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Salvianolic acid B inhibits platelets as a P2Y12 antagonist and PDE inhibitor: Evidence from clinic to laboratory
Thrombosis Research 134 (2014) 866–876
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Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
Salvianolic acid B inhibits platelets as a P2Y12 antagonist and PDE inhibitor: Evidence from clinic to laboratory Lei Liu a,1, Jian Li a, Yan Zhang b, Shenghui Zhang b, Jianqin Ye b, Zhichao Wen a, Jianping Ding c, Satya P. Kunapuli d, Xinping Luo a,⁎, Zhongren Ding b,⁎⁎ a
Department of Cardiology, Huashan Hospital, Fudan University Key Laboratory of Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai, China Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China d Department of Physiology, and Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, USA b c
a r t i c l e
i n f o
Article history: Received 4 April 2014 Received in revised form 5 June 2014 Accepted 13 July 2014 Available online 17 July 2014 Keywords: acute coronary syndrome antiplatelet P2Y12 receptor antagonist phosphodiesterase inhibitor salvianolate
a b s t r a c t Salviae miltiorrhiza (Danshen) has been used for thousands of years in China and some other Asian countries to treat atherothrombotic diseases. Salvianolate which consists of three water-soluble ingredients purified from Salviae miltiorrhiza, has been approved by Chinese SFDA to treat coronary artery disease. So far, there is no evidence clearly showing the clinical efficiency of salvianolate and the underlying mechanism. This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome and explore the underlying mechanism. We evaluated the effects of salvianolate on platelets in patients with acute coronary syndrome by measuring ADP-induced PAC-1 binding and P-selectin expression on platelets. Salvianolate significantly potentiated the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of salvianolatic acid B (Sal-B), the major component which composes 85% of salvianolate. Sal-B inhibits human platelet activation induced by multiple agonists in vitro by inhibiting phosphodiesterase (PDE) and antagonizing P2Y12 receptor. For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12 antagonism which may account for the clinical antiplatelet effects of salvianolate. Our results suggest that Sal-B may substitute salvianolate for clinical use. © 2014 Elsevier Ltd. All rights reserved.
Introduction Arterial thrombotic diseases, such as heart attack and stroke, are the leading cause of morbidity and mortality worldwide. Platelet activation triggered by atherosclerotic plaque disruption or endothelium injury caused by percutaneous coronary intervention (PCI) and the consequent intravascular arterial thrombogenesis is the common pathological basis
Abbreviations: Sal-B, salvianolatic acid B; VASP, vasodilator-stimulated phosphoprotein; AFM, atomic force microscopy; PDE, phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine; cAMP, 3,5-cyclic adenosine monophosphate; aspirin, acetylsalicylic acid; PRP, platelet-rich plasma; HPLC, high-performance liquid chromatography. ⁎ Correspondence to: X. Luo, Department of Cardiology, Huashan Hospital, Fudan University Shanghai Medical College, Shanghai 200040, China. Tel.: +86 21 52887165; fax: +86 21 64037268. ⁎⁎ Correspondence to: Z. Ding, Key Laboratory of Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai 200032, China. Tel.: +86 21 54237896; fax: +86 21 64033738. E-mail addresses: [email protected] (X. Luo), [email protected] (Z. Ding). 1 Present address: Department of Cardiology, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai 201508, China.
http://dx.doi.org/10.1016/j.thromres.2014.07.019 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
of heart attack and stroke; therefore, antiplatelet drugs are effective for prevention and treatment of coronary artery disease (CAD) and stroke. Currently, cyclooxygenase inhibitor aspirin, thienopyridine P2Y12 receptor antagonists clopidogrel and prasugrel, fibrinogen receptor antagonists, and phosphodiesterase (PDE) inhibitor cilostazol, are the mainly used antiplatelet drugs. Among these antiplatelet drugs for arterial thrombotic diseases, aspirin and P2Y12 receptor antagonists are most successfully and most widely used for coronary heart disease and stroke [1], while the PDE inhibitor cilostazol is used mainly in peripheral arterial occlusion and is under clinical trials in patients undergoing PCI in combination with clopidogrel and aspirin [2–5]. Though the current antiplatelet drugs are proven to be beneficial to patients with coronary heart disease, stroke and peripheral arterial disease, morbidity and mortality are still high, and novel antiplatelet agents with improved efficacy and safety are still needed. Traditional Chinese medicine (TCM) Salviae miltiorrhiza (Dangshen) has been used clinically for the treatment of cardiovascular diseases in China and some other Asian countries for thousands of years. Salvianolate, also known as depsides salts, the active components extracted from Salviae miltiorrhiza, was approved by Chinese SFDA in 2005 and widely used in clinical practice to treat CAD. However, there are few evidences
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clearly showing its clinical efficiency as most clinically used TCMs, and the underlying mechanism is also not clear. Salvianolate is the mixture of salvianolic acid B (Sal-B, ≥ 85%), salvianolic acid A (Sal-A, ≥ 1.9%) and rosmarinic acid (RA, ≥ 10.1%) [6]. Huang et al have found that Sal-A inhibits human platelet activation in vitro via PI3K inhibition [7]. As the most abundant ingredient of salvianolate, Sal-B has also been reported to inhibit rat platelet activation by targeting integrin α2β1 [8]. Given the pivotal roles of platelets in CAD and the proven benefits of antiplatelet drugs in treating CAD, we hypothesize that salvianolate may exert its clinical effects via platelet inhibition in CAD patients. In this study, we evaluated the effects of salvianolate on platelets in acute coronary syndrome (ACS) patients and found that it potentiates the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of Sal-B, the major component of salvianolate and demonstrated that Sal-B inhibited platelet activation induced by multiple agonists by inhibiting PDE and antagonizing P2Y12 receptor.
