Antisense oligonucleotides and nucleic acids generate hypersensitive platelets

Alexander Zaslavsky; Mackenzie Adams; Xiu Cao; Adriana Yamaguchi; James Henderson; Peter Busch-Østergren; Aaron Udager; Sethuramasundaram Pitchiaya; Benjamin Tourdot; Tadas Kasputis; Samuel J Church; Samantha K Lee; Sydney Ohl; Shivam Patel; Todd M Morgan; Ajjai Alva; Thomas W Wakefield; Zachery Reichert; Michael Holinstat; Ganesh S Palapattu

doi:10.1016/j.thromres.2021.01.006

Antisense oligonucleotides and nucleic acids generate hypersensitive platelets

Thromb Res. 2021 Apr:200:64-71. doi: 10.1016/j.thromres.2021.01.006. Epub 2021 Jan 18.

Authors

Alexander Zaslavsky¹, Mackenzie Adams², Xiu Cao², Adriana Yamaguchi³, James Henderson², Peter Busch-Østergren⁴, Aaron Udager⁵, Sethuramasundaram Pitchiaya⁶, Benjamin Tourdot⁷, Tadas Kasputis², Samuel J Church², Samantha K Lee², Sydney Ohl², Shivam Patel², Todd M Morgan², Ajjai Alva⁸, Thomas W Wakefield⁹, Zachery Reichert¹⁰, Michael Holinstat³, Ganesh S Palapattu¹¹

Affiliations

¹ Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Urology, University of Michigan Medical School, Ann Arbor, MI, USA. Electronic address: azaslavs@med.umich.edu.
² Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Urology, University of Michigan Medical School, Ann Arbor, MI, USA.
³ Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
⁴ Department of Urology, Herlev and Gentofte Hospital, Herlev, Denmark.
⁵ Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
⁶ Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Pathology, University of Michigan, Ann Arbor, MI, USA; Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, USA.
⁷ Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA.
⁸ Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Internal Medicine-Division of Hematology/Oncology, University of Michigan Medical School, Ann Arbor, MI, USA.
⁹ Section of Vascular Surgery, Department of Surgery, Conrad Jobst Vascular Research Laboratories, University of Michigan Medical School, Ann Arbor, MI, USA.
¹⁰ Department of Internal Medicine-Division of Hematology/Oncology, University of Michigan Medical School, Ann Arbor, MI, USA.
¹¹ Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Urology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Urology, Medical University of Vienna, Vienna, Austria. Electronic address: gpalapat@med.umich.edu.

Abstract

Introduction: Despite the great promise for therapies using antisense oligonucleotides (ASOs), their adverse effects, which include pro-inflammatory effects and thrombocytopenia, have limited their use. Previously, these effects have been linked to the phosphorothioate (PS) backbone necessary to prevent rapid ASO degradation in plasma. The main aim of this study was to assess the impact of the nucleic acid portion of an ASO-type drug on platelets and determine if it may contribute to thrombosis or thrombocytopenia.

Methods: Platelets were isolated from healthy donors and men with advanced prostate cancer. Effects of antisense oligonucleotides (ASO), oligonucleotides, gDNA, and microRNA on platelet activation and aggregation were evaluated. A mouse model of lung thrombosis was used to confirm the effects of PS-modified oligonucleotides in vivo.

Results: Platelet exposure to gDNA, miRNA, and oligonucleotides longer than 16-mer at a concentration above 8 mM resulted in the formation of hypersensitive platelets, characterized by an increased sensitivity to low-dose thrombin (0.1 nM) and increase in p-Selectin expression (6-8 fold greater than control; p < 0.001). The observed nucleic acid (NA) effects on platelets were toll-like receptor (TLR) -7 subfamily dependent. Injection of a p-Selectin inhibitor significantly (p = 0.02) reduced the formation of oligonucleotide-associated pulmonary microthrombosis in vivo.

Conclusion: Our results suggest that platelet exposure to nucleic acids independent of the presence of a PS modification leads to a generation of hypersensitive platelets and requires TLR-7 subfamily receptors. ASO studies conducted in cancer patients may benefit from testing the ASO effects on platelets ex vivo before initiation of patient treatment.

Keywords: Antisense oligonucleotides; Platelets; Prostate Cancer; Toll-like receptors.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Blood Platelets
Humans
Mice
Nucleic Acids*
Oligonucleotides, Antisense
Pharmaceutical Preparations*
Phosphorothioate Oligonucleotides

Substances

Nucleic Acids
Oligonucleotides, Antisense
Pharmaceutical Preparations
Phosphorothioate Oligonucleotides

Abstract

Publication types

MeSH terms

Substances

Grants and funding