| Platelets, anucleated cells that originate from bone marrow megakaryocytes, are essential for primary hemostasis in the blood. In addition, a role for platelets in pathogenesis of a number of cardiovascular events is recognized. Increasing evidence indicates that enhanced platelet activation plays a critical role in initiation and development of atherothrombotic diseases. Accordingly, to prevent and treat thrombosis and vascular diseases, antiplatelet therapy may be a potential strategy. Many antiplatelet agents such as aspirin (an inhibitor of cyclooxygenase (COX)), clopidogrel, and ticlopidine (adenosine diphosphate receptor antagonists acting at the purinergic receptors of P2Y), have been reported to be beneficial to patients suffering from cardiovascular disorders. However, some drawbacks of these drugs such as aspirin resistance, the risk of neutropenia, bleeding, and thrombocytopenia, were occasionally observed. Therefore, development of more safe and effective antiplatelet agents may be a promising approach for prevention and treatment of atherothrombosis.Steroidal saponins are widely distributed in plants and have many pharmacologic actions and biological activities, such as antiplatelet activity, antitumor activity, and antidementia activity. They are an important class of natural products which is composed of a C-27 aglycone moiety and sugar chains of one or more monosaccharides. These compounds are classified as spirostanol glycosides with a sugar chain at C3 position, and furostanol saponins with two sugar chains at both C3 and C26 positions respectively. They have considerable potential as pharmaceutical and/or nutraceutical agents in natural or synthetic form. Steroidal glycosides, from a variety of sources, have been reported to have platelet inhibitory or aggregative activity, such as sarsasapogenin glycosides extracted from the rhizome of Anemarrhenae asphodeloides Bge (Liliaceae), has shown strong anti-platelet activity. But until now, little is known about molecular mechanism for the anti-platelet activity .Our aim is to determine the mechanism of steroidal saponins of anti-platelet activity. We first screened 6 natural steroidal saponins isolated from the root of Anemarrhenae asphodeloides Bge (Liliaceae) for their effects on rat platelet aggregation. We found that Timosaponin AⅢexhibited stronger inhibitory effects on platelet aggregation than other compounds, Further structure–activity assay revealed that the activity of Timosaponin AⅢin inhibiting platelet aggregation was not only attributed in part to the number, the length and the type of sugar side chains attached by a glycoside at C-3, but also related with the aglycon part. In the in vivo study, Timosaponin AⅢprolonged mouse tail bleeding time using bleeding time model and protected mice against fatal thrombosis in a concentration dependent manner. These results indicated that Timosaponin AⅢmay be appropriate probe to study the molecular mechanism of steroid saponins for antiplatelet activity.We further investigated the characteristic of Timosaponin AⅢfor anti-platelet activity. Platelets react to a variety of biologically active substances (agonists) with partial or full activation characterized by shape change, release of granule contents, and aggregation. Conversely, other agents (antagonists) induce a state of reduced responsiveness to agonists. In our study, we demonstrated that Timosaponin AⅢinhibited the processes of platelet activation, such as platelet aggregation, platelet shape change, plateletαIIbβ3 activation, granule secretion and protein phosphorylation. Timosaponin AⅢinhibited the U46619-,arachidonic acid-, collagen-, and ADP-induced platelet aggregation in a concentration-dependent manner, but the sensitivity of platelet agonists varied to the inhibitory effect of Timosaponin AⅢ, U46619- and arachidonic acid -mediated platelet aggregation being more sensitive, suggesting that Timosaponin AⅢmay interfere with the TXA2 synthesis or its action directly. We observed an interesting phenomenon that Timosaponin AⅢby itself caused a shape change without aggregation. prostaglandin E1, A synthetic compound,inhibits platelets activity by stimulating the Gs-coupled prostacyclin IP receptor leading to the activation of adenylyl cyclase and production of cAMP. Cyclic AMP is a control molecule in platelets that interrupts multiple signaling pathways. Elevation cAMP levels can inhibit most platelet responses. However, Timosaponin AⅢdid not significantly increase the level cAMP in this study. Then, Timosaponin AⅢis also a potent platelet inhibitor as PGE1, but inhibits platelet activity independent of elevation cAMP levels of platelets.The PKC family has long been know to involved in a number of platelet activity processes, most importantly aggregation and secretion. PMA can induce aggregation and secretion by activating PKC. Timosaponin AⅢdid not inhibited PMA–induced aggregation and secretion , suggesting Timosaponin AⅢmay inhibit the upstream signals of PKC. Thrombin interacts with platelet through a specific receptor belonging to the class or superfamily of receptors that are coupled to G proteins and phospholipase C, producing diacylglycerol, which stimulates protein kinase C that is closely linked to secretion. Activation of platelets by thrombin depends on TXA2 formation only at lower concentrations; at higher concentrations the ability of thrombin to activate platelets is independent of TXA2. In the present study, a higher concentration of thrombin was selected to induce platelet aggregation, and Timosaponin AⅢdidn't inhibit thrombin-induced aggregation and secretion. The inhibitory effect of Timosaponin AⅢwas selective for U46619 that activated TXA2-mediated platelet aggregation.The ERKs are phosphorylated, and presumably activated, by various agonists such as thrombin, PMA and U46619, but their role and relevance in platelet function remain unclear. Our study further confirms ERK 1/2 activation in response to platelet stimulation with agonists. Moreover, we have demonstrated that Timosaponin AⅢis efficient inhibitors of ERK 1/2 phosphorylation induced by U46619. This inhibitory effect could involve blockage of signaling downstream TP, since these Timosaponin AⅢhave a minor effect on TXA2-independent thrombin-induced ERK1/2 phosphorylation and PMA-induced ERK phosphorylation.TXA2 is a metabolite of arachidonic acid with a chemical half-life of about 30 s. TXA2 released from activated platelets binds to TXA2 receptors and causes platelet shape change and aggregation as a positive feedback mediator. TXA2 receptor interacts with heterotrimeric G proteins Gq and G12/13. Gq-mediated phospholipase Cβactivation appears to play a central role in platelet aggregation and secretion, whereas G12/13-signaling pathway induces platelet shape change, involving Rho/Rho kinase-mediated phosphorylation of the myosin light chain. Our further research found Timosaponin AⅢselectively blockaded TP-mediated Gq-signaling pathway, and had no effect on G12/13-signaling pathway. We observed U73122, a potent inhibitor of PLCβ, inhibited Timosaponin AⅢ-induced shape change, suggesting Timosaponin AⅢbeing a partial agonist by activating Gq. Finally, we found that Timosaponin AⅢhas a synergistic action with SQ29548, a selective TXA2 receptor antagonist, on U46619-induced platelet aggregation.In conclusion, our present study first used Timosaponin AⅢ, isolated from the root of Anemarrhenae asphodeloides Bge (Liliaceae) , as an ideal probe to explore the molecular mechanism of anti-platelet activity of steroid saponins.. Based on signal pathway, we supposed that candidate target of Timosaponin AⅢon anti-platelet activity might be TXA2 receptor . |
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