Pennogenin glycosides with a spirostanol structure are strong platelet agonists: structural requirement for activity and mode of platelet agonist synergism

Background: Steroidal saponins have long attracted scientific attention, due to their structural diversity and significant biological activities. For example, total steroidal saponins extracted from the rhizome of Paris polyphyllaSm. var. yunnanensis(TSSPs) constitute an effective treatment for abnormal uterine bleeding. Objective: To determine the active constituents in TSSPs and elucidate the mechanisms that underlie their in vivopharmacologic actions on hemostasis. Methods: Steroidal saponins were purified by chromatography, and their effects upon hemostasis and platelet function were evaluated by tail bleeding time in mice and rats, aggregometry, flow cytometry and Western blotting. Results: TSSPs promoted hemostasis in vivoand dose-dependently induced rat or human platelet aggregation in vitro. Using bioassay-guided separation, four known pennogenin glycosides with a spirostanol structure were identified as the active ingredients of TSSPs. A structure–activity assay showed that the aglycone and sugar moieties of pennogenin glycosides are both essential for their aggregatory activity. Their synergistic actions on platelet aggregation were observed with pennogenin glycosides and with other known platelet agonists, suggesting that these glycosides are platelet agonists. Aggregation in response to the pennogenin glycosides involved αIIbβ3activation, was inhibited by cAMP, was dependent upon extracellular calcium, secreted ADP and thromboxane synthesis, and was mediated by phosphatidylinositol-3-kinase. Conclusion: We identified pennogenin glycosides with a spirostanol structure as the active ingredients of Paris polyphyllaSm. var. yunnanensisin promoting hemostasis in vivo. Their mode of their action on platelets suggests that they represent a new type of platelet agonist.