Asebogenin suppresses thrombus formation via inhibition of Syk phosphorylation

Abstract

Background and Purpose Thrombosis is a major cause of morbidity and mortality worldwide. Platelet activation by exposed collagen through glycoprotein VI (GPVI) and formation of neutrophil extracellular traps (NETs) are critical pathogenic factors for arterial and venous thrombosis. Both events are regulated by spleen tyrosine kinase (Syk)‐mediated signalling events. Asebogenin is a dihydrochalcone whose pharmacological effects remain largely unknown. This study aims to investigate the antithrombotic effects of asebogenin and the underlying molecular mechanisms. Experimental Approach Platelet aggregation was assessed using an aggregometer. Platelet P‐selectin exposure, integrin activation and calcium mobilization were determined by flow cytometry. NETs formation was assessed by SYTOX Green staining and immunohistochemistry. Quantitative phosphoproteomics, microscale thermophoresis, in vitro kinase assay and molecular docking combined with dynamics simulation were performed to characterize the targets of asebogenin. The in vivo effects of asebogenin on arterial thrombosis were investigated using FeCl3‐induced and laser‐induced injury models, whereas those of venous thrombosis were induced by stenosis of the inferior vena cava. Key Results Asebogenin inhibited a series of GPVI‐induced platelet responses and suppressed NETs formation induced by proinflammatory stimuli. Mechanistically, asebogenin directly interfered with the phosphorylation of Syk at Tyr525/526, which is important for its activation. Further, asebogenin suppressed arterial thrombosis demonstrated by decreased platelet accumulation and fibrin generation and attenuated venous thrombosis determined by reduced neutrophil accumulation and NETs formation, without increasing bleeding risk. Conclusion and Implications Asebogenin exhibits potent antithrombotic effects by targeting Syk and is a potential lead compound for the development of efficient and safe antithrombotic agents.

Low P-Selectin Glycoprotein Ligand-1 Expression in Neutrophils Associates with Disease Activity and Deregulated NET Formation in Systemic Lupus Erythematosus

“…Syk has been implicated in NET generation [ 14 , 15 , 16 ]. Given that PSGL-1 interaction with P-selectin induces Syk activation [ 13 ] and L-Selectin contributes to full activation of neutrophils, when interacting with P-selectin [ 33 ], we first studied the expression level of L-Selectin in the cell membrane and the level of pSyk in basal experimental conditions of neutrophils from HD and SLE patients.…”

“…We also analyzed the susceptibility of neutrophils from lupus patients to generate NETs in our different experiment conditions. Furthermore, we analyzed the level of Syk activation, given that different reports described that the activation of the tyrosine kinase Syk by different stimuli was implicated in the generation of NETs [ 14 , 15 , 16 ]. In basal conditions, neutrophils from iSLE patients, both the basal susceptibility to NET generation and the amount of DNA extruded in the NETs were similar to HD ( Figure 3 ).…”

“…These interactions are also responsible for the activation of integrins for leukocyte firm adhesion before tissue extravasation [ 12 ]. Furthermore, our group described that during these interactions, PSGL-1 triggers a signaling cascade initiated by Syk [ 13 ], a tyrosine kinase involved in triggering the generation of NETs [ 14 , 15 , 16 ]. In monocyte-derived-dendritic cells, PSGL-1 signaling cascade promotes the capacity of these cells to induce the differentiation of naïve T cells to T-regulatory (Treg) cells [ 17 ].…”

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment

“…Human peripheral neutrophils were isolated from venous blood using Polymorphprep by gradient centrifugation as described previously. 31 Informed consent was obtained from the donors, and the procedure was approved by the Ethics Review Board of Tongji Medical College, HUST, and in accordance with the Declaration of Helsinki. The volunteers include both men and women.…”

Platelet–Neutrophil Crosstalk in Thrombosis

“…Similarly, experimental mouse stroke models suggest that DNase alone or in combination with thrombolytic agents also have promising outcomes for stroke treatment [ 82 , 92 , 93 ]. More recently, asebogenin, a Syk phosphorylation inhibitor, was shown to reduce arterial thrombosis by decreasing platelet accumulation and fibrin formation, as well as venous thrombosis, by reducing neutrophil recruitment and subsequent NET formation [ 184 ]. However, all these approaches to target NET formation might equally affect physiological NETosis, which may impair the normal innate immune response.…”