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and CD62P expression were subsequently measured using a FACS (FACSCalibur, Becton Dickinson). Results are expressed as percent of events designated positive for the marker of interest [10]. Preparation of Platelet Rich Plasma (PRP) and Washed Platelets from Normal Humans All experiments using human samples were performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board, Fudan University. The healthy volunteers (staff or students at Fudan University) without taking aspirin or other nonsteroidal anti-inflammatory drugs for at least two weeks were recruited after the informed consent was obtained. Blood (36 ml) was drawn into tubes containing 6 ml ACD (85 mM sodium citrate, 71.38 mM citric acid, and 27.78 mM glucose) solution. PRP and washed platelets were prepared as reported before [11–13].
Methods
Platelet Aggregation Assay
Study Design to Evaluate the Antiplatelet Effects of Salvianolate in ACS Patients
Aggregation of 0.5 ml human washed platelets in response to agonists or antagonists was analyzed using a lumi-aggregometer (Model 400VS; Chrono-Log, Havertown, PA, USA) under stirring condition (900 rpm) at 37 °C as reported before [11–14]. Platelet aggregation was initiated by addition of agonists with or without preincubating platelets with Sal-B (HPLC N 99%, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China) for 3 min. Each sample was allowed to aggregate for at least 3 min. The baseline was set using Tyrode’s buffer as blank.
Sixty three consecutive patients (Supplemental Table 1) were included after obtaining informed consent in abidance with the Declaration of Helsinki. Patients between 18 - 75 years old with chest pain suggestive of new-onset ACS were eligible, according to the 2007 ACC/ AHA guideline [9]. All patients enrolled were admitted to the Department of Cardiology in Huashan Hospital, Fudan University. Exclusion criteria were previous diagnosis of ACS before and relapse, long-term use of antiplatelet or anticoagulant therapy, receiving or scheduled use of platelet GP IIb/IIIa antagonist, and history of coagulopathy. Patients were randomly divided into two groups (Supplemental Table 1): the salvianolate group was given salvianolate (Shanghai Green Valley Pharmaceutical Co., Ltd, Shanghai, China) 200 mg/day intravenously in addition to standard treatment (according to the practice guideline of ACC/AHA) [9], and the control group was given standard treatment only (Fig. 1A). Salvianolate drip ended after one week. Current guidelines were followed for patient management and therapy, including percutaneous coronary interventions and thrombolysis. Drug therapy includes aspirin (Bay-aspirin) and clopidogrel (Plavix) (the loading dose of 300 mg, followed by a regimen therapy of 100 mg/d and 75 mg/d, respectively), statins (Lipitor 20 mg/d), LMWH (4000 IU twice daily, injected subcutaneously for 3 days) (Fig. 1A). To evaluate platelet activation status, PAC-1 binding and CD62P expression on platelet surface were determined by flow cytometry (FACS) using blood collected before and after antiplatelet treatment 7 days later, according to the protocol shown in Fig. 1A. PAC-1 Binding and CD62P Expression Assay in Whole Blood from ACS Patients Whole blood FACS analysis of PAC-1 binding and CD62P expression on platelet surface was taken before and 7 days after antiplatelet treatment. PAC-1 binding and CD62P expression were determined on resting and ADP-activated platelets from ACS patients, using monoclonal antibodies PAC-1 and anti-CD62P for activated αIIbβ3 and P-selectin, respectively, as reported before [10]. Blood was collected from an antecubital vein into Venous Blood Collection tubes containing 0.5 mL buffered sodium citrate (equiv. To 3.2% sodium citrate, Franklin Lakes, NJ, USA). The first 2 mL of blood were discarded. Isotonic HEPES Tyrode buffer was used to dilute the blood. Resting and platelets activated by ADP (10 μM) for 3 min were incubated with FITC-conjugated PAC-1 (Santa Cruz, San Diego, CA, USA) and PE-conjugated anti-CD62P (AbD Serotec, Oxford, UK) antibodies in the dark at room temperature for 15 min without stirring. PAC-1 binding
