Platelet P2Y12 receptors are involved in the haemostatic effect of notoginsenoside Ft1, a saponin isolated from Panax notoginseng

Abstract

Saponins isolated from Panax notoginseng (Burk.) F.H. Chen have been shown to relieve thrombogenesis and facilitate haemostasis. However, it is not known which saponin accounts for this haemostatic effect. Hence, in the present study we aimed to identify which saponins contribute to its haemostatic activity and to elucidate the possible underlying mechanisms. Platelet aggregation was analysed using a platelet aggregometer. Prothrombin time, activated partial thromboplastin time and thrombin time were measured using a blood coagulation analyser, which was further corroborated with bleeding time and thrombotic assays. The interaction of notoginsenoside Ft1 with the platelet P2Y₁₂ receptor was determined by molecular docking analysis, cytosolic Ca(2+) and cAMP measurements, and phosphorylation of PI3K and Akt assays. Among the saponins examined, Ft1 was the most potent procoagulant and induced dose-dependent platelet aggregation. Ft1 reduced plasma coagulation indexes, decreased tail bleeding time and increased thrombogenesis. Moreover, it potentiated ADP-induced platelet aggregation and increased cytosolic Ca(2+) accumulation, effects that were attenuated by clopidogrel. Molecular docking analysis suggested that Ft1 binds to platelet P2Y₁₂ receptors. The increase in intracellular Ca(2+) evoked by Ft1 in HEK293 cells overexpressing P2Y₁₂ receptors could be blocked by ticagrelor. Ft1 also affected the production of cAMP and increased phosphorylation of PI3K and Akt downstream of P2Y₁₂ signalling pathways. Ft1 enhanced platelet aggregation by activating a signalling network mediated through P2Y₁₂ receptors. These novel findings may contribute to the effective utilization of this compound in the therapy of haematological disorders.