Sequence-specific 2'-O-methoxyethyl antisense oligonucleotides activate human platelets through glycoprotein VI, triggering formation of platelet-leukocyte aggregates

Abstract

Antisense oligonucleotides (ASOs) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2’MOE) ASOs have reported dose- and sequencespecific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count. Phenotype 2 is rare, severe thrombocytopenia. This paper will focus on the underlying cause of the more common Phenotype 1, investigating the effects of ASOs on platelet production and platelet function. Five phosphorothioate ASOs were included: three 2’MOE sequences; 487660 (no effects on platelet count), 104838 (associated with Phenotype 1), 501861 (effects unknown) and two CpG sequences; 120704 and ODN 2395 (known to activate platelets). Human cord blood-derived megakaryocytes were treated with these ASOs to study effects on proplatelet production. Platelet activation (surface P-selectin) and platelet-leukocyte aggregates (PLAs) were analyzed in ASO-treated blood from healthy human volunteers. None of the ASOs inhibited proplatelet production from human megakaryocytes. All the ASOs were shown to bind to the platelet receptor glycoprotein VI (GPVI, KD ~0.2-1.5μM). CpG ASOs had the highest affinity to GPVI and the most potent platelet activating effects and PLA formation. 2’MOE ASO 487660 had no detectable platelet effects, while 2’MOE ASOs 104838 and 501861 triggered moderate platelet activation and SYK-dependent formation of PLAs. Donors with higher platelet GPVI levels had larger ASO-induced platelet activation. Sequence-dependent ASO-induced platelet activation and PLAs may explain Phenotype 1- moderate drops in platelet count. Platelet GPVI levels could be useful as a screening tool to identify patients at higher risk of ASO-induced platelet side effects.

Platelet Activation by Antisense Oligonucleotides (ASOs) in the Göttingen Minipig, including an Evaluation of Glycoprotein VI (GPVI) and Platelet Factor 4 (PF4) Ontogeny

“…Accordingly, no increase in P-selectin surface expression was observed in vitro, and no specific PF4-ASO antibodies were detected in this study, suggesting that no direct platelet activation occurred and no induction of the HITlike humoral response developed in the study animals. The same sequence (ISIS104838) and other 2 MOE-modified and PS ASOs activated human platelets in vitro (release of alpha granule markers and P-selectin surface expression) through the GPVI binding mechanism [27]. On the other hand, a previous study in adult Göttingen minipigs treated with various LNA-based PS ASOs did not show a fall in platelet count following four weeks of treatment [29].…”

“…Due to the polyanionic nature of ASOs with PS backbone linkages, their nonspecific affinity to plasma and cellular surface proteins is enhanced [24][25][26]. Therefore, one mechanism by which platelets can be activated directly by ASOs is through nonspecific binding to platelet collagen receptor glycoprotein VI (GPVI), which then leads to platelet aggregation [22,27,28]. Moreover, ASOs can bind to platelet factor 4 (PF4), inducing a humoral response to the ASO-PF4 complex, similar to heparin-induced thrombocytopenia (HIT) [23,28].…”

“…Moreover, ASOs can bind to platelet factor 4 (PF4), inducing a humoral response to the ASO-PF4 complex, similar to heparin-induced thrombocytopenia (HIT) [23,28]. Incubation of human platelets with ODN2395, a proinflammatory oligonucleotide with PS backbone modification without sugar moiety modification, causes direct platelet activation (P-selectin surface expression and alpha granule content release) and subsequent platelet aggregation [22,27,28]. The PS backbone modification of ODN2395 is the central mediator of its platelet-activating effects through GPVI receptor activation and enhances its responsiveness via GPVI clustering/dimerization [22].…”

Considerations in the Preclinical Assessment of the Safety of Antisense Oligonucleotides

“…Thrombocytopenia, that is, decrease in platelet counts, has been occasionally reported in preclinical models (rodents and NHPs) and in three recent clinical trials following treatment with ONDs, in particular PS-ASO (volanesorsen, inotersen, and drisapersen) [ 84 ]. Two phenotypes of platelet count decrease have been distinguished: phenotype 1 characterized by a moderate but not clinically severe drop in platelet count and the rarer phenotype 2 of severe thrombocytopenia [ 85 ]. In contrast with PS-ASO, severe thrombocytopenia has not been reported for siRNA drugs, neither in preclinical studies nor in clinical trials, but encapsulation of siRNA in lipid nanoparticles has been shown to cause decrease in platelet counts in rats, presumably induced by the cationic lipid molecules themselves [ 86 , 87 ].…”

“…More recently, the sequence-specific binding of OND (PS-ASO specifically) to platelet glycoprotein VI was shown to activate human platelets triggering formation of platelet–leukocyte aggregates [ 85 ]. These findings also highlight the possibility of a genetic susceptibility component given that donors with higher platelet glycoprotein VI levels had greater OND-induced platelet activation.…”