Assay of PAC-1 Binding on Platelets from Normal Humans Whole blood from normal humans was incubated with Sal-B at different concentrations. PAC-1 binding on the surface of resting or ADP activated platelets was determined by FACS as described above. [Ca2+]i Measurements Human PRP was prepared as described above. 1 mg Fluo-3/AM (Biotium, Hayward, CA, USA) was dissolved in DMSO with 20% F-127 as 1 mM stock solution [15]. Platelets pelleted from PRP were resuspended in Ca2+-free Tyrode’s solution, and then incubated with 2 μM Fluo-3/AM for 30 min at 37 °C. After washing twice, the fluo-3-loaded platelets were finally suspended in Tyrode’s buffer containing 0.5 mM EGTA, at a density of 3 × 108 platelets/ml. The fluo-3-loaded platelets were preincubated with Sal-B at 37 °C for 3 min before the addition of the platelet agonists. Fluorescence (excitation 505 nm, emission 530 nm) was measured with a fluorescence spectrophotometer (Model F4500; Hitachi, Tokyo, Japan) [15]. Western Blotting Analysis of VASP (Vasodilator-Stimulated Phosphoprotein) Phosphorylation in Normal Human Platelets Washed platelets was prepared as described above and treated or untreated with 5 μM prostaglandin E1 (PGE1) at 37 °C for 30 min [16], followed by incubation with Sal-B (70, 140, 280 μM) or AR-C69931MX (100 nM, a gift from AstraZeneca, Loughborough, UK) under stirring condition (900 rpm) for 3 min. After stimulation with 10 μM ADP for 3 min the reaction was stopped by addition of 5 x sample buffer and boiled for 5 min. Platelet lysates were further subjected to western blot analysis of VASP phosphorylation using anti-pSer157-VASP (Cell signalling, Beverly, MA, USA)[17,18]. The optical density of the bands was measured using Image J (National Institutes of Health, Bethesda, MD).
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Fig. 1. Study protocol for (A) blood sampling and (B) FACS assay of PAC-1 binding and CD62P expression before and after antiplatelet therapy in ACS patients. Panel B is a typical dot figure showing antiplatelet treatment significantly inhibits CD62P expression and PAC-1 binding on both resting and ADP-activated platelets from patients, while salvianolate group having more significant inhibition on CD62P expression and PAC-1 binding on ADP-activated platelets in salvianolate group (anti-CD62P: 46.2/36.2; PAC-1: 53.2/39.3) compared with control group.
Measurement of cAMP in Platelets Intracellular cAMP of stimulated platelets was measured using chromatography as described before [11,14]. After incubation with Sal-B or AR-C69931MX for 30 s, ADP 10 μM was added to activate platelets for 2.5 min. Levels of cAMP were determined and cAMP conversion from ATP was calculated using the following formula: cAMP conversion from ATP = [3H]cAMP/([3H]ATP + [3H]cAMP) x 103. We also assayed cAMP in resting platelets using commercially available cAMP 125I radioimmunoassay kits (Isotype Laboratory of Shanghai University of Traditional Chinese Medicine, Shanghai, China) as reported previously [11,19]. Assay of the Activity of Phosphodiesterase (PDE) Extracted from Human Platelets Human platelet PDE extracts were prepared as previously reported [11,12]. The assay for cAMP-PDE activity was performed using the
method previously reported [12,20]. The inhibition of PDE activity was calculated using the following formula: % inhibition of PDE activity = (1 - converted cAMP in enzyme reaction system treated by different agents/converted cAMP in untreated enzyme reaction system) x 100%.
Interaction Between ADP and P2Y12 Receptor with Atomic Force Microscopy (AFM) Effects of Sal-B on the interaction between ADP and P2Y12 receptors stably expressed in Chinese hamster ovary (CHO-K1) cells were measured using a MFP-3D-BIO atomic force microscope (Asylum Research, Santa Barbara, CA, USA), integrated with an IX71 inverted microscope (Olympus, Tokyo, Japan) as previously reported [11,12]. P2Y12 receptor antagonist AR-C69931MX (100 nM) was used as positive control while P2Y1 receptor antagonist MRS2179 (100 μM, Tocris Bioscience, Ellisville, MO, USA) was used as negative control.
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Platelet Spreading Experiment
Sal-B Inhibits Platelet Aggregation and GPIIb/IIIa Activation Induced by ADP
Glass coverslips were coated with 10 μg/mL fibrinogen in 0.1 M NaHCO3 (pH 8.3) at 4 °C overnight. Human washed platelets (2 × 107/ mL) preincubated with or without inhibitors were allowed to adhere and spread on the fibrinogen-coated glass coverslips at 37 °C for 90 min. After washing with PBS, attached platelets were fixed with freshly prepared 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 min, stained with fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR, USA), and viewed as previously described [21,22].
