TR-05265; No of Pages 9 Thrombosis Research xxx (2013) xxx–xxx
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Regular Article
Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis Qi Zhou a, Lei Jiang a, Chunhua Xu a, Dongjiao Luo a, Chunlai Zeng c, Pu Liu a, Ming Yue a, Yangyang Liu a, Xiaosheng Hu b, Hu Hu a,⁎ a b c
Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, China Department of Cardiology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Department of Cardiology, Lishui Central Hospital, Lishui, China
a r t i c l e
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Article history: Received 22 July 2013 Received in revised form 19 October 2013 Accepted 20 October 2013 Available online xxxx Keywords: Platelet Thrombin ERK Ginsenoside Rg1 Arterial thrombosis
a b s t r a c t Introduction: Derived from the root of Panax ginseng C.A.Mey, Panax notoginsenosides (PNS) is a widely used herbal medicine to treat atherothrombotic diseases in Asian medicine. Ginsenoside Rg1 is one of the main compounds responsible for the pharmaceutical actions of PNS. As platelets play pivotal roles in atherothrombogenesis, we therefore studied the effect of Rg1 on platelet activation and its underlying mechanisms. Materials and Methods: Human platelets are obtained from healthy subjects. Platelet activation and the inhibition of Rg1 were assessed by Born aggregometer, flow cytmetry, flow chamber and western blot. The in vivo thrombosis model was induced by 10% FeCl3 on mesenteric arterioles of wild type B57/b6 mice. Results: Rg1 significantly inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, e.g., aggregation rate stimulated by 0.1 U mL-1 thrombin was decreased 46% by Rg1. Rg1 also reduced thrombin (0.1 U mL-1)-enhanced fibrinogen binding and P-selectin expression of single platelet by 81% and 66%, respectively. Rg1 affected αIIbβ3-mediated outside-in signaling as demonstrated by diminished platelet spreading on immobilized fibrinogen. Rg1 also decreased the rate of clot retraction in platelet rich plasma. Furthermore, Rg1 decreased platelet adhesion on collagen surface under a shear rate correlated to the arterial flow (1000 s-1) by approximately 70%. Western blot showed that Rg1 potently inhibited ERK phosphrylation. The in vitro findings were further evaluated in the mouse model of in vivo arterial thrombosis, and Rg1 was found to prolong the mesenteric arterial occlusion time (34.9 ± 4.1 min without and 64.3 ± 4.9 min with Rg1; p b 0.01). Conclusions: Rg1 inhibits platelet activation via the inhibition of ERK pathway, and attenuates arterial thrombus formation in vivo. © 2013 Elsevier Ltd. All rights reserved.
Introduction Platelet activation plays pivotal roles in atherothrombogenesis [1]. Antiplatelet treatment, e.g., the cyclooxygenase (COX) inhibitor aspirin and the P2Y12 inhibitor clopidelgrel, has been established as a cornerstone in the management of thrombotic and cardiovascular disease [2,3]. There are, however, limitations associated with current antiplatelet treatment, such as increased risk of bleeding complications and drug resistance commonly associated with aspirin and clopidelgrel
Abbreviations: COX, cyclooxygenase; CDDPs, Compound Danshen Dripping Pills; SAA, salvianolic acid A; PNS, panax notoginsenosides; Rg1, ginsenoside Rg1; PARs, proteinaseactivated receptors; PLCβ, phospholipase C β; PKC, protein kinase C; MAPKs, mitogenactivated protein kinases; BSA, bovine serum albumin; PBS, phosphate-buffered saline; SDS-PAGE, SDS polyacrylamide gel electrophoresis; TXA2, thromboxane A2. ⁎ Corresponding author at: Laboratory for Thrombosis and Haemostasis, Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China. Tel.: +86 571 882 085 17; fax: +86 571 882 081 97. E-mail address: [email protected] (H. Hu).
[4–6]. Searching for novel classes of antiplatelet agents has thus become an important measure to improve the current antiplatelet therapies. In addition to chemical synthesis of newer generation of conventional antiplatelet drugs, there is a special interest on natural compounds present in dietary and medication plants exhibiting antiplatelet/ thrombotic properties. Resveratrol residing in red wines is a wellknown example which has been shown with inhibitory effects on platelet activity by many studies [7]. Compound Danshen Dripping Pills (CDDPs) is a herb formula widely used in the clinical management of thrombotic diseases in Chinese traditional medicine [8–10]. The major active ingredients of CDDPs include extracts from Savia miltiorrhiza and Panax Notoginseng, both herbs are known for “promoting circulation and remove blood stasis” [11]. We have previously reported a potent antiplatelet compound salvianolic acid A (SAA), which is isolated from Savia miltiorrhiza [12]. It is presently unclear the antiplatelet properties of extracts from Panax Notoginseng. Extracts from Panax Notoginseng are collectively named Panax notoginsenosides (PNS), which consist of ginsenosides Rg1, Rb1 and notoginsenoside R1, with Rg1 being the most abundant
0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.10.032
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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[13,14]. Previous study has shown that Rg1 can reduce side effects while maintain the efficacy of glucocorticoid in inflammatory diseases [15]. Rg1 has also been reported to facilitate neural differentiation of mouse embryonic stem cells [16]. However, effects and mechanism of Rg1 on platelet activation has not been investigated. Thrombin is one of the most potent platelet activator and plays a central role in both haemostasis and thrombosis [17,18]. Upon vessel wall injury, after the initial formation of platelet monolayer, thrombin serves as an essential mediator for the recruitment of additional platelets from the circulation to support the extension of thrombus and the resultant vessel occlusion. The effects of thrombin are mediated through G protein-coupled proteinase-activated receptors (PARs) [19–22]. PAR1 and PAR4 are present on human platelets. Both PAR1 and PAR4 are coupled to Gq to activate phospholipase C β (PLCβ), which can induce the activation of protein kinase C (PKC) and subsequent mitogenactivated protein kinase (MAPK) cascade [23]. PAR1 and PAR4 can also be coupled to G12/13 to activate Rho/Rho kinase [24]. Furthermore, these two can activate the Gi pathway through released ADP binding to P2Y12 receptor [25–27]. Thrombin per se and the key molecules of its signaling pathways have always been important targets in antithrombotic practice [21,28]. In the present study, we investigated the effect of Rg1 on platelet activation in response to thrombin, as well as the potential influence of Rg1 on the cellular signaling pathways. We demonstrated that Rg1 significantly inhibited thrombin-induced platelet activation in vitro and arterial thrombosis in a mouse model in vivo. We have also shown that Rg1 may act via inhibiting platelet PKC-MAPK pathway. Materials and methods Study subjects The study protocol was approved by the Ethic Committee of the Zhejiang University. The non-smoking healthy subjects (aged 18 to 28 years) were recruited from the staff and students at the Zhejiang University School of Medicine. Informed consent was obtained from all the volunteers. Animal experiments were carried out in compliance with the guidelines of the International Society on Thrombosis and Haemostasi [29], and were approved by the Board of Animal Study of Zhejiang University. Reagents Rg1 was purchased from the Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). SAA was purchased from the Division of Chinese Material Medica and Natural Products, National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). Thrombin, apyrase, indomethacin, HEPES and bovine serum albumin (BSA) were all purchased from Sigma (St Louis, MO, USA). The MEK inhibitor U0126 and PKC pan inhibitor Ro-31-8220 were from Cell Signaling Technology (Beverly, MA, USA). The fluorescence dye calcien was from Invitrogen (Eugene, Oregen, USA). For flow cytometric analysis, platelets were identified with the fluorescein isothiocyanate (FITC)-conjugated anti-CD42a monoclonal antibody (mAb) Beb 1 (Becton Dickinson; San Jose, CA, USA). The platelet activation markers, fibrinogen binding and P-selectin expression, were monitored with FITC-conjugated polyclonal anti-human fibrinogen antibody (DAKO, Glostrup, Denmark) and the R-phycoerythrin (RPE)conjugated anti-CD62 mAb AC1.2 (Becton Dickinson), respectively. For immunoblotting, p44/42 MAPK (ERK1/2) (137F5) rabbit mAb and phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (E10) mouse mAb, p38 MAPK antibody and phospho-p38 MAPK (Thr180/Thr182) (28B10) mouse mAb, Akt antibody and phospho-Akt (Ser473) (587 F11) mouse mAb, phosphor-(ser) PKC substrate antibody and β-actin (8H10D10) mouse mAb were all from Cell Signaling Technology (Beverly, MA, USA).
