Abstract
Tussilagone is a sesquiterpenoid extracted from Tussilago farfara and is used as an oriental medicine for asthma and bronchitis. Although previous studies have shown that tussilagone has an inhibitory effect on platelet aggregation, no studies have been performed to investigate its precise effect on platelets, and the underlying mechanism remains unclear. In the present study, we showed that tussilagone inhibited platelet aggregation induced by collagen, thrombin and ADP, as well as platelet release induced by collagen and thrombin, in mice. Tussilagone decreased P-selectin expression and αIIbβ3 activation (JON/A binding) in activated platelets, which indicated that tussilagone inhibited platelet activation. Moreover, tussilagone suppressed platelet spreading on fibrinogen and clot retraction. The levels of phosphorylated Syk, PLCγ2, Akt, GSK3β, and MAPK (ERK1/2 and P38) and molecules associated with GPVI downstream signaling were downregulated in the presence of tussilagone. In addition, tussilagone prolonged the occlusion time in a mouse model of FeCl3-induced carotid artery thrombosis and had no effect on mouse tail bleeding time. These results indicate that tussilagone inhibits platelet function in vitro and in vivo and that the underlying mechanism involves the Syk/PLCγ2-PKC/MAPK and PI3K-Akt-GSK3β signaling pathways downstream of GPVI. This research suggests that tussilagone is a potential candidate antiplatelet drug for the prevention of thrombosis.
Highlights
Platelets play important roles in hemostasis, thrombosis, inflammation, immunity, tumor metastasis and cardiovascular diseases, such as heart failure, ischemic stroke, and acute coronary syndrome [1,2,3,4,5,6]
Incubation of washed platelets or platelet-rich plasma (PRP) with tussilagone (10, 20, or 40 μM) for 5 min led to a concentration-dependent inhibitory effect on platelet aggregation induced by collagen (0.8 μg/mL) (55.7 ± 1.5, 47.3 ± 4.7, and 18.7 ± 1.3%, respectively), thrombin (0.08 U/mL) (62.3 ± 3.8, 47.7 ± 5.5, and 18.0 ± 1.2%, respectively) and adenosine diphosphate (ADP) (8 μM) (44.0 ± 1.5, 20.7 ± 2.2, and 11.0 ± 1.2%, respectively) (Figure 2)
The results showed that tussilagone (10, 20, or 40 μM) had a significant inhibitory effect on platelet ATP release induced by collagen (61.2 ± 2.3, 44.0 ± 4.5, and 24.7 ± 2.3%, respectively) and thrombin (60.0 ± 2.9, 41.7 ± 2.0, and 27.3 ± 1.8%, respectively) (Figures 3A,B)
Summary
Platelets play important roles in hemostasis, thrombosis, inflammation, immunity, tumor metastasis and cardiovascular diseases, such as heart failure, ischemic stroke, and acute coronary syndrome [1,2,3,4,5,6]. Recent studies highlighted the importance of platelet glycoprotein (GP) VI receptor, widely known as the major receptor for collagen, and binds to laminins, immobilized fibrinogen and fibrin [7, 8]. Binding of GPVI to collagen induces tyrosine phosphorylation of the immune-receptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail of the FcRγ chain, leading to a tyrosine phosphorylationregulated cascade that involves Syk and PLCγ2 [9]. GPVI blockade has been demonstrated to have efficient antithrombotic potential and show beneficial effects in other diseases [10]
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