Sal-B is the major component of salvianolate, composes 85% of salvianolate. After showing the antiplatelet effects of salvianolate in ACS patients, we sought to investigate the role of Sal-B on human platelet activation. A plasma concentration ranging from 8 to 280 μM has been reported in rat [23], therefore the antiplatelet role of Sal-B was tested in this concentration range. In the range of 8 to 280 μM, Sal-B concentration-dependently inhibited platelet aggregation induced by 10 μM ADP in aspirintreated human washed platelets with IC50 of 55.5 μM (Fig. 2B and C). Interestingly, we have shown that Sal-B and salvianolate equivalently inhibit platelet aggregation induced by ADP (Supplemental Fig. 1). The inhibitory effect of Sal-B on ADP-induced platelet activation was further confirmed in whole blood from normal humans by FACS analysis of PAC-1 binding (Fig. 2D). At higher concentrations (140 and 280 μM), the antiplatelet effects of Sal-B are even more profound than 100 nM AR-C69931MX, a P2Y12 receptor antagonist under clinical trials as antiplatelet drugs.
Statistics For ex vivo measurements, baseline platelet glycoprotein data were analyzed for differences by analysis of variance (ANOVA) with the use of treatment and sex as factors. Data were expressed as the mean ± SD for continuous variables and as frequencies and percentages for categorical variables. Wilcoxon test was used to compare continuous variables, and a Chi square test or Fisher’s exact test was used to compare the categorical variables. For in vitro measurements, data were expressed as mean ± SEM. Differences between the groups were analyzed by one-way ANOVA followed by a Newman-Keuls test using GraphPad Prism version 5.0 unless otherwise stated. P b 0.05 was considered to be statistically significant. Results Patient Characteristics Sixty seven patients with chest pain between November 1, 2010 and April 31, 2011 were consecutively enrolled, among which 4 patients died of non-cardiac reasons were excluded. A total of 63 subjects fulfilled the study (Supplemental Table 1), the average age in salvianolate group (n = 32) and control group (n = 31) is 66 ± 9 and 66 ± 11 years old, respectively. There were no differences in demographic characteristics and baseline values between 2 groups (Supplemental Table 1). Salvianolate Potentiates the Antiplatelet Effects of Aspirin Plus Clopidogrel in ACS Patients There were no significant differences in PAC-1 binding and CD62P expression on the surface of resting and ADP-activated platelets between the 2 groups before antiplatelet treatment (Fig. 1B and Table 1). Antiplatelet treatment significantly inhibits both PAC-1 binding and CD62P expression on the surface of resting and ADP-activated platelets in both groups (Fig. 1B and Table 1). Compared with control group, addition of salvianolate attenuated PAC-1 binding on ADP-activated platelets from ACS patients more significantly (47.0 ± 10.0 % vs 52.1 ± 6.2 %, P b 0.05). CD62P expression on ADP-activated platelets was also attenuated more significantly in salvianolate group compared with control group (39.5 ± 8.3 % vs 45.0 ± 6.7%, P b 0.01) (Table 1). These results indicate that addition of salvianolate to the standard dual antiplatelet therapy further enhances the antiplatelet effects in ACS patients.
Sal-B Inhibits Platelet Aggregation Induced by Thrombin, Arachidonic Acid, Collagen and U46619 Platelet activation involves complex signal transduction cascades mediated by multiple agonists including ADP, thrombin, collagen, and thromboxane A2. ADP receptor P2Y12 contributes to platelet activation induced by other platelet agonists [24]. Hence, after showing excellent antiplatelet efficacy of Sal-B on human platelets activated by ADP, we further explored the antiplatelet effects of Sal-B on platelet activation induced by thrombin, arachidonic acid, collagen, and U46619. As shown in Fig. 2E, in the range of 8 - 280 μM, Sal-B also abolished or drastically inhibited human platelet aggregation induced by thrombin, arachidonic acid, collagen, and U46619. Sal-B Inhibits Platelet Spreading Platelet aggregation is the result of fibrinogen binding to the activated integrin GPIIb/IIIa mediated by inside-out signal pathways. Upon GPIIb/IIIa binding to fibrinogen, it triggers outside-in signaling, causing platelet spreading and clot retraction which play a crucial role in thrombosis. We have shown that Sal-B inhibits platelet aggregation induced by multiple agonists and integrin GPIIb/IIIa activation induced by ADP, the typical inside-out signaling events. We next explored whether SalB also influence platelet outside-in signaling. As shown in Fig. 3, Sal-B 280 μM dramatically inhibited platelet spreading on immobilized fibrinogen, similarly to LY294002 (Calbiochem, San Diego, CA, USA), a PI3K inhibitor, which has been reported to inhibit platelet spreading [7,22]. Sal-B did not inhibit platelet spreading at 140 μM, a concentration that dramatically inhibited platelet aggregation and PAC-1 binding (Fig. 3). Sal-B Increases cAMP Level in Resting and ADP-Stimulated Platelets Sal-B inhibits platelet aggregation induced by multiple agonists including ADP, thrombin, collagen, and U46619 with more potent