Human blood collection and preparation of washed platelets and red blood cells All blood doners had antecubital veins that allowed a clean venepuncture, and denied taking any medication during the 2 weeks preceding venepuncture. Blood was drawn without stasis into the siliconized vacutainers containing 1/9 (v/v) 3.8% sodium citrate. For preparation of washed platelets, blood was anticoagulanted with 75 mM sodium citrate, 39 mM citric acid, and 135 mM dextrose, PH 6.5 (ACD), and centrifuged at 150 ×g for 20 min; the platelet-rich plasma (PRP) was then collected. The PRP was diluted three-fold in ACD, and centrifuged for 10 min at 800 ×g. The platelet pellet was then resuspended in Tyrode’s buffer (137 mM NaCl, 12 mM NaHCO3, 2 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 5.5 mM glucose, 5 mM HEPES, and 0.35% BSA) at a concentration of 250 × 106 platelets mL-1. CaCl2 (2 mM) was added prior to the agonist stimulation. To prepare washed red blood cells, the remaining blood after PRP collection, consisting mainly of red blood cells, was washed with Tyrode’s buffer containing ACD and centrifugated three times at 800×g for 10min to remove plasma proteins. Flow cytometric analysis Flow cytometric analyses of platelet P-selectin expression and fibrinogen binding have been described previously [30]. Sample analysis was performed using a Beckman Coulter FC-500 flow cytometer (Beckman Coulter, Hialeah, FL, USA). Platelet P-selectin expression and fibrinogen binding were reported as the percentages of P-selectin-positive and fibrinogen bindingpositive cells, respectively, in the platelet population. Born aggregometry Agonist-induced platelet aggregation was measured using a ChronoLog aggregometer. Washed human platelets (0.25 mL; 250 × 106 mL-1) were preincubated with vehicle (HEPES buffer; 150 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM HEPES) or Rg1 (0.4, 2 and 4 mg mL-1) for 10 min at 37 °C without stirring. The agonists were then added with stirring at 900 rpm. Platelet adhesion on collagen-coated surface under flow conditions The experiments were performed using a previously described method [31], with minor modifications. Briefly, glass coverslips (24 × 50 mm; EUPOTUBO, Amadora, Portugal) were cleaned overnight with a dichromic acid solution, rinsed with distilled water, and dried before coating. Collagen was coated at a density of 0.08 μg cm-2 on coverslips overnight at 4 °C. The flow chamber was then assembled so that the coverslip formed the lower surface of the chamber. A peristaltic pump (Masterflex L/S; Cole-Parmer, Vernon Hills, IL, USA) was used to aspirate blood through the flow chamber. The fluorescence dye calcien (10 μg mL-1) was added to the washed platelets. After 20 min of incubation, calcein-labeled platelets were resuspended and mixed with washed red blood cells to form reconstituted blood with a platelet concentration of 250 × 106 mL-1 and a hemocrit of 45%. The reconstituted blood was incubated at 37 °C for 10 min in the absence or presence of Rg1 (4 mg mL-1) or U0126 (20 μM). Blood was perfused over the coverslip for 5 min under a shear rate correlated to the arterial flow (1000 s-1). The perfusion was live-monitored with a fluorescence microscope (Nikon TE-2000S; Nikon, Melville, NY, USA) equipped with a Nikon DS-2MBWc-U1 CCD camera (Nikon). After the termination of perfusion, the flow chamber was washed with phosphate-buffered saline (PBS) for 2 min to remove non-adherent cells. The images from at least 10 independent fields per flow experiment were recorded and analyzed by using IMAGE-PRO PLUS software (Media Cybernetics, Bethesda, MD, USA).
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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Fig. 1. Effects of Rg1 on platelet aggregation. (A, B) Washed human platelets (250 × 106 mL-1) were preincubated for 10 min with Rg1 (4 mg mL-1) or vehicle (HEPES buffer). Platelet aggregation was initiated with thrombin (0.05 U mL-1 or 0.1 U mL-1), ADP (10 μM or 20 μM), collagen (1 μg mL-1 or 2 μg mL-1) and U46619 (0.3 μM or 3 μM). (C, D) Aggregation of Rg1treated (4 mg mL-1) or vehicle-treated washed human platelets was induced by thrombin (0.1 U mL-1), and in the presence or absence of apyrase (1 U mL-1) or indomethacin (10 μM). (E) Washed human platelets were preincubated with Rg1 (4 mg mL-1), SAA (0.1 mg mL-1) or the combination of Rg1 and SAA for 10 min, and then stimulated with thrombin (0.1 U mL-1). Mean ± SEM of platelet aggregation percentage from four experiments are plotted in the bars charts; * P b 0.05, ** P b 0.01 as compared with control.
Platelet spreading on fibrinogen
Immunoblotting
Glass coverslips (24 × 50 mm; EUPOTUBO) were coated with 20 ug mL fibrinogen in 0.1 M NaHCO3 (pH 8.3) at 4 °C overnight. Washed platelets (200 × 105 mL-1) preincubated with (4 mg mL-1 Rg1 or 20 μM U0126; 10 min at 37 °C) or without inhibitors were allowed to spread on the fibrinogen-coated surfaces at 37 °C for 90 min. After three washes with PBS, the cells were fixed, permeabilized, and stained with fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR, USA). Adherent platelets were viewed with an inverted fluorescence microscope (Nikon TE-2000S) using a PLAN FLUOR lens (× 100/1.30 numerical aperture oil objective). Images were acquired using a Nikon DS-2MBWc-U1 CCD camera. The spreading area of individual platelet was assessed using IMAGE-PRO PLUS software (Media Cybernetics).
Aliquots of washed platelets (0.25 mL; 250 × 106 mL- 1) treated with or without Rg1 (4 mg mL- 1; 10-min preincubation) were stimulated with agonists at 37 °C. The reaction was stopped by the addition of sodium dodecylsulfate (SDS) sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue; pH 6.8). Samples were boiled for 5 min at 95 °C before storage at - 20 °C. Sample aliquots (40μL) were loaded, and the proteins were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to a poly (vinylidence difluoride) (PVDF) membrane and subjected to western blotting. ERK and phospho-ERK, p38 and phospho-p38, Akt and phospho-Akt, phospho-PKC substrate and β-actin were detected using corresponding antibodies (all from
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Cell Signaling Technology, Beverly, MA, USA). After incubation with the corresponding secondary antibodies goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680 (LI-COR Biosciences, Lincoln, NE, USA), densitometric band scanning was performed using an Odyssey® infrared imaging system (LI-COR Biosciences).
To study the effect of Rg1 on thrombus formation, a bolus (10mgkg-1) of Rg1 or vehicle (HEPES buffer) was applied through the catheterized jugular vein 5 min prior to the vessel wall injury. Fluorescently labeled platelets were also preincubated with Rg1 (4 mg mL-1; 10 min) before injection.
Platelet-mediated clot retraction
Statistics
Platelet-rich plasma (PRP) was preincubated with inhibitors for 10 mins at 37 °C. Coagulation was induced with 0.2 U mL-1 thrombin, and clots were allowed to retract at 37 °C and photographed at various time points.
The data are presented as mean ± SEM (standard errors of the mean). Individual measurement of Rg1 effect was assessed with Wilcoxon’s signed rank test, and comparisons of vessel occlusion times were made using Mann-Whitney statistical tests. P b 0.05 was considered to be statistically significant.
