The mechanism of thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides

In this study, we investigated thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides (Chol-ASO). First, we evaluated platelet activation induced by Chol-ASO in mice by flow cytometry after administration of platelet-rich plasma (PRP). An increase in the number of large particle-size events with platelet activation was detected in the Chol-ASO-treated group. In a smear study, numerous platelets were observed to attach to nucleic acid-containing aggregates. A competition binding assay showed that the conjugation of cholesterol to ASOs increased their affinity for glycoprotein VI. Platelet-free plasma was then mixed with Chol-ASO to form aggregates. The assembly of Chol-ASO was confirmed by dynamic light scattering measurements in the concentration range in which the formation of aggregates with plasma components was observed. In conclusion, the mechanism by which Chol-ASOs causes thrombocytopenia is proposed to be as follows: (1) Chol-ASOs form polymers, (2) the nucleic acid portion of the polymers interacts with plasma proteins and platelets, which cross-links them to form aggregates, and (3) platelets bound to aggregates become activated, resulting in platelet aggregation, leading to a decrease in platelet count in vivo. The details of the mechanism revealed in this study could contribute to creating safer oligonucleotide therapies without the risk of thrombocytopenia. •Cholesterol-conjugated antisense oligonucleotides (Chol-ASO) may cause thrombocytopenia.•Chol-ASO binds to itself to form homopolymers•We show that platelets attach to Chol-ASO aggregates, leading to a decrease in platelet count in mice