Table 1 Antiplatelet treatment inhibits platelet function in ACS patients as evaluated by FACS analysis of PAC-1 binding and CD62P expression on platelet surface. PAC-1 positive (%) before treatment control group (n = 31) salvianolate group (n = 32)
resting ADP-activated resting ADP-activated
9.7 69.9 10.2 71.8
± ± ± ±
3.9 8.0 6.4 12.7
anti-CD62P positive (%) after treatment 5.5 52.1 6.2 47.0
± ± ± ±
#
1.4 6.2## 2.8# 10.0#⁎
before treatment
after treatment
7.8 60.0 7.0 59.0
4.7 45.0 4.5 39.5
± ± ± ±
2.8 10.5 3.5 13.5
± ± ± ±
1.7# 6.7# 2.3# 8.3#⁎⁎
Antiplatelet treatment for control group is standard of care, salvianolate treatment group received standard of care plus salvianolate. #P b 0.05, ##P b 0.01 compared with before treatment; ⁎P b 0.05 (47.0 ± 10.0 in salvianolate group compared with 52.1 ± 6.2 in control group), ⁎⁎P b 0.01 (39.5 ± 8.3 in salvianolate group compared with 45.0 ± 6.7 in control group).
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Fig. 2. Sal-B concentration-dependently inhibits human platelet aggregation induced by ADP, thrombin, arachidonic acid (AA), collagen and U46619. A, Structure of Sal-B and Sal-A. B, Sal-B concentration-dependently inhibits ADP (10 μM) induced platelet aggregation in aspirin-treated human washed platelets. C, Concentration-response curve of Sal-B inhibition on platelet aggregation induced by ADP 10 μM in aspirin-treated washed human platelets. Platelets were preincubated with saline, Sal-B (10 μM) or AR-C69931 MX (100 nM) followed by stimulation with ADP (10 μM). Tracings shown are representative of at least three experiments using platelets from different donors. 0.9% saline was used as a vehicle control. D, Sal-B concentrationdependently inhibits PAC-1 binding on platelets in human blood measured by FACS analysis. Data were expressed as mean ± SEM representing three separate experiments measured in duplicate. E, Sal-B was preincubated with human washed platelets for 3 min before stimulation with thrombin (0.05 U/ml), AA (0.5 mM), collagen (2 μg/ml) and U46619 (1 μM). Tracings shown are representative of at least 3 experiments using platelets from different donors. 0.9% saline was used as vehicle control.
inhibition on ADP-induced aggregation (Fig. 2), suggesting that Sal-B may target ADP receptors to exert its antiplatelet effects. Sal-B inhibits ADP-induced platelet aggregation without influence on platelet shape change (Fig. 2B), indicating that Sal-B may target P2Y12 receptor rather than P2Y1 [25]. To elucidate the antiplatelet mechanism of Sal-B, we first measured the effects of Sal-B on cAMP level in platelets stimulated with ADP to explore its possible P2Y12 antagonizing role. As shown in Fig. 4A, in the antiplatelet concentration range (35 – 280 μM), Sal-B concentration-dependently inhibited ADP-induced decrease in cAMP levels, consistent with its putative P2Y12 antagonizing role. At 280 μM, Sal-B exhibited similar effects as AR-C69931MX, a P2Y12 receptor
antagonist. Higher concentrations of Sal-B (140 and 280 μM) also significantly elevated intracellular cAMP levels of resting platelets as forskolin did (Fig. 4B), indicating that Sal-B may also exert its antiplatelet effects via mechanisms besides P2Y12 antagonism. Sal-B Enhances VASP Phosphorylation in Platelets Stimulated with ADP VASP is the major substrate of PKA (cAMP-dependent protein kinase), which prefers to phosphorylate Ser157 of the 3 phosphorylation sites on VASP [17]. Similar to cAMP decrease, P2Y12- dependent inhibition by ADP of phosphorylation of VASP is another specific assay to
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Fig. 3. Sal-B inhibits platelet spreading on fibrinogen-coated surface. Human washed platelets were seeded on fibrinogen-coated cover slips in the presence of Sal-B 280 μM, PI3-kinase inhibitor LY294002 (100 nM) or vehicle control. Platelets were stained with FITC-phalloidin and observed under a 40 x objective with magnification of 5 (upper panel). Further amplified single platelets in the squares from upper panel are shown in bottom panel. Results shown are representative of 3 experiments using platelets from different donors run in duplicate.
reflect P2Y12 receptor activation status [26,27] and is widely used to measure the effects of antiplatelet drugs targeting P2Y12 receptor [28]. Similar to AR-C69931, at 140 and 280 μM Sal-B concentrationdependently inhibited ADP-induced reduction of VASP phosphorylation on Ser157 (Fig. 4C), in line with its ability to inhibit platelet aggregation and cAMP decrease induced by ADP. Taken together, these results suggest that P2Y12 receptor antagonism is involved in the antiplatelet roles of Sal-B.