Preparation and labeling of washed mouse platelets
Results
Mice, at 8-12 weeks of age, were bled from the inferior vena cava under chloraldurate anesthesia (400 mg kg-1 intraperitoneally). Mouse blood was collected in a 1: 10 volume of 3.2% sodium citrate containing 200 μg mL-1 hirudin. PRP was obtained by centrifugation at 800 ×g for 30 s, and then at 5 min at 150 ×g. The PRP was diluted in ACD and washed for 5 min. The platelet pellet was then resuspended in Ca2+free Tyrode’s buffer containing 10 μg mL-1 calcein, and incubated at 37 °C for 20 min. The labeled platelets were washed and resuspended at 500 × 106 mL -1.
Effects of Rg1 on thrombin induced platelet aggregation
10% FeCl3 induced thrombosis Wide-type mice (21.8 ± 0.4 g, n = 12) were anesthetized with chloraldurate, and the jugular vein was catheterized with an intramedic PE10 canule (Becton Dicknson). After laparotomy, the mouse intestines were exposed, and the mesentery was spread on the translucent table of the Nikon fluorescence microscope (TE-2000S) equipped with a DS2MBWc-U1 CCD camera (Nikon). An arteriole (60-80 μm in diameter) and its accompanying venule were selected under the microscope. Fluorescently labeled murine platelets (0.2 mL; 500 × 106 mL-1) were injected into the catheterized jugular vein. Injury of mesenteric arterioles was induced by topical application of 10% FeCl3 (Sigma, St Louis, MO, USA). A 2 × 5 mm strip of filter paper (Whatman) saturated with 10% FeCl3 solution was applied to the surface of mice mesenteric arterioles and venules for 2 minutes followed by rinsing with PBS. Vessels were monitored and occlusion time was defined as cessation of blood flow lasted longer than 1 minute due to an occlusive thrombus in the injured arteriole.
First, we examined the effect of Rg1 on different agonists-induced platelet aggregation. Rg1 inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, with the most prominent inhibition shown on thrombin-induced platelet aggregation (Fig. 1A,B). For example, thrombin (0.05 U mL-1) induced platelet aggregation rate was reduced by Rg1 (4 mg mL-1) from 87.5 ± 4.4% to 27.3 ± 5.6% (n = 4; P b 0.05), same concentration of Rg1 decreased platelet aggregation rate by thrombin (0.1 U mL-1) from 94.7 ± 3.9% to 57.0 ± 3.1% (n = 4; P b 0.01). Furthermore, apyrase and indomethacin were used to exclude the possibility that the effect of Rg1 on thrombin-induced platelet aggregation was due to the inhibition of ADP secretion or thromboxane A2 (TXA2) generation. Rg1 further inhibited thrombin-induced platelet aggregation in the presence of apyrase or indomethacin, suggesting an ADP- and TXA2-independent effect of Rg1 (Fig. 1C,D). Interestingly, a synergistic inhibitory effect was found when Rg1 was combined with SAA, a compound we previously identified to negatively regultate PI3K pathway [12]. Shown in Fig. 1E, combination of Rg1 and SAA nearly abolished thrombin (0.1 U mL-1) induced platelet aggregation, which was inhibited poorly by SAA alone. Influence of Rg1 on thrombin-induced single platelet activation The influence of Rg1 on single-platelet activation was further investigated using flow cytometry. Low (0.05 U mL-1) and high (0.1 U mL-1) concentration of thrombin increased platelet fibrinogen binding, a marker reflecting platelet aggregability, from 2.5 ± 0.5% at
Fig. 2. Effects of Rg1 on single platelet activation. Platelet-rich plasma (PRP) was preincubated with or without Rg1 (4 mg mL-1; 22 °C; 10 min) in the presence of fluorescent P-selectin and fibrinogen antibodies. Samples were then challenged with thrombin (0.05 U mL-1 or 0.1 U mL-1), and incubated for a further 10 min. Fibrinogen binding (A) and P-selectin expression (B) of single platelet were monitored by flow cytometry. Data plotted are mean ± SEM; n = 4; * P b 0.05, ** P b 0.01 without vs. with Rg1 treatment.
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baseline to 65.3 ± 12.4% and 83.7 ± 8.7% (Fig. 2A), respectively (n = 4; P b 0.05 and P b 0.01, respectively). The increases were reduced to 6.3 ± 5.2% and 2.2 ± 1.6% (Fig. 2A) in the presence of 4 mg mL-1 Rg1. Similar inhibitory effects of Rg1 were seen on thrombin-induced platelet P-selectin expression, which reflects platelet α-granule secretion (Fig. 2B). Influence of Rg1 on platelet intracellular signaling To investigate the intracellular signaling targets of Rg1, two important platelet signaling pathways were monitored: PI3K pathway and MAPK pathway (p38 phosphorylation or ERK phosphorylation). Here, washed human platelets were preincubated with or without Rg1 (4 mg mL-1, 37 °C, 10 min) before being challenged by thrombin. Western blotting showed that thrombin markedly enhanced phosphorylation of the enzymes (Fig. 3A). Rg1 had no influence on Akt or p38 phosphorylation, but markedly inhibited ERK phosphorylation induced by thrombin, indicating Rg1 action on thrombin-induced ERK pathway (Fig. 3A,B). To further confirm that Rg1 may act on ERK pathway, the MEK inhibitor U0126 was employed. Rg1 and U0126 showed nearly identical inhibitory effects on thrombin-induced ERK phosphorylation. Moreover, Rg1 and U0126 demonstrated no additive inhibition when combined (Fig. 3B). PKC was known to be important during thrombin-induced ERK phosphorylation [23,32]. Consistent with previous studies, our data confirmed that the PKC pan inhibitor Ro-31-8220 inhibited the activation of ERK (Fig. 3C). We also found that thrombin-induced PKC-substrate phosphorylation was partial and total inhibited by Rg1 and Ro-31-8220, respectively. The combination of Rg1 and Ro31-8220 did not show any additive inhibition (Fig. 3D). These results demonstrated that Rg1 affected PKC/ERK pathway in thrombinactivated platelets. Effects of Rg1 on platelet adhesion under flow Perfusion of reconstituted blood over collagen-coated surfaces under a shear rate correlated to the arterial flow (1000 s-1) led to significant platelet adhesion. The adhesion was inhibited by Rg1 and, to a slightly milder degree, by U0126 (Fig. 4A), as shown by reduced densities of adherent platelets (Fig. 4B). However, the combination of Rg1 and U0126 did not strengthen the inhibitory effect. Effects of Rg1 on platelet spreading on immobilized fibrinogen We further determined whether Rg1 could affect outside-in signaling, a process that is dependent on ERK (Fig. 5). In the absence of Rg1, the average surface coverage of spread platelets was 44.5 ± 8.7 μm2. Rg1 significantly inhibited platelet spreading and reduced the surface coverage to 21.0 ± 3.8 μm2, and Rg1 and U0126 showed similar reductions. The combination of Rg1 and U0126 did not show any additive inhibition. Fig. 3. Influences of Rg1 on platelet intracellular signaling. (A) Washed human platelets (250 × 106 mL-1) were preincubated with or without Rg1 (4 mg mL-1), and then stimulated with thrombin (0.1 U mL-1) with stirring at 900 rpm in an aggregometer at 37 °C. Platelets were lysed, and immunoblotted using the corresponding antibodies recognizing total or phosphorylated enzymes: p38 and Akt. (B) Platelets were preincubated with either Rg1 (4 mg mL-1), U0126 (20 μM) or the combination of the two, and then stimulated with thrombin (0.05 U mL-1 or 0.1 U mL-1). Phosphorylation of ERK was detected by western blotting. (C) Platelets were preincubated with either Rg1 (4 mg mL-1), Ro-31-8220 (10 μM) or the combination of the two, and then stimulated with thrombin (0.05 U mL-1 or 0.1 U mL-1). Phosphorylation of ERK was detected by Western blotting. (D) Platelets were preincubated with either Rg1 (4 mg mL-1), Ro-31-8220 (10 μM) or the combination of the two, and then stimulated with thrombin (0.05 U mL-1 or 0.1 U mL-1). Phosphorylation of PKC substrate was detected by Western blotting. Representative results were from at least three independent experiments with similar results.
