GSK669, a NOD2 receptor antagonist, inhibits thrombosis and oxidative stress via targeting platelet GPVI

Guanxing Pan; Lin Chang; Jianjun Zhang; Yangyang Liu; Liang Hu; Si Zhang; Jian Zhang; Jianlin Qiao; Žiga Jakopin; Hu Hu; Jianzeng Dong; Zhongren Ding

doi:10.1016/j.bcp.2020.114315

GSK669, a NOD2 receptor antagonist, inhibits thrombosis and oxidative stress via targeting platelet GPVI

Biochem Pharmacol. 2021 Jan:183:114315. doi: 10.1016/j.bcp.2020.114315. Epub 2020 Nov 3.

Authors

Guanxing Pan¹, Lin Chang¹, Jianjun Zhang¹, Yangyang Liu², Liang Hu², Si Zhang¹, Jian Zhang³, Jianlin Qiao⁴, Žiga Jakopin⁵, Hu Hu⁶, Jianzeng Dong², Zhongren Ding⁷

Affiliations

¹ Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
² Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450018, China.
³ Department of Pathophysiology, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China.
⁴ Department of Hematology, Blood Disease Institute, Xuzhou Medical University, Xuzhou 221004, China.
⁵ Faculty of Pharmacy, University of Ljubljana, Askerceva 7, SI-1000 Ljubljana, Slovenia.
⁶ Department of Pathophysiology, Zhejiang University School of Medicine, Hangzhou 310012, China.
⁷ Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450018, China.

PMID: 33152345
DOI: 10.1016/j.bcp.2020.114315

Abstract

Background and purpose: Previously, we discovered that the activation of nucleotide-binding oligomerization domain 2 (NOD2) enhances platelet activation. We here investigated the antiplatelet and antithrombotic potential of GSK669, a NOD2 antagonist.

Experimental approach: Effects of GSK669 on platelet functions, reactive oxygen species (ROS) and proinflammatory cytokine generation were detected. NOD2-/- platelets were used to confirm GSK669 target. The interaction between GSK669 and glycoprotein VI (GPVI) was detected using surface plasmon resonance (SPR) spectroscopy. GPVI downstream signaling was examined by Western blot. The antithrombotic and antioxidative effects were investigated using mouse mesenteric arteriole thrombosis model and pulmonary embolism model.

Key results: GSK669 significantly inhibits platelet proinflammatory cytokine release induced by muramyl dipeptide, platelet aggregation, ATP release, and ROS generation induced by collagen and collagen related peptide (CRP). Platelet spreading and clot retraction are also inhibited. GSK669 also decreases collagen-induced phosphorylation of Src, Syk, PLCγ2, and Akt. The antiplatelet effect of GSK669 is NOD2-independent and mediated by GPVI antagonism. Consistent with its antiplatelet activity as a GPVI antagonist, GSK669 inhibits platelet adhesion on collagen in flow condition. Notably, GSK669 inhibits mouse mesenteric arteriole thrombosis similarly to aspirin without bleeding. The antithrombotic effect of GSK669 is further confirmed in the pulmonary embolism model; decreased malonaldehyde (MDA) and increased superoxide dismutase (SOD) levels in mouse plasma reveal a significant antioxidant effect of GSK669.

Conclusion and implications: Beyond its anti-inflammatory effect as a NOD2 antagonist, GSK669 is also an efficient and safe antiplatelet agent combined with antioxidant effect by targeting GPVI. An antiplatelet agent bearing antioxidative and anti-inflammatory activities without bleeding risk may have therapeutic advantage over current antiplatelet drugs for atherothrombosis.

Keywords: Anti-inflammatory; Antioxidative; Antithrombotic; GPVI; GSK669; NOD2.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Anti-Inflammatory Agents / administration & dosage
Anti-Inflammatory Agents / metabolism
Blood Platelets / drug effects*
Blood Platelets / metabolism
Drug Delivery Systems / methods
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
Nod2 Signaling Adaptor Protein / antagonists & inhibitors*
Nod2 Signaling Adaptor Protein / metabolism
Oxidative Stress / drug effects*
Oxidative Stress / physiology
Platelet Aggregation Inhibitors / administration & dosage*
Platelet Aggregation Inhibitors / metabolism
Platelet Membrane Glycoproteins / antagonists & inhibitors*
Platelet Membrane Glycoproteins / metabolism
Thrombosis / drug therapy*
Thrombosis / metabolism

Substances

Anti-Inflammatory Agents
Nod2 Signaling Adaptor Protein
Nod2 protein, mouse
Platelet Aggregation Inhibitors
Platelet Membrane Glycoproteins
platelet membrane glycoprotein VI