Pharmacological effects and mechanism of Ilexsaponin A1 in modulating platelet function

Ilex pubescens Hook. & Arn. is a traditional Chinese medicine for promoting blood circulation. Ilexsaponin A1 (IsA), a monomer of the compound, exhibits pro-angiogenic, anti-apoptotic and anti-inflammatory activities. Nevertheless, the pharmacological effects and specific mechanisms by which IsA affects platelets remain unknown.

This study aims to investigate the antiplatelet effects of IsA and the underlying molecular mechanisms.

Platelet aggregation and ATP release were assessed using platelet aggregometry. Flow cytometry was employed to evaluate the exposure of P-selectin, integrin α_Ⅱbβ₃ activation and calcium mobilization. Fluorescence microscopy was applied to observe platelet spreading. Clot retraction was imaged by digital camera. Protein phosphorylation regulation of major signaling pathways in platelets was determined by immunoblotting analysis. Doppler flowmetry was used to investigate the in vivo effect of IsA on FeCl₃-induced carotid artery injury model. Tail vein transection was used to measure bleeding time.

IsA dose-dependently inhibited platelet aggregation and ATP release induced by collagen, U46619, thrombin and ADP. It also suppressed thrombin-induced P-selectin exposure and PAC-1 binding. Furthermore, IsA inhibited intracellular Ca²⁺ mobilization and the inward flow of extracellular Ca²⁺. It also influenced integrin α_Ⅱbβ₃ outside-in signaling pathways, including the inhibition of platelet spreading, clot retraction and phosphorylation of outside-in signaling molecules. In addition, IsA suppressed the phosphorylation of Syk-PLCγ2, PI3K-Akt-GSK3β and MAPKs proteins, which are downstream effectors of the collagen and thrombin receptors.

IsA inhibited platelet function and thrombus formation. This has potential to be developed into a novel therapeutic agent for the treatment of thrombotic diseases.