Effect of Rg1 on clot retraction Consistently, retraction of the fibrin clot in PRP, which also requires functional αIIbβ3 alongside tight membrane-cytoskeleton interactions, was significantly delayed by Rg1. The maximal clot retraction occurred at 45 min after addition of thrombin (0.2 U mL-1). In contrast, platelets treated with Rg1 (4 mg mL-1) failed to form clot retraction at 45 min and only partial retraction was observed 60 min after thrombin stimulation (Fig. 6). Influence of Rg1 on arterial thrombosis The effect of Rg1 on thrombus formation in vivo was investigated using a 10% FeCl3 induced mesenteric arterial thrombosis model in
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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Fig. 4. Effects of Rg1 on platelet adhesion to collagen-coated surfaces. Calcein-labelled platelets (250 × 106 mL-1) were added into reconstituted blood (red blood cell resuspended with Tyrodes’ buffer containing 1% albumin; hematocrit 45%). The reconstituted blood was incubated at 37 °C for 10 min with either Rg1 (4 mg mL-1), U0126 (20 μM) or the combination of the two. The reconstituted blood was then perfused over collagen-coated (0.08 μg mm-2) coverslips for 5 min under the shear rate (1000 s-1), and this was followed by washing with PBS to remove non-adherent cells. Adherent platelets were counted under a fluorescence microscope. (A) Representative images from at least three independent experiments with similar results. Mean ± SEM of adherent platelets per field (%) are plotted in (B). * P b 0.05, ** P b 0.01 as compared with control.
Fig. 5. Effects of Rg1 on platelet spreading on fibrinogen-coated surface. Washed platelets (200 × 105 mL-1) were preincubated with either Rg1 (4 mg mL-1), U0126 (20 μM) or the combination of the two for 10 min at 37 °C. Platelets were allowed to spread on fibrinogen-coated slides for 90 min, then fixed with paraformaldehyde to stop spreading. Platelets were subsequently labeled with fluorescein isothiocyanate-conjugated phalloidin, and photographed under a fluorescence microscope. (A) Representative images from at least three independent experiments with similar results. Mean ± SEM of the average surface area of individual platelets are plotted in (B). * P b 0.05 as compared with control.
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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Fig. 6. Effects of Rg1 on clot retraction. Platelet-rich plasma (PRP) was preincubated with or without Rg1 (4 mg mL-1) for 10 min at 37 °C. Coagulation was induced with 0.2 U mL-1 thrombin, and clots were allowed to retract at 37 °C and photographed. Representative images were from at least three independent experiments with similar results.
wild type mice. Washed and calcein-labeled platelets were preincubated with or without Rg1 (4 mgmL-1; 10 min) and then injected into the mice through catheterized jugular vein immediately before vessel injury. Thrombus formation was monitored in real time until vessel occlusion under an intravital fluorescence microscope. The thrombus complete occlusion was often seen in the arterioles approximately 34.9 ± 4.1 min after vessel injury (Fig. 7A). In the mice treatment with Rg1 (10 mg kg-1 bolus), vessel injury-induced platelet adhesion and thrombus formation were markedly attenuated, with the arterial occlusion time usually being 64.3 ± 4.9 min after vessel injury (Fig. 7B).
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indicate that Rg1 is an important contributor of the antithrombotic properties of Panax ginseng. Rg1 may have the potential to be further developed into a new generation of antiplatelet and antithrombotic drug. It is worth of mentioning that a recent study has shown that Rp1, the derivative of ginsenoside Rg3, has the similar inhibitory effects in platelet activation as Rg1 [33]. Rg1 strongly inhibits thrombin-induced platelet activation. This inhibition still occurs in the presence of apyrase or indomethacin, which indicates that ADP secretion or TXA2 generation are less likely the main mechanisms influenced by Rg1. However, our data suggested that both inside-out and outside-in signaling of αIIbβ3 are compromised by Rg1 treatment, as demonstrated by the decreased fibrinogen binding, impaired platelet spreading on immobilized fibrinogen surface, and delayed clot retraction by Rg1. Further investigation of the molecular mechanisms revealed that Rg1 may have targeted PKC-MAPK pathway. Indeed, human platelets contain several members of MAPK family, including p38, JNK and ERKs (ERK1/2) [34,35], and previous studies using the p38, ERK, JNK inhibitors or JNK-deficient mice have demonstrated the prominent roles of MAPKs in platelet activation induced by thrombin [36–39]. However, our data ruled out the possibility that MAPK p38 was the target of Rg1, based on the fact that Rg1 failed to influence p38 phosphorylation level induced by thrombin stimulation. Several lines of evidences in this study support that Rg1 may target on ERKs. First, the phosphorylation of PKC substrates and MAPK ERK2 is significantly decreased by Rg1. Second, MEK inhibitor U0126 and Rg1 showed similar but nonadditive inhibitory effects on platelet adhesion. Furthermore, previous study using U0126 has demonstrated inhibition towards platelet aggregation, clot retraction, and platelet spreading on fibrinogen, all of these functions are inhibited by Rg1 in the present study [37]. Despite that PI3K pathway has been suggested to regulate MAPKs activation, Rg1 does not seem to inhibit PI3K as judged by the intact Akt phosphorylation level. Herb formulae have been considered the quintessence of Chinese traditional medicine. The identification of the active ingredients and elucidation of their cellular mechanisms are essential steps to understand this ancient medical practice. The findings, on the other hand, hold great promises to the discovery of novel therapeutic agents. It is of interest to note that components within a formula are usually complementary to each other in functions. Savia miltiorrhiza and Panax Notoginseng have a long history of being used in combination in the management of cardiovascular and thrombotic diseases. We have previously showed that SAA, a compound from Savia miltiorrhiza, inhibited platelet activation via PI3K, without affecting MAPK activation [12]. Conversely, the present study reported that Rg1, extracted from Panax Notoginseng, exerts the anti-platelet effects via inhibiting MAPK, but not PI3K. Intriguingly, such a synergistic inhibition of two signaling pathways has produced a better antiplatlet effect (Fig. 1E). In conclusion, Rg1 inhibits platelet activation via the inhibition of PKC and ERK pathway and attenuates arterial thrombus formation in vivo. It thus may be developed as a potential novel therapeutic agent. Our study also suggests that traditional Chinese herb medicine could serve as a good resource for the screening and development of novel therapeutic agents for thrombotic disorders.
Discussion Conflict of interest statement The present study has shown that Rg1 inhibits thrombin-induced platelet activation and decreases platelet adhesion on collagen-coated surface under flow conditions. The inhibitory effect of Rg1 is possibly achieved by interfering platelet PKC-MAPK pathway, as evidenced by decreased ERK and PKC substrate phosphrylation. The in vivo effect of Rg1 was further confirmed in a mice mesenteric arterial thrombosis model. Rg1 exists in abundance in the medicinal herb Panax ginseng C.A.Mey. The antiplatelet properties of Rg1 demonstrated in this study
The authors declare no competing interests.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81170478, 81370618), National Basic Research Program of China (2012CB966603).
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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Fig. 7. Effects of Rg1 on arterial thrombus formation in vivo in wild-type mice. (A) Representative images and time course of thrombus formation induced by 10% FeCl3 injury of wide-type mouse intestinal arterioles, in the absence or presence of Rg1 (bolus 10 mg kg-1). Selected arterioles irrigating the cecum had a diameter of 60-100 μm. Arrows indicate irreversible arteriolar vessel occlusion. A indicates arteriole. (B) Dot plots of arteriolar occlusion time from five to ten experiments. Two-tailed P-values were calculated by two-column MannWhitney comparison (** P b 0.01).
