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Panax notoginseng (Burk.) F.H. Chen, known as Sanqi in China, is regarded as a trauma panacea, which is famous for its bidirectional therapeutic effect on haematological diseases, having both a haemostatic and anti-thrombotic action. Panax notoginseng saponins (PNS), including ginsenosides and notoginsenosides, are thought to account for the anti-thrombotic functions of the herb. Numerous studies have shown that PNS as a whole decreases platelet superficial activation, inhibits platelet adhesion and aggregation, prevents thrombosis and improves the microcirculation, and it has been widely used clinically in China (Mo et al., 1989; Ma and Xiao, 1998; Li et al., 2007; Wang et al., 2008; Lau et al., 2009). Saponin molecules found in P. notoginseng, such as ginsenoside Rg1, Rp1, Rh1, F1 and 2A, have been reported to inhibit platelet aggregation and thrombus formation when used alone (Ng, 2006; Endale et al., 2012). Dencichine, a non-saponin molecule present in the raw extract of P. notoginseng, is famous for its haemostatic effect. Total PNS has also been demonstrated to have a haemostatic effect when used externally (White et al., 2000), but, until now, few studies have shown that saponin accounts for the procoagulant effect of total PNS.
The process of blood coagulation involves platelet aggregation and interactions among multiple coagulation factors (De Cristofaro and De Candia, 2003; Joseph and Alpert, 2003; Davi and Patrono, 2007; Clemetson, 2010). The interplay between platelets and injured endothelial cells, combined with the modulation of other factors, triggers platelet aggregation and contributes to a series of events in the coagulation cascade leading to thrombin generation and fibrin clot formation that ultimately arrests bleeding (Park et al., 1996). Therefore, platelets play a vital role in the process of haemostasis. Defects in platelet function are believed to be the cause of many cardiovascular and cerebrovascular diseases, including bleeding (haemorrhage), stroke, cerebral thrombus and coronary artery disease (White et al., 2000; Chan et al., 2002; Ueno et al., 2011).
ADP has been identified as an important regulator of platelet function and exerts its effect via P2Y1 and P2Y12 receptors (Cunningham et al., 2013). The P2Y12 receptor is one of the GPCRs. When activated by ADP, the P2Y12 receptor on the platelet triggers a series of downstream events leading to platelet aggregation, shape change and dense granule secretion (Dorsam and Kunapuli, 2004), and therefore is a critical regulator of haemostasis and thrombosis.
In our studies, we used an in vitro assay to evaluate the effects of 11 types of saponins found in P. notoginseng on platelet aggregation. The results revealed that notoginsenoside Ft1 (Ft1) that distinctively exists in P. notoginseng facilitates platelet aggregation significantly. Further studies showed that the haemostatic effect of Ft1 is closely associated with P2Y12 receptors on platelets. Molecular docking analysis indicated the possible binding of Ft1 to P2Y12 receptors, which was confirmed in HEK293 cells overexpressing these receptors. Furthermore, Ft1 modulated the signal pathway molecules, such as PI3K and Akt, downstream of the activation of P2Y12 receptors. These findings, which suggest that platelet P2Y12 receptors are actively engaged in the procoagulant effect of Ft1, will benefit basic science and aid in the development of effective therapies for haematological disorders.
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In the present study, we evaluated the effects of 11 types of saponins found in P. notoginseng on platelet aggregation. Compared with the control, most of the saponins examined at 200 μM showed an anti-aggregatory effect on platelets; they markedly decreased ADP-induced platelet aggregation (P < 0.001, Figure 1). These results are consistent with previous findings, which showed that overall total PNS has an anti-thrombogenic effect induced by inhibiting platelet aggregation and adhesion (Mo et al., 1989; Liao and Li, 1997; Ma and Xiao, 1998) and increasing fluidity of platelet membranes. In contrast, total PNS also induces a haemostatic effect when used externally (White et al., 2000). However, our study is the first one to identify the exact procoagulant components of PNS. In our research, three types of notoginsenosides (i.e. Ft1, Fe and protopanaxadiol) enhanced platelet aggregation significantly, this has never been reported elsewhere.
Although Ft1 was first identified in 2006, few pharmacological studies have been conducted on it except for the recently reported angiogenetic effect observed by our group. As Ft1 displayed the most prominent effect on acceleration of platelet aggregation, we investigated it further to elucidate its procoagulant effect and possible underlying mechanisms. As illustrated in Figure 2, Ft1 dose- dependently induced platelet aggregation. However, the response window was very narrow as the difference in the concentration needed to induce the peak response from that producing a depressed response was less than 15 μM. Because total PNS has been used widely as an anti-thrombotic medicine in China (Lei and Chiou, 1986), precautions should be taken to control its Ft1 content so as to avoid an unwanted thrombogenic effects.
Both APTT and PT are the performance indicators for coagulation pathways. But the pathways measured are quite different. APTT is a measure of the intrinsic pathway, whereas PT is an indicator of the extrinsic pathway. TT is used to assess the effectiveness of fibrinolytic therapy. In our study, Ft1 shortened all the coagulation indexes (i.e. APTT, PT and TT) in both rat and human plasma (Figures 2 and 3), which indicated its strong procoagulant efficacy. To further confirm this effect, thrombogenesis and tail bleeding time assays were carried out in vivo. As expected, Ft1 shortened bleeding time significantly but increased thrombus formation in rats (Figure 4). These results provide strong evidence that Ft1 has a haemostatic effect.