Even at 280 μM, Sal-B only slightly inhibited ADP-evoked Ca2+ rise. In contrast, P2Y1 receptor antagonist 100 μM MRS2179 totally abolished Ca2+ rise. P2Y12 receptor was reported to sustain the increased Ca2+ level [29], in agreement with this, Sal-B accelerated Ca2 + decay (Fig. 6B and C), consistent with its P2Y12 antagonizing role. These results indicate that Sal-B antagonize ADP receptor P2Y12 rather than P2Y1.
Sal-B Inhibits the Interaction Between ADP and P2Y12 Receptor Measured by AFM
The ability of Sal-B to enhance cAMP in resting platelets implicates another mechanism in addition to P2Y12 antagonism that might contribute to its antiplatelet effects. We extracted PDE from human platelets and investigated the effects of Sal-B on PDE activity by measuring residual cAMP in a reaction mixture containing Sal-B, PDE extracts and PDE substrate cAMP using HPLC. As shown in Fig. 7, at concentrations ranging from 70 to 280 μM, Sal-B concentration-dependently inhibited the activity of platelet PDE (Fig. 7), in line with its cAMP-enhancing effect in resting platelets (Fig. 4B). Compared with IBMX, a non-specific PDE inhibitor, the PDE inhibition activity is weak even at a high concentration of 280 μM. Furthermore, in the range of 70 - 280 μM, Sal-B increased VASP phosphorylation in human platelets (Fig. 7C), further confirming that Sal-B is a PDE inhibitor.
Sal-B reverses ADP-induced decrease of intracellular cAMP and VASP phosphorylation in platelets (Fig. 4A and C) strongly suggests that Sal-B may work as a P2Y12 antagonist to exert its antiplatelet roles. To provide direct evidence that Sal-B blocks ADP binding to P2Y12 receptor, the interaction between ADP and single P2Y12-expressing CHO-K cell was assayed in the presence of Sal-B by direct force measurement using AFM. As shown in Fig. 5, similar to P2Y12 receptor antagonist ARC69931MX, 280 μM Sal-B remarkably suppressed the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells. In contrast, MRS2179, a P2Y1 antagonist of ADP receptors, did not affect the interaction between ADP and P2Y12 receptor. Sal-B inhibits the interaction between ADP and P2Y12 receptor confirmed its P2Y12 antagonist activity as an antiplatelet drug. Effects of Sal-B on Platelet Intracellular Ca2+ Mobilization We investigated whether Sal-B affects P2Y1-Gq pathway by assaying ADP-induced Ca2+ mobilization. As shown in Fig. 6, in the absence of extracellular Ca2 +, 10 μM ADP elicited rapid Ca2 + rise, which then returned to baseline as a result of Ca2 + re-uptake or extrusion. After preincubating with Sal-B 140 μM, ADP elicited similar rapid Ca2+ rise.
Sal-B Inhibits the Activity of PDE from Human Platelets
Discussion In this study, we first investigated the antiplatelet effects of salvianolate in ACS patients. We found that addition of salvianolate to the standard antiplatelet therapy could further enhance platelet inhibition in ACS patients. To our knowledge, this is the first report to show that salvianolate potentiates the antiplatelet effects of the standard antiplatelet therapy in ACS patients ex vivo. We further investigated the antiplatelet effects and the underlying mechanism of Sal-B, the major component of salvianolate. We found that Sal-B inhibited platelet
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Fig. 4. Effects of Sal-B on platelet cAMP and VASP phosphorylation in washed human platelets. A & B, Sal-B concentration-dependently increases intracellular cAMP in ADP-stimulated (A) and resting (B) human platelets. Data are expressed as mean ± SEM representing three separate experiments measured in duplicate. C, Sal-B concentration-dependently increases VASP phosphorylation in washed human platelets stimulated with ADP. After preincubation with 5 μM prostaglandin E1, platelets were incubated with vehicle, AR-C69931MX (ARC) or Sal-B and further stimulated for 5 min with 10 μM ADP. Equal amounts of proteins were separated by SDS-PAGE, western blotted, and probed for anti-phospho-VASP or antiGAPDH antibody. The results shown are representative of 3 experiments using platelets from different donors.
activation induced by multiple agonists, antagonized ADP binding to P2Y12 receptor, and inhibited platelet PDE activity at the antiplatelet concentrations. Clinical trials have demonstrated that antiplatelet drugs including aspirin, clopidogrel and GP IIb/IIIa inhibitors provide substantial therapeutic benefits in patients with ACS [30,31]. After platelet stimulation, GP IIb/IIIa serves as an anchoring site for soluble fibrinogen and von Willebrand factor (vWF), and P-selectin released from platelet granules to the surface membrane permits platelet binding to white blood cells and endothelium [32]. Therefore, ACS patients may benefit from salvianolate given in combination with the standard antiplatelet therapy, which warrants further study.