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Regular Article
Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis Qi Zhou a, Lei Jiang a, Chunhua Xu a, Dongjiao Luo a, Chunlai Zeng c, Pu Liu a, Ming Yue a, Yangyang Liu a, Xiaosheng Hu b, Hu Hu a,⁎ a b c
Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, China Department of Cardiology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China Department of Cardiology, Lishui Central Hospital, Lishui, China
a r t i c l e
i n f o
Article history: Received 22 July 2013 Received in revised form 19 October 2013 Accepted 20 October 2013 Available online xxxx Keywords: Platelet Thrombin ERK Ginsenoside Rg1 Arterial thrombosis
a b s t r a c t Introduction: Derived from the root of Panax ginseng C.A.Mey, Panax notoginsenosides (PNS) is a widely used herbal medicine to treat atherothrombotic diseases in Asian medicine. Ginsenoside Rg1 is one of the main compounds responsible for the pharmaceutical actions of PNS. As platelets play pivotal roles in atherothrombogenesis, we therefore studied the effect of Rg1 on platelet activation and its underlying mechanisms. Materials and Methods: Human platelets are obtained from healthy subjects. Platelet activation and the inhibition of Rg1 were assessed by Born aggregometer, flow cytmetry, flow chamber and western blot. The in vivo thrombosis model was induced by 10% FeCl3 on mesenteric arterioles of wild type B57/b6 mice. Results: Rg1 significantly inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, e.g., aggregation rate stimulated by 0.1 U mL-1 thrombin was decreased 46% by Rg1. Rg1 also reduced thrombin (0.1 U mL-1)-enhanced fibrinogen binding and P-selectin expression of single platelet by 81% and 66%, respectively. Rg1 affected αIIbβ3-mediated outside-in signaling as demonstrated by diminished platelet spreading on immobilized fibrinogen. Rg1 also decreased the rate of clot retraction in platelet rich plasma. Furthermore, Rg1 decreased platelet adhesion on collagen surface under a shear rate correlated to the arterial flow (1000 s-1) by approximately 70%. Western blot showed that Rg1 potently inhibited ERK phosphrylation. The in vitro findings were further evaluated in the mouse model of in vivo arterial thrombosis, and Rg1 was found to prolong the mesenteric arterial occlusion time (34.9 ± 4.1 min without and 64.3 ± 4.9 min with Rg1; p b 0.01). Conclusions: Rg1 inhibits platelet activation via the inhibition of ERK pathway, and attenuates arterial thrombus formation in vivo. © 2013 Elsevier Ltd. All rights reserved.
Introduction Platelet activation plays pivotal roles in atherothrombogenesis [1]. Antiplatelet treatment, e.g., the cyclooxygenase (COX) inhibitor aspirin and the P2Y12 inhibitor clopidelgrel, has been established as a cornerstone in the management of thrombotic and cardiovascular disease [2,3]. There are, however, limitations associated with current antiplatelet treatment, such as increased risk of bleeding complications and drug resistance commonly associated with aspirin and clopidelgrel
Abbreviations: COX, cyclooxygenase; CDDPs, Compound Danshen Dripping Pills; SAA, salvianolic acid A; PNS, panax notoginsenosides; Rg1, ginsenoside Rg1; PARs, proteinaseactivated receptors; PLCβ, phospholipase C β; PKC, protein kinase C; MAPKs, mitogenactivated protein kinases; BSA, bovine serum albumin; PBS, phosphate-buffered saline; SDS-PAGE, SDS polyacrylamide gel electrophoresis; TXA2, thromboxane A2. ⁎ Corresponding author at: Laboratory for Thrombosis and Haemostasis, Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China. Tel.: +86 571 882 085 17; fax: +86 571 882 081 97. E-mail address: [email protected] (H. Hu).
[4–6]. Searching for novel classes of antiplatelet agents has thus become an important measure to improve the current antiplatelet therapies. In addition to chemical synthesis of newer generation of conventional antiplatelet drugs, there is a special interest on natural compounds present in dietary and medication plants exhibiting antiplatelet/ thrombotic properties. Resveratrol residing in red wines is a wellknown example which has been shown with inhibitory effects on platelet activity by many studies [7]. Compound Danshen Dripping Pills (CDDPs) is a herb formula widely used in the clinical management of thrombotic diseases in Chinese traditional medicine [8–10]. The major active ingredients of CDDPs include extracts from Savia miltiorrhiza and Panax Notoginseng, both herbs are known for “promoting circulation and remove blood stasis” [11]. We have previously reported a potent antiplatelet compound salvianolic acid A (SAA), which is isolated from Savia miltiorrhiza [12]. It is presently unclear the antiplatelet properties of extracts from Panax Notoginseng. Extracts from Panax Notoginseng are collectively named Panax notoginsenosides (PNS), which consist of ginsenosides Rg1, Rb1 and notoginsenoside R1, with Rg1 being the most abundant
0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.10.032
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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[13,14]. Previous study has shown that Rg1 can reduce side effects while maintain the efficacy of glucocorticoid in inflammatory diseases [15]. Rg1 has also been reported to facilitate neural differentiation of mouse embryonic stem cells [16]. However, effects and mechanism of Rg1 on platelet activation has not been investigated. Thrombin is one of the most potent platelet activator and plays a central role in both haemostasis and thrombosis [17,18]. Upon vessel wall injury, after the initial formation of platelet monolayer, thrombin serves as an essential mediator for the recruitment of additional platelets from the circulation to support the extension of thrombus and the resultant vessel occlusion. The effects of thrombin are mediated through G protein-coupled proteinase-activated receptors (PARs) [19–22]. PAR1 and PAR4 are present on human platelets. Both PAR1 and PAR4 are coupled to Gq to activate phospholipase C β (PLCβ), which can induce the activation of protein kinase C (PKC) and subsequent mitogenactivated protein kinase (MAPK) cascade [23]. PAR1 and PAR4 can also be coupled to G12/13 to activate Rho/Rho kinase [24]. Furthermore, these two can activate the Gi pathway through released ADP binding to P2Y12 receptor [25–27]. Thrombin per se and the key molecules of its signaling pathways have always been important targets in antithrombotic practice [21,28]. In the present study, we investigated the effect of Rg1 on platelet activation in response to thrombin, as well as the potential influence of Rg1 on the cellular signaling pathways. We demonstrated that Rg1 significantly inhibited thrombin-induced platelet activation in vitro and arterial thrombosis in a mouse model in vivo. We have also shown that Rg1 may act via inhibiting platelet PKC-MAPK pathway. Materials and methods Study subjects The study protocol was approved by the Ethic Committee of the Zhejiang University. The non-smoking healthy subjects (aged 18 to 28 years) were recruited from the staff and students at the Zhejiang University School of Medicine. Informed consent was obtained from all the volunteers. Animal experiments were carried out in compliance with the guidelines of the International Society on Thrombosis and Haemostasi [29], and were approved by the Board of Animal Study of Zhejiang University. Reagents Rg1 was purchased from the Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). SAA was purchased from the Division of Chinese Material Medica and Natural Products, National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, China). Thrombin, apyrase, indomethacin, HEPES and bovine serum albumin (BSA) were all purchased from Sigma (St Louis, MO, USA). The MEK inhibitor U0126 and PKC pan inhibitor Ro-31-8220 were from Cell Signaling Technology (Beverly, MA, USA). The fluorescence dye calcien was from Invitrogen (Eugene, Oregen, USA). For flow cytometric analysis, platelets were identified with the fluorescein isothiocyanate (FITC)-conjugated anti-CD42a monoclonal antibody (mAb) Beb 1 (Becton Dickinson; San Jose, CA, USA). The platelet activation markers, fibrinogen binding and P-selectin expression, were monitored with FITC-conjugated polyclonal anti-human fibrinogen antibody (DAKO, Glostrup, Denmark) and the R-phycoerythrin (RPE)conjugated anti-CD62 mAb AC1.2 (Becton Dickinson), respectively. For immunoblotting, p44/42 MAPK (ERK1/2) (137F5) rabbit mAb and phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (E10) mouse mAb, p38 MAPK antibody and phospho-p38 MAPK (Thr180/Thr182) (28B10) mouse mAb, Akt antibody and phospho-Akt (Ser473) (587 F11) mouse mAb, phosphor-(ser) PKC substrate antibody and β-actin (8H10D10) mouse mAb were all from Cell Signaling Technology (Beverly, MA, USA).
