyte aggregates and to what degree this is paired with clinical reductions in platelet count.
Our data showing that pre-treatment with a SYK inhibitor was able to fully reverse ASO-induced platelet activation and platelet-leukocyte aggregate formation, speculatively highlights the potential of using a clinically available SYK inhibitor, for instance fostamatinib,33 to treat ASO-induced platelet side effects. This would need to be investigated further.
Flierl et al. were the first to identify that ODN 2395 binds to and activates GPVI on platelets.14 In the current study, we confirmed these findings and added the novel finding that 2’MOE ASO also bind to GPVI. Overall, the 2’MOE ASO had lower affinity to GPVI compared to the CpG ASO, which is consistent with their weaker platelet activating effects. In general it has been shown that the stronger affinity with which PS ASO bind, the larger the conformational change of the protein.34 Hence, CpG ASO, with their greater affinity than 2’MOE ASO for GPVI, may induce a more substantial conformational change in the GPVI protein, affecting for instance GPVI clustering/dimerization,14 explaining the greater potency of CpG ASO to activate platelets.
Interestingly, the 2’MOE ASO 487660, which has not been shown to lower platelet counts in monkeys, did not affect any of the platelet function tests we performed in this study. Hence, the platelet effects observed with 104838 and 501861 appear to be sequence-dependent rather than due to their 2’MOE-backbone chemistry. 487660 did however still bind to human GPVI with a sim- ilar affinity as that of the other 2’MOE ASO, 104838 and 501861. This finding highlights the importance of func- tional in vitro platelet studies beyond ASO-GPVI binding assays in cell-free environments. The discrepancy by which 2’MOE ASO 487660 can bind GPVI but does not activate platelets may be explained by its greater affinity to bind human serum albumin, compared to the other 2’MOE ASO investigated, which may keep 487660 more bound to plasma proteins. 2’MOE ASO 487660 may also interact differently with the GPVI receptor as there appeared to be less surface and internalized 2’MOE ASO 487660 staining in platelets in the electron-microscopy images.
There is a degree of subject variability in platelet count reductions following ASO treatment in monkeys and humans.8,10,16 We also noticed a high degree of variability in responsiveness to ASO in our in vitro studies of blood from healthy human donors. Although the sample was small (n=7), there was a strong positive correlation between an individual’s platelet GPVI levels and their platelet responsiveness to both 2’MOE (104838 and 501861) and CpG ASO-induced activation. Platelet GPVI levels have been shown to vary in healthy individuals35 and to be increased in different disease states such as obe- sity.36 Platelet GPVI levels could potentially be useful as a screening tool (used before commencement of treatment) to identify at-risk patients who may be more susceptible to platelet side effects of some sequence-specific GPVI- activating ASO.
We have shown that 2’MOE ASO that have been asso- ciated with phenotype 1 may not simply be reducing platelet counts, but can also have direct effects on platelets, triggering interactions between platelets and immune cells.
In summary, we have defined new mechanisms by which 2’MOE ASO-based drugs affect human platelets, which may yield new strategies to avoid ASO sequences with unfavorable platelet effects.
Disclosures
JEI has financial interest in and is a founder of Platelet BioGenesis, a company that aims to produce donor-independent human platelets from human-induced pluripotent stem cells at scale.TheinterestsofJEIwerereviewedandaremanagedbythe Brigham and Women’s Hospital and Partners HealthCare. The remaining authors declare that they have no conflicts of interest.
Contributions
MHLS supervised the study, performed research, analyzed data and wrote the manuscript; PV, IT, HR, GC, AW, HG, JM, RO, BE, and JT performed research, analyzed data and edited the manuscript; JWS, SPH and PN analyzed data and edited the manuscript; JEI supervised the study, analyzed data and edit- ed the manuscript.
Funding
This work was funded by a corporate sponsored research agreement between Ionis Pharmaceuticals Inc. and Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict-of-interest policies.
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