As the major component of salvianolate, Sal-B constitutes 85% of salvianolate. Previously Sal-B has been reported to inhibit platelet adhesion to collagen [8,33] and platelet aggregation induced by collagen and ADP in rat platelets [8,34]. Consistent with these results, in the present study, we demonstrated that Sal-B effectively inhibited platelet activation elicited by multiple agonists including collagen, ADP, thrombin, arachidonic acid, and U46619 in human platelets. Importantly, we demonstrated that salvianolate and Sal-B exhibited the almost equivalent inhibitory effects on platelet aggregation induced by ADP (Supplemental Fig. 2), indicating that salvianolate inhibits platelet aggregation mainly through Sal-B. We also found that Sal-B inhibited platelet spreading on immobilized fibrinogen and ADP-induced PAC-1 binding
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Fig. 5. Sal-B inhibits interaction between ADP and P2Y12 receptor. A, Typical atomic force microscopy force-displacement measurements of the interaction between ADP and P2Y12 receptors expressed in CHO-K1 cells in the presence of different ADP receptor antagonists, Sal-B or vehicle control. The measurements were required with 100 pN indentation force, 0.2 s contact time, and a cantilever retraction speed of 3 μm/s. B, Histograms of individual unbinding forces of ADP and P2Y12 in the presence of different ADP receptor antagonists, Sal-B or vehicle control from force displacement measurements. The y-axis plots the number of force transitions detected and x-axis is the unbinding force (pN). C, Statistical analysis of unbinding forces of ADP and P2Y12 in the presence of different ADP receptor antagonists, Sal-B (filled bars) or vehicle control (blank bars). AR-C69931MX (100 nM), MRS2179 (100 μM) and Sal-B (280 μM) were used. Data are expressed as mean ± SEM of 100 measurements in panel A.
in human whole blood, which further confirmed the antiplatelet role of Sal-B on human platelets. The effective concentrations of Sal-B range from 8 to 280 μM, which is in line with antiplatelet concentrations previously reported in rats [8,34] and is within the blood drug concentration range of in rats [35]. Platelet activation is a complex process involving multiple agonists and signaling pathways. Among the numerous signaling molecules regulating platelet activation, haemostasis and thrombosis, P2Y12 receptor plays a central role [24] and has been one of the most successful antiplatelet targets. cAMP is also an important signaling molecule in platelet activation, and increased platelet cAMP level negatively regulates platelet function. PDE inhibitors, which increase cAMP by inhibiting cAMP degrading, inhibit platelet activation elicited by all agonists. In this
study, we found that Sal-B inhibits platelet activation induced by a broad range of agonists, consistent with the central role of P2Y12 receptor in platelet activation [24] and the broad spectrum antiplatelet activity of PDE inhibitors. Our results confirm and further extend the previous finding that Sal-B inhibits rat platelet aggregation induced by ADP [34]. Concomitant activation of Gq-coupled P2Y1 and Gi-coupled P2Y12 is essential for ADP induced platelet aggregation [25]. P2Y1 induces Ca2+ mobilization, platelet shape change, and reversible platelet aggregation, while P2Y12 inhibits adenylyl cyclase, decreases cAMP, amplifies P2Y1induced platelet aggregation, leading to irreversible aggregation. P2Y12 also amplifies platelet activation induced by other agonists, plays a central role in platelet activation [24], and is one of the most successful
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Fig. 6. Effects of Sal-B on ADP-induced Ca2+ mobilization in platelets. Sal-B accelerates Ca2+ decay at 140 and 280 μM and only slightly inhibits Ca2+ rise in human platelets stimulated with ADP 10 μM. Intracellular Ca2+ mobilization was assayed as the Ca2+ fluorescence in Fluo-3-loaded human platelets in the absence of extracellular Ca2+. Results shown are representative of three experiments using platelets from different donors.