Human blood collection and preparation of washed platelets and red blood cells All blood doners had antecubital veins that allowed a clean venepuncture, and denied taking any medication during the 2 weeks preceding venepuncture. Blood was drawn without stasis into the siliconized vacutainers containing 1/9 (v/v) 3.8% sodium citrate. For preparation of washed platelets, blood was anticoagulanted with 75 mM sodium citrate, 39 mM citric acid, and 135 mM dextrose, PH 6.5 (ACD), and centrifuged at 150 ×g for 20 min; the platelet-rich plasma (PRP) was then collected. The PRP was diluted three-fold in ACD, and centrifuged for 10 min at 800 ×g. The platelet pellet was then resuspended in Tyrode’s buffer (137 mM NaCl, 12 mM NaHCO3, 2 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 5.5 mM glucose, 5 mM HEPES, and 0.35% BSA) at a concentration of 250 × 106 platelets mL-1. CaCl2 (2 mM) was added prior to the agonist stimulation. To prepare washed red blood cells, the remaining blood after PRP collection, consisting mainly of red blood cells, was washed with Tyrode’s buffer containing ACD and centrifugated three times at 800×g for 10min to remove plasma proteins. Flow cytometric analysis Flow cytometric analyses of platelet P-selectin expression and fibrinogen binding have been described previously [30]. Sample analysis was performed using a Beckman Coulter FC-500 flow cytometer (Beckman Coulter, Hialeah, FL, USA). Platelet P-selectin expression and fibrinogen binding were reported as the percentages of P-selectin-positive and fibrinogen bindingpositive cells, respectively, in the platelet population. Born aggregometry Agonist-induced platelet aggregation was measured using a ChronoLog aggregometer. Washed human platelets (0.25 mL; 250 × 106 mL-1) were preincubated with vehicle (HEPES buffer; 150 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM HEPES) or Rg1 (0.4, 2 and 4 mg mL-1) for 10 min at 37 °C without stirring. The agonists were then added with stirring at 900 rpm. Platelet adhesion on collagen-coated surface under flow conditions The experiments were performed using a previously described method [31], with minor modifications. Briefly, glass coverslips (24 × 50 mm; EUPOTUBO, Amadora, Portugal) were cleaned overnight with a dichromic acid solution, rinsed with distilled water, and dried before coating. Collagen was coated at a density of 0.08 μg cm-2 on coverslips overnight at 4 °C. The flow chamber was then assembled so that the coverslip formed the lower surface of the chamber. A peristaltic pump (Masterflex L/S; Cole-Parmer, Vernon Hills, IL, USA) was used to aspirate blood through the flow chamber. The fluorescence dye calcien (10 μg mL-1) was added to the washed platelets. After 20 min of incubation, calcein-labeled platelets were resuspended and mixed with washed red blood cells to form reconstituted blood with a platelet concentration of 250 × 106 mL-1 and a hemocrit of 45%. The reconstituted blood was incubated at 37 °C for 10 min in the absence or presence of Rg1 (4 mg mL-1) or U0126 (20 μM). Blood was perfused over the coverslip for 5 min under a shear rate correlated to the arterial flow (1000 s-1). The perfusion was live-monitored with a fluorescence microscope (Nikon TE-2000S; Nikon, Melville, NY, USA) equipped with a Nikon DS-2MBWc-U1 CCD camera (Nikon). After the termination of perfusion, the flow chamber was washed with phosphate-buffered saline (PBS) for 2 min to remove non-adherent cells. The images from at least 10 independent fields per flow experiment were recorded and analyzed by using IMAGE-PRO PLUS software (Media Cybernetics, Bethesda, MD, USA).
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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Fig. 1. Effects of Rg1 on platelet aggregation. (A, B) Washed human platelets (250 × 106 mL-1) were preincubated for 10 min with Rg1 (4 mg mL-1) or vehicle (HEPES buffer). Platelet aggregation was initiated with thrombin (0.05 U mL-1 or 0.1 U mL-1), ADP (10 μM or 20 μM), collagen (1 μg mL-1 or 2 μg mL-1) and U46619 (0.3 μM or 3 μM). (C, D) Aggregation of Rg1treated (4 mg mL-1) or vehicle-treated washed human platelets was induced by thrombin (0.1 U mL-1), and in the presence or absence of apyrase (1 U mL-1) or indomethacin (10 μM). (E) Washed human platelets were preincubated with Rg1 (4 mg mL-1), SAA (0.1 mg mL-1) or the combination of Rg1 and SAA for 10 min, and then stimulated with thrombin (0.1 U mL-1). Mean ± SEM of platelet aggregation percentage from four experiments are plotted in the bars charts; * P b 0.05, ** P b 0.01 as compared with control.
Platelet spreading on fibrinogen
Immunoblotting
Glass coverslips (24 × 50 mm; EUPOTUBO) were coated with 20 ug mL fibrinogen in 0.1 M NaHCO3 (pH 8.3) at 4 °C overnight. Washed platelets (200 × 105 mL-1) preincubated with (4 mg mL-1 Rg1 or 20 μM U0126; 10 min at 37 °C) or without inhibitors were allowed to spread on the fibrinogen-coated surfaces at 37 °C for 90 min. After three washes with PBS, the cells were fixed, permeabilized, and stained with fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR, USA). Adherent platelets were viewed with an inverted fluorescence microscope (Nikon TE-2000S) using a PLAN FLUOR lens (× 100/1.30 numerical aperture oil objective). Images were acquired using a Nikon DS-2MBWc-U1 CCD camera. The spreading area of individual platelet was assessed using IMAGE-PRO PLUS software (Media Cybernetics).
Aliquots of washed platelets (0.25 mL; 250 × 106 mL- 1) treated with or without Rg1 (4 mg mL- 1; 10-min preincubation) were stimulated with agonists at 37 °C. The reaction was stopped by the addition of sodium dodecylsulfate (SDS) sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue; pH 6.8). Samples were boiled for 5 min at 95 °C before storage at - 20 °C. Sample aliquots (40μL) were loaded, and the proteins were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to a poly (vinylidence difluoride) (PVDF) membrane and subjected to western blotting. ERK and phospho-ERK, p38 and phospho-p38, Akt and phospho-Akt, phospho-PKC substrate and β-actin were detected using corresponding antibodies (all from
-1
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Cell Signaling Technology, Beverly, MA, USA). After incubation with the corresponding secondary antibodies goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680 (LI-COR Biosciences, Lincoln, NE, USA), densitometric band scanning was performed using an Odyssey® infrared imaging system (LI-COR Biosciences).
To study the effect of Rg1 on thrombus formation, a bolus (10mgkg-1) of Rg1 or vehicle (HEPES buffer) was applied through the catheterized jugular vein 5 min prior to the vessel wall injury. Fluorescently labeled platelets were also preincubated with Rg1 (4 mg mL-1; 10 min) before injection.
Platelet-mediated clot retraction
Statistics
Platelet-rich plasma (PRP) was preincubated with inhibitors for 10 mins at 37 °C. Coagulation was induced with 0.2 U mL-1 thrombin, and clots were allowed to retract at 37 °C and photographed at various time points.
The data are presented as mean ± SEM (standard errors of the mean). Individual measurement of Rg1 effect was assessed with Wilcoxon’s signed rank test, and comparisons of vessel occlusion times were made using Mann-Whitney statistical tests. P b 0.05 was considered to be statistically significant.