Both P2Y1 and P2Y12 receptors mediate ADP-induced platelet aggregation. However, they play different roles in the process. ADP initiates platelet aggregation and platelet shape change via P2Y1 recptors, but activation of P2Y12 receptors leads to amplification, sustained aggregation and secretion (Hechler et al., 1998; Storey et al., 2000). Unlike the wide distribution of P2Y1 receptors (Ralevic and Burnstock, 1998), the P2Y12 receptor is found only on the platelet surface and in the brain (Murugappa and Kunapuli, 2006). Therefore, blockade of the P2Y12 receptor is a powerful anti-platelet strategy in the treatment and prevention of arterial thrombosis. Antagonists or inhibitors targeting P2Y12 receptors such as clopidogrel, prasugrel and TIC have already been used clinically or are undergoing clinical trials (Patel et al., 2013). In our experiments, both clopidogrel and TIC were used as reference compounds not only as the negative control for Ft1, but were also employed as P2Y12 antagonists to probe the binding target of Ft1. In most cases, such as in vivo thrombogenesis and bleeding time assays, clopidogrel showed opposite effects to Ft1. These results provide evidence that Ft1 has a P2Y12 agonist-like effect.
To elucidate the mechanism of the effects of Ft1 on haemostasis, a molecular docking model was established based on rat P2Y12 receptors. The binding sites or active centre of rat P2Y12 receptors has not been well characterized, However, a few studies have investigated the ligand-binding domain of the human P2Y12 receptor (Hoffmann et al., 2008; Mao et al., 2010). As reported, transmembrane (TM)3, TM6 and TM7 regions are involved in agonist binding, especially the polar amino acids such as Arg and Lys. In addition, the extracellular loop 2 has been suggested to be associated with nucleobase recognition and to load the agonist into the binding pocket (Moro et al., 1999; Hillmann et al., 2009). As the structures of the nucleotide-like GPCRs are similar, 3QAK with agonist in the active centre was selected as a template to build the model of rat P2Y12 receptor. The residues around the agonist within 7 Å were selected as a binding pocket, which was quite similar to that of the human P2Y12 receptor reported previously (Van Giezen et al., 2009). The binding cavity, especially the interior part, was composed of hydrophobic residues such as Phe83, Phe110, Tyr115, Ile118, Trp250, Phe258 and His259. On the other side of the cavity, several polar and charged amino acids such as Arg262, Glu279, Asn280 and Lys286 were also involved into the build-up of the pocket. The molecular docking study with Ft1 disclosed that the molecule interacted with many residues such as Phe83, Ile87, Asp90, Phe110, Phe183, Leu184, Tyr265, Asn280 and Lys286 in the pocket. The interaction may change the conformation of the P2Y12 receptor and thus facilitate downstream signalling pathways that contribute to platelet aggregation.
In agreement with the molecular docking results, further intracellular measurements of calcium and cAMP levels in platelets indicated the binding of Ft1 to P2Y12 recptors. Fluorescent indicators have been widely used for calcium measurements and can be classified into two categories: qualitative indicators and quantitative indicators (Zhou and Mao, 2007). In the current study, we selected fluo-3, one of the typical qualitative indictors excited by visible lights, to investigate any alterations in cytosolic calcium elicited by Ft1. Fluo-3 has a high affinity for free calcium and is, therefore, sensitive to and reflects changes in the concentration of calcium. As shown in Figure 5C, Ft1 markedly elevated the intracellular concentration of calcium and this effect was blocked by CIH. In HEK293 cells overexpressing rat P2Y12 receptors, Ft1 evoked a marked increase in the calcium accumulation and this effect was blocked by TIC. Furthermore, Ft1 had an inhibitory effect on cAMP production in platelets that could be abolished by TIC. Ft1 also increased, in a time-dependent manner, the phosphorylation of PI3K and Akt, the signalling molecules downstream of P2Y12 activation. All of the above results further confirm that Ft1 binds to the P2Y12 receptor.
More importantly, the haemostatic mechanism of Ft1 is different from that of the currently used haemostatic drugs such as etamsylate, amniomethylbenzoic acid, transmic acid and adrenosin. For example, etamsylate enhances the crosstalk among platelets, leukocytes and the vascular wall via membrane P-selectin-PSGL-1 interactions under conditions of vascular injury. Adrenosin increases the resistance of microvessels and, therefore, prevents haemorrhage by disrupting the permeability of microvessels. Amniomethylbenzoic acid and transmic acid inhibit many activators of plasminogen and prevent its conversion to plasmin. In contrast, Ft1 exerts its haemostatic effects by accelerating platelet aggregation through binding to P2Y12 receptors. However, at the current stage, further studies need to be carried out to determine if Ft1 can be developed into a new type of haemostatic drug.
In conclusion, of the eleven types of PNS investigated Ft1 was found to be the most potent at enhancing ADP-induced platelet aggregation and promoting blood haemostasis. P2Y12 receptors on platelets were shown to play an important role in mediating the haemostatic effect of Ft1. This is the first time that the exact procoagulant saponins responsible for the haemostatic effects of P. notoginseng have been identified. This has provided us with selective P2Y12 receptor agonists for use in basic research and also potential drugs for clinical therapy of haemorrhage.