Ginsenoside Rg5 allosterically interacts with P2RY and ameliorates deep venous thrombosis by counteracting neutrophil NETosis and inflammatory response.

Background: Deep venous thrombosis (DVT) highly occurs in patients with severe COVID-19 and probably accounted for their high mortality. DVT formation is a time-dependent inflammatory process in which NETosis plays an important role. However, whether ginsenoside Rg5 from species of genus could alleviate DVT and its underlying mechanism has not been elucidated.

Methods: The interaction between Rg5 and P2RY was studied by molecular docking, molecular dynamics, surface plasmon resonance (SPR), and molecular biology assays. The preventive effect of Rg5 on DVT was evaluated in inferior vena cava stasis-induced mice, and immunocytochemistry, Western blot, and calcium flux assay were performed in neutrophils from bone marrow to explore the mechanism of Rg5 in NETosis P2RY.

Results: Rg5 allosterically interacted with P2RY, formed stable complex, and antagonized its activity residue E188 and R265. Rg5 ameliorated the formation of thrombus in DVT mice; accompanied by decreased release of Interleukin (IL)-6, IL-1β, and tumor necrosis factor-α in plasma; and suppressed neutrophil infiltration and neutrophil extracellular trap (NET) release. In lipopolysaccharide- and platelet-activating factor-induced neutrophils, Rg5 reduced inflammatory responses inhibiting the activation of ERK/NF-κB signaling pathway while decreasing cellular Ca concentration, thus reducing the activity and expression of peptidyl arginine deiminase 4 to prevent NETosis. The inhibitory effect on neutrophil activity was dependent on P2RY.

Conclusions: Rg5 could attenuate experimental DVT by counteracting NETosis and inflammatory response in neutrophils P2RY, which may pave the road for its clinical application in the prevention of DVT-related disorders.