Preparation and labeling of washed mouse platelets
Results
Mice, at 8-12 weeks of age, were bled from the inferior vena cava under chloraldurate anesthesia (400 mg kg-1 intraperitoneally). Mouse blood was collected in a 1: 10 volume of 3.2% sodium citrate containing 200 μg mL-1 hirudin. PRP was obtained by centrifugation at 800 ×g for 30 s, and then at 5 min at 150 ×g. The PRP was diluted in ACD and washed for 5 min. The platelet pellet was then resuspended in Ca2+free Tyrode’s buffer containing 10 μg mL-1 calcein, and incubated at 37 °C for 20 min. The labeled platelets were washed and resuspended at 500 × 106 mL -1.
Effects of Rg1 on thrombin induced platelet aggregation
10% FeCl3 induced thrombosis Wide-type mice (21.8 ± 0.4 g, n = 12) were anesthetized with chloraldurate, and the jugular vein was catheterized with an intramedic PE10 canule (Becton Dicknson). After laparotomy, the mouse intestines were exposed, and the mesentery was spread on the translucent table of the Nikon fluorescence microscope (TE-2000S) equipped with a DS2MBWc-U1 CCD camera (Nikon). An arteriole (60-80 μm in diameter) and its accompanying venule were selected under the microscope. Fluorescently labeled murine platelets (0.2 mL; 500 × 106 mL-1) were injected into the catheterized jugular vein. Injury of mesenteric arterioles was induced by topical application of 10% FeCl3 (Sigma, St Louis, MO, USA). A 2 × 5 mm strip of filter paper (Whatman) saturated with 10% FeCl3 solution was applied to the surface of mice mesenteric arterioles and venules for 2 minutes followed by rinsing with PBS. Vessels were monitored and occlusion time was defined as cessation of blood flow lasted longer than 1 minute due to an occlusive thrombus in the injured arteriole.
First, we examined the effect of Rg1 on different agonists-induced platelet aggregation. Rg1 inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, with the most prominent inhibition shown on thrombin-induced platelet aggregation (Fig. 1A,B). For example, thrombin (0.05 U mL-1) induced platelet aggregation rate was reduced by Rg1 (4 mg mL-1) from 87.5 ± 4.4% to 27.3 ± 5.6% (n = 4; P b 0.05), same concentration of Rg1 decreased platelet aggregation rate by thrombin (0.1 U mL-1) from 94.7 ± 3.9% to 57.0 ± 3.1% (n = 4; P b 0.01). Furthermore, apyrase and indomethacin were used to exclude the possibility that the effect of Rg1 on thrombin-induced platelet aggregation was due to the inhibition of ADP secretion or thromboxane A2 (TXA2) generation. Rg1 further inhibited thrombin-induced platelet aggregation in the presence of apyrase or indomethacin, suggesting an ADP- and TXA2-independent effect of Rg1 (Fig. 1C,D). Interestingly, a synergistic inhibitory effect was found when Rg1 was combined with SAA, a compound we previously identified to negatively regultate PI3K pathway [12]. Shown in Fig. 1E, combination of Rg1 and SAA nearly abolished thrombin (0.1 U mL-1) induced platelet aggregation, which was inhibited poorly by SAA alone. Influence of Rg1 on thrombin-induced single platelet activation The influence of Rg1 on single-platelet activation was further investigated using flow cytometry. Low (0.05 U mL-1) and high (0.1 U mL-1) concentration of thrombin increased platelet fibrinogen binding, a marker reflecting platelet aggregability, from 2.5 ± 0.5% at
Fig. 2. Effects of Rg1 on single platelet activation. Platelet-rich plasma (PRP) was preincubated with or without Rg1 (4 mg mL-1; 22 °C; 10 min) in the presence of fluorescent P-selectin and fibrinogen antibodies. Samples were then challenged with thrombin (0.05 U mL-1 or 0.1 U mL-1), and incubated for a further 10 min. Fibrinogen binding (A) and P-selectin expression (B) of single platelet were monitored by flow cytometry. Data plotted are mean ± SEM; n = 4; * P b 0.05, ** P b 0.01 without vs. with Rg1 treatment.
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
Q. Zhou et al. / Thrombosis Research xxx (2013) xxx–xxx
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baseline to 65.3 ± 12.4% and 83.7 ± 8.7% (Fig. 2A), respectively (n = 4; P b 0.05 and P b 0.01, respectively). The increases were reduced to 6.3 ± 5.2% and 2.2 ± 1.6% (Fig. 2A) in the presence of 4 mg mL-1 Rg1. Similar inhibitory effects of Rg1 were seen on thrombin-induced platelet P-selectin expression, which reflects platelet α-granule secretion (Fig. 2B). Influence of Rg1 on platelet intracellular signaling To investigate the intracellular signaling targets of Rg1, two important platelet signaling pathways were monitored: PI3K pathway and MAPK pathway (p38 phosphorylation or ERK phosphorylation). Here, washed human platelets were preincubated with or without Rg1 (4 mg mL-1, 37 °C, 10 min) before being challenged by thrombin. Western blotting showed that thrombin markedly enhanced phosphorylation of the enzymes (Fig. 3A). Rg1 had no influence on Akt or p38 phosphorylation, but markedly inhibited ERK phosphorylation induced by thrombin, indicating Rg1 action on thrombin-induced ERK pathway (Fig. 3A,B). To further confirm that Rg1 may act on ERK pathway, the MEK inhibitor U0126 was employed. Rg1 and U0126 showed nearly identical inhibitory effects on thrombin-induced ERK phosphorylation. Moreover, Rg1 and U0126 demonstrated no additive inhibition when combined (Fig. 3B). PKC was known to be important during thrombin-induced ERK phosphorylation [23,32]. Consistent with previous studies, our data confirmed that the PKC pan inhibitor Ro-31-8220 inhibited the activation of ERK (Fig. 3C). We also found that thrombin-induced PKC-substrate phosphorylation was partial and total inhibited by Rg1 and Ro-31-8220, respectively. The combination of Rg1 and Ro31-8220 did not show any additive inhibition (Fig. 3D). These results demonstrated that Rg1 affected PKC/ERK pathway in thrombinactivated platelets. Effects of Rg1 on platelet adhesion under flow Perfusion of reconstituted blood over collagen-coated surfaces under a shear rate correlated to the arterial flow (1000 s-1) led to significant platelet adhesion. The adhesion was inhibited by Rg1 and, to a slightly milder degree, by U0126 (Fig. 4A), as shown by reduced densities of adherent platelets (Fig. 4B). However, the combination of Rg1 and U0126 did not strengthen the inhibitory effect. Effects of Rg1 on platelet spreading on immobilized fibrinogen We further determined whether Rg1 could affect outside-in signaling, a process that is dependent on ERK (Fig. 5). In the absence of Rg1, the average surface coverage of spread platelets was 44.5 ± 8.7 μm2. Rg1 significantly inhibited platelet spreading and reduced the surface coverage to 21.0 ± 3.8 μm2, and Rg1 and U0126 showed similar reductions. The combination of Rg1 and U0126 did not show any additive inhibition. Fig. 3. Influences of Rg1 on platelet intracellular signaling. (A) Washed human platelets (250 × 106 mL-1) were preincubated with or without Rg1 (4 mg mL-1), and then stimulated with thrombin (0.1 U mL-1) with stirring at 900 rpm in an aggregometer at 37 °C. Platelets were lysed, and immunoblotted using the corresponding antibodies recognizing total or phosphorylated enzymes: p38 and Akt. (B) Platelets were preincubated with either Rg1 (4 mg mL-1), U0126 (20 μM) or the combination of the two, and then stimulated with thrombin (0.05 U mL-1 or 0.1 U mL-1). Phosphorylation of ERK was detected by western blotting. (C) Platelets were preincubated with either Rg1 (4 mg mL-1), Ro-31-8220 (10 μM) or the combination of the two, and then stimulated with thrombin (0.05 U mL-1 or 0.1 U mL-1). Phosphorylation of ERK was detected by Western blotting. (D) Platelets were preincubated with either Rg1 (4 mg mL-1), Ro-31-8220 (10 μM) or the combination of the two, and then stimulated with thrombin (0.05 U mL-1 or 0.1 U mL-1). Phosphorylation of PKC substrate was detected by Western blotting. Representative results were from at least three independent experiments with similar results.
