Abstract
The thromboxane (Tx) A2 pathway is a major contributor to the amplification of initial platelet activation and is therefore a key drug target. To identify potent small-molecule inhibitors of the thromboxane prostaglandin (TP) receptor, we screened a small steroidal saponin library using U46619-induced rat platelet aggregation assays. Timosaponin AIII (TAIII) was identified as a potent inhibitor of U46619-induced rat platelet aggregation and exhibited superior selectivity for the TP receptor versus other G protein-coupled receptors and a PKC activator. TAIII inhibited U46619-induced rat platelet aggregation independent of increases in cAMP and cGMP and the inhibition of TxA2 production. Both PKC and PLC activators restored TAIII-inhibited platelet aggregation, whereas TAIII did not inhibit platelet aggregation induced by co-activation of the G12/13 and Gz pathways. Furthermore, TAIII did not affect the platelet shape change or ROCK2 phosphorylation evoked by low-dose U46619. In vivo, TAIII prolonged tail bleeding time, reduced the mortality of animals with acute pulmonary thromboembolism and significantly reduced venous thrombus weight. Our study suggests that TAIII, by preferentially targeting Gq-mediated PLC/PKC signaling from the TP receptor, induces stronger in vitro antiplatelet activity and in vivo antithrombotic effects and may be an excellent candidate for the treatment of thrombotic disorders.
Highlights
Peroxidation of membrane phospholipids in platelets, cells of blood vessels and monocytes/macrophages, such as isoprostanes and hydroxyeicosatetraenoic acids (HETEs), are all TP receptor ligands[6,7]; TP receptor antagonists have certain pharmacological advantages over aspirin in that they block the effect of thromboxane A2 (TxA2) on platelets and inhibit the deleterious effects of other TP ligands, such as isoprostanes and HETEs
The biotransformation metabolite compound 2, a stereoisomer of Timosaponin AIII (TAIII) with a β-ranged CH3-21, and compound 4, produced by selective hydrolysis of β-D-glucopyranosyl from the β-D-galactopyranoside of TAIII, both exhibited little inhibitory activity. These results suggest that the spirostan-type 3-O-glycoside structure of TAIII is important for antiplatelet aggregation activity and that 15-OH and 2-OH can reduce this activity
At the same concentration, thrombin- and PMA-induced activation of ERK1/2 was not affected by TAIII. These results further confirmed that TAIII is a selective inhibitor of TxA2-induced platelet activation
Summary
Peroxidation of membrane phospholipids in platelets, cells of blood vessels and monocytes/macrophages, such as isoprostanes and hydroxyeicosatetraenoic acids (HETEs), are all TP receptor ligands[6,7]; TP receptor antagonists have certain pharmacological advantages over aspirin in that they block the effect of TxA2 on platelets and inhibit the deleterious effects of other TP ligands, such as isoprostanes and HETEs. Steroidal saponins are complex compounds featuring a steroid attached to a carbohydrate moiety. They are natural surfactants and detergents and possess a broad range of biological and pharmacological properties, such as hypocholesterolemic, anti-tumor, antidiabetic, anti-inflammatory and antifungal activity[10]. We screened a small steroidal saponin library isolated from Chinese medicinal herbs using a turbidimetric assay based on U46619 (a TxA2 analogue)-induced rat platelet aggregation. This screen identified Timosaponin AIII (TAIII) as a selective inhibitor of the TxA2 receptor. The antithrombotic activity and synergism of TAIII with known antiplatelet agents may facilitate the development of novel antithrombotic agents
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