title: Targeting of C-type lectin-like receptor 2 or P2Y12 for the prevention of platelet activation by immunotherapeutic CpG oligodeoxynucleotides.
abstract: Essentials CpG oligodeoxynucleotide (ODN) immuotherapeutics cause undesired platelet activating effects. It is crucial to understand the mechanisms of these effects to identify protective strategies. CpG ODN-induced platelet activation depends on C-type lectin-like receptor 2 (CLEC-2) and P2Y12. Targeting CLEC-2 or P2Y12 fully prevents CpG ODN-induced platelet activation and thrombosis.
  
  
  
  Background Synthetic phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) show potent immunostimulatory properties that are widely exploited in clinical trials of anticancer treatment. Unexpectedly, a recent study indicated that CpG ODNs activate human platelets via the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor glycoprotein VI. Objective To further analyze the mechanisms of CpG ODN-induced platelet activation and identify potential inhibitory strategies. Methods In vitro analyses were performed on human and mouse platelets, and on cell lines expressing platelet ITAM receptors. CpG ODN platelet-activating effects were evaluated in a mouse model of thrombosis. Results We demonstrated platelet uptake of CpG ODNs, resulting in platelet activation and aggregation. C-type lectin-like receptor 2 (CLEC-2) expressed in DT40 cells bound CpG ODNs. CpG ODN uptake did not occur in CLEC-2-deficient mouse platelets. Inhibition of human CLEC-2 with a blocking antibody inhibited CpG ODN-induced platelet aggregation. CpG ODNs caused CLEC-2 dimerization, and provoked its internalization. They induced dense granule release before the onset of aggregation. Accordingly, pretreating platelets with apyrase, or inhibiting P2Y12 with cangrelor or clopidogrel, prevented CpG ODN platelet-activating effect. In vivo, intravenously injected CpG ODN interacted with platelets adhered to mouse injured endothelium, and promoted thrombus growth, which was inhibited by CLEC-2 deficiency or by clopidogrel. Conclusions CLEC-2 and P2Y12 are required for CpG ODN-induced platelet activation and thrombosis, and might be targeted to prevent adverse events in patients at risk.
authors.affiliation: Laboratory of Thrombosis and Hemostasis and Valvular Heart Disease, GIGA-Cardiovascular Sciences, Department of Cardiology, University of Liège, CHU Sart-Tilman, Liège, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Delierneux
authors.affiliation: Laboratory of Thrombosis and Hemostasis and Valvular Heart Disease, GIGA-Cardiovascular Sciences, Department of Cardiology, University of Liège, CHU Sart-Tilman, Liège, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Donis
authors.affiliation: Laboratory of Thrombosis and Hemostasis and Valvular Heart Disease, GIGA-Cardiovascular Sciences, Department of Cardiology, University of Liège, CHU Sart-Tilman, Liège, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Servais
authors.affiliation: Laboratory of Thrombosis and Hemostasis and Valvular Heart Disease, GIGA-Cardiovascular Sciences, Department of Cardiology, University of Liège, CHU Sart-Tilman, Liège, Belgium.
authors.forename: O
authors.initials: O
authors.lastname: Wéra
authors.affiliation: Department of Laboratory Hematology, CHU Sart-Tilman, Liège, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Lecut
authors.affiliation: Immunology and Infectious Diseases Unit, GIGA-Signal Transduction, University of Liège, Liège, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Vandereyken
authors.affiliation: Laboratory of Thrombosis and Hemostasis and Valvular Heart Disease, GIGA-Cardiovascular Sciences, Department of Cardiology, University of Liège, CHU Sart-Tilman, Liège, Belgium.
authors.forename: L
authors.initials: L
authors.lastname: Musumeci
authors.affiliation: Immunology and Infectious Diseases Unit, GIGA-Signal Transduction, University of Liège, Liège, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Rahmouni
authors.affiliation: Luxembourg Center for Systems Biomedicine, University of Luxembourg, Luxembourg City, Luxembourg.
authors.forename: J
authors.initials: J
authors.lastname: Schneider
authors.affiliation: Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany.
authors.forename: J A
authors.initials: JA
authors.lastname: Eble
authors.affiliation: Laboratory of Thrombosis and Hemostasis and Valvular Heart Disease, GIGA-Cardiovascular Sciences, Department of Cardiology, University of Liège, CHU Sart-Tilman, Liège, Belgium.|Gruppo Villa Maria Care and Research, Anthea Hospital, Bari, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Lancellotti
authors.affiliation: Laboratory of Thrombosis and Hemostasis and Valvular Heart Disease, GIGA-Cardiovascular Sciences, Department of Cardiology, University of Liège, CHU Sart-Tilman, Liège, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Oury
mesh_terms: D000276:Adjuvants, Immunologic / Q000378:metabolism / Q000633:toxicity; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D004195:Disease Models, Animal; D006801:Humans; D061625:Immunoreceptor Tyrosine-Based Activation Motif; D037181:Lectins, C-Type / Q000172:deficiency / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D054735:Phosphorothioate Oligonucleotides / Q000276:immunology / Q000378:metabolism / Q000633:toxicity*; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology*; D058925:Receptors, Purinergic P2Y12 / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013927:Thrombosis / Q000097:blood / Q000276:immunology / Q000517:prevention & control; D013997:Time Factors
chemical_list: D000276:Adjuvants, Immunologic; D000906:Antibodies; C529518:CLEC-2 protein, mouse; C104778:CLEC2B protein, human; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; C548576:P2RY12 protein, human; C548577:P2ry12 protein, mouse; D054735:Phosphorothioate Oligonucleotides; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D058921:Purinergic P2Y Receptor Antagonists; D058925:Receptors, Purinergic P2Y12; C098341:platelet membrane glycoprotein VI
keywords: CLEC-2 protein; CPG-ODN; P2Y12 purinoceptor; immunotherapy; platelet activation