Effect of Rg1 on clot retraction Consistently, retraction of the fibrin clot in PRP, which also requires functional αIIbβ3 alongside tight membrane-cytoskeleton interactions, was significantly delayed by Rg1. The maximal clot retraction occurred at 45 min after addition of thrombin (0.2 U mL-1). In contrast, platelets treated with Rg1 (4 mg mL-1) failed to form clot retraction at 45 min and only partial retraction was observed 60 min after thrombin stimulation (Fig. 6). Influence of Rg1 on arterial thrombosis The effect of Rg1 on thrombus formation in vivo was investigated using a 10% FeCl3 induced mesenteric arterial thrombosis model in
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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Fig. 4. Effects of Rg1 on platelet adhesion to collagen-coated surfaces. Calcein-labelled platelets (250 × 106 mL-1) were added into reconstituted blood (red blood cell resuspended with Tyrodes’ buffer containing 1% albumin; hematocrit 45%). The reconstituted blood was incubated at 37 °C for 10 min with either Rg1 (4 mg mL-1), U0126 (20 μM) or the combination of the two. The reconstituted blood was then perfused over collagen-coated (0.08 μg mm-2) coverslips for 5 min under the shear rate (1000 s-1), and this was followed by washing with PBS to remove non-adherent cells. Adherent platelets were counted under a fluorescence microscope. (A) Representative images from at least three independent experiments with similar results. Mean ± SEM of adherent platelets per field (%) are plotted in (B). * P b 0.05, ** P b 0.01 as compared with control.
Fig. 5. Effects of Rg1 on platelet spreading on fibrinogen-coated surface. Washed platelets (200 × 105 mL-1) were preincubated with either Rg1 (4 mg mL-1), U0126 (20 μM) or the combination of the two for 10 min at 37 °C. Platelets were allowed to spread on fibrinogen-coated slides for 90 min, then fixed with paraformaldehyde to stop spreading. Platelets were subsequently labeled with fluorescein isothiocyanate-conjugated phalloidin, and photographed under a fluorescence microscope. (A) Representative images from at least three independent experiments with similar results. Mean ± SEM of the average surface area of individual platelets are plotted in (B). * P b 0.05 as compared with control.
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
Q. Zhou et al. / Thrombosis Research xxx (2013) xxx–xxx
Fig. 6. Effects of Rg1 on clot retraction. Platelet-rich plasma (PRP) was preincubated with or without Rg1 (4 mg mL-1) for 10 min at 37 °C. Coagulation was induced with 0.2 U mL-1 thrombin, and clots were allowed to retract at 37 °C and photographed. Representative images were from at least three independent experiments with similar results.
wild type mice. Washed and calcein-labeled platelets were preincubated with or without Rg1 (4 mgmL-1; 10 min) and then injected into the mice through catheterized jugular vein immediately before vessel injury. Thrombus formation was monitored in real time until vessel occlusion under an intravital fluorescence microscope. The thrombus complete occlusion was often seen in the arterioles approximately 34.9 ± 4.1 min after vessel injury (Fig. 7A). In the mice treatment with Rg1 (10 mg kg-1 bolus), vessel injury-induced platelet adhesion and thrombus formation were markedly attenuated, with the arterial occlusion time usually being 64.3 ± 4.9 min after vessel injury (Fig. 7B).
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indicate that Rg1 is an important contributor of the antithrombotic properties of Panax ginseng. Rg1 may have the potential to be further developed into a new generation of antiplatelet and antithrombotic drug. It is worth of mentioning that a recent study has shown that Rp1, the derivative of ginsenoside Rg3, has the similar inhibitory effects in platelet activation as Rg1 [33]. Rg1 strongly inhibits thrombin-induced platelet activation. This inhibition still occurs in the presence of apyrase or indomethacin, which indicates that ADP secretion or TXA2 generation are less likely the main mechanisms influenced by Rg1. However, our data suggested that both inside-out and outside-in signaling of αIIbβ3 are compromised by Rg1 treatment, as demonstrated by the decreased fibrinogen binding, impaired platelet spreading on immobilized fibrinogen surface, and delayed clot retraction by Rg1. Further investigation of the molecular mechanisms revealed that Rg1 may have targeted PKC-MAPK pathway. Indeed, human platelets contain several members of MAPK family, including p38, JNK and ERKs (ERK1/2) [34,35], and previous studies using the p38, ERK, JNK inhibitors or JNK-deficient mice have demonstrated the prominent roles of MAPKs in platelet activation induced by thrombin [36–39]. However, our data ruled out the possibility that MAPK p38 was the target of Rg1, based on the fact that Rg1 failed to influence p38 phosphorylation level induced by thrombin stimulation. Several lines of evidences in this study support that Rg1 may target on ERKs. First, the phosphorylation of PKC substrates and MAPK ERK2 is significantly decreased by Rg1. Second, MEK inhibitor U0126 and Rg1 showed similar but nonadditive inhibitory effects on platelet adhesion. Furthermore, previous study using U0126 has demonstrated inhibition towards platelet aggregation, clot retraction, and platelet spreading on fibrinogen, all of these functions are inhibited by Rg1 in the present study [37]. Despite that PI3K pathway has been suggested to regulate MAPKs activation, Rg1 does not seem to inhibit PI3K as judged by the intact Akt phosphorylation level. Herb formulae have been considered the quintessence of Chinese traditional medicine. The identification of the active ingredients and elucidation of their cellular mechanisms are essential steps to understand this ancient medical practice. The findings, on the other hand, hold great promises to the discovery of novel therapeutic agents. It is of interest to note that components within a formula are usually complementary to each other in functions. Savia miltiorrhiza and Panax Notoginseng have a long history of being used in combination in the management of cardiovascular and thrombotic diseases. We have previously showed that SAA, a compound from Savia miltiorrhiza, inhibited platelet activation via PI3K, without affecting MAPK activation [12]. Conversely, the present study reported that Rg1, extracted from Panax Notoginseng, exerts the anti-platelet effects via inhibiting MAPK, but not PI3K. Intriguingly, such a synergistic inhibition of two signaling pathways has produced a better antiplatlet effect (Fig. 1E). In conclusion, Rg1 inhibits platelet activation via the inhibition of PKC and ERK pathway and attenuates arterial thrombus formation in vivo. It thus may be developed as a potential novel therapeutic agent. Our study also suggests that traditional Chinese herb medicine could serve as a good resource for the screening and development of novel therapeutic agents for thrombotic disorders.
Discussion Conflict of interest statement The present study has shown that Rg1 inhibits thrombin-induced platelet activation and decreases platelet adhesion on collagen-coated surface under flow conditions. The inhibitory effect of Rg1 is possibly achieved by interfering platelet PKC-MAPK pathway, as evidenced by decreased ERK and PKC substrate phosphrylation. The in vivo effect of Rg1 was further confirmed in a mice mesenteric arterial thrombosis model. Rg1 exists in abundance in the medicinal herb Panax ginseng C.A.Mey. The antiplatelet properties of Rg1 demonstrated in this study
The authors declare no competing interests.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81170478, 81370618), National Basic Research Program of China (2012CB966603).
Please cite this article as: Zhou Q, et al, Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis, Thromb Res (2013), http://dx.doi.org/ 10.1016/j.thromres.2013.10.032
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Fig. 7. Effects of Rg1 on arterial thrombus formation in vivo in wild-type mice. (A) Representative images and time course of thrombus formation induced by 10% FeCl3 injury of wide-type mouse intestinal arterioles, in the absence or presence of Rg1 (bolus 10 mg kg-1). Selected arterioles irrigating the cecum had a diameter of 60-100 μm. Arrows indicate irreversible arteriolar vessel occlusion. A indicates arteriole. (B) Dot plots of arteriolar occlusion time from five to ten experiments. Two-tailed P-values were calculated by two-column MannWhitney comparison (** P b 0.01).
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