antiplatelet drug targets. In this study we found that Sal-B inhibits platelet activation via P2Y12 antagonism as evidenced by: 1) it concentrationdependently inhibited ADP-induced cAMP decrease in platelets; 2) it concentration-dependently inhibited ADP-induced VASP phosphorylation decrease in platelets; 3) it did not affect Ca2+ mobilization amplitude, but accelerated the decay of the elevated Ca2 + in platelets stimulated with ADP, consistent with the previous report that P2Y12 sustains the increased Ca2+ level [29] while P2Y1 elicits Ca2+ mobilization; 4) it inhibited the interaction between ADP and P2Y12. The fact that Sal-B does not influence ADP-induced platelet shape change and Ca2+ mobilization amplitude rules out P2Y1 as its antiplatelet target. Sal-B increased cAMP in resting platelets cannot be explained by P2Y12 antagonism, suggesting the involvement of an alternative mechanism underlying the antiplatelet effects of Sal-B, which could be adenylyl cyclase activation or PDE inhibition. Direct assay of the activity of PDE extracted from human platelets confirmed that Sal-B inhibits the activity of PDE concentration-dependently in its antiplatelet concentration range (Fig. 7). Sal-B inhibiting platelets as a PDE inhibitor is in agreement with the previous report that Injectio Salvia Miltiorrhizae inhibits human platelet activation by increasing platelet cAMP [36]. However, even at 280 μM, the concentration Sal-B remarkably inhibited platelet activation, PDE is only partly inhibited (42% inhibition), indicating that PDE inhibition only partly contributes to the antiplatelet effects of Sal-B with the rest effects attributed to P2Y12 antagonism. Taken together, our results indicate that Sal-B exerts its antiplatelet effects via P2Y12 antagonism and PDE inhibition, similarly to the BF compounds
previously reported in our lab [11,12]. Interestingly, the two BF compounds share similar structure, while Sal-B is structurely distinctive. Sal-B has been reported to target α2β1 [8,33]. We found that Sal-B did not affect anti-α2β1 antibody binding to integrin α2β1 of human platelets at 280 μM, the concentration that dramatically inhibits platelet activation (Supplemental Fig. 2). Therefore, we think that α2β1antagonism does not contribute the antiplatelet and antithrombotic effects of Sal-B. Multiple signaling pathways contribute to platelet activation and thrombosis, therefore combined antiplatelet therapy targeting different pathways is recommended. Currently dual antiplatelet therapy with aspirin plus thienopyridine P2Y12 receptor antagonists is widely used, while triple antiplatelet therapy (dual antiplatelet therapy plus PDE inhibitor cilostazol) is under intensive evaluation with improved efficacy and safety reported recently [2,37,38]. Consistently, in this study we demonstrated that addition of salvianolate to the standard dual antiplatelet therapy (aspirin plus clopidogrel) enhanced platelet inhibition without affecting hemostasis in ACS patients, which can be attributed to the dual antiplatelet activity (targeting P2Y12 and PDE) of Sal-B. Platelet outside-in signaling pathway is mainly involved in thrombosis rather than hemostasis of platelet function. Targeting platelet outside-in signaling pathway represents a more attractive approach to develop effective and safe antiplatelet agents [21]. In this study, we found that Sal-B dramatically inhibited platelet spreading on immobilized fibrinogen at high concentration (280 μM) (Fig. 3). However, this inhibitory effect was not observed at the lower concentrations profoundly inhibiting platelet aggregation. In concert with our findings,
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Fig. 7. Sal-B inhibits cAMP-phosphodiesterase activity in human platelet extracts and increases VASP phosphorylation in washed human platelets. A, Sal-B concentration-dependently inhibits the activity of PDE extracted from human platelets. Data are expressed as mean ± SEM representing three separate experiments. B, Representative HPLC tracings of residual cAMP in the presence of PDE and various inhibitors. C, Sal-B increases VASP phosphorylation in washed human platelets. Platelets were incubated with vehicle, Sal-B or IBMX for 5 min. Equal amounts of proteins were separated by SDS-PAGE, western blotted, and probed for anti-phospho-VASP or anti-GAPDH antibody. All concentrations of Sal-B increase VASP phosphorylation compared with the vehicle control using t-test. The results shown are representative of 4 experiments using platelets from different donors.
salvianolate acid A, another antiplatelet component in salvianolate, also inhibits platelet spreading at high concentration (202 μM) [7]. We have shown that salvianolate potentiated platelet inhibition in ACS patients receiving dual antiplatelet therapy without affecting hemostasis in the present study. Whether the inhibition of Sal-B on platelet spreading contributes to the antiplatelet and antithrombotic effects of salvianolate without affecting hemostasis remains to be elucidated. In conclusion, for the first time, we show that salvianolate potentiates the antiplatelet effects of the standard antiplatelet therapy without affecting hemostasis in ACS patients. We also find that Sal-B, which constitutes 85% of salvianolate, inhibits platelet activation via P2Y12 antagonism and PDE inhibition, which may account for the clinical antiplatelet effects of salvianolate. Whether Sal-B can substitute salvianolate for clinical use deserves further investigation. Conflict of Interest Statement None of the authors have any conflict of interests. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2014.07.019.
Acknowledgements This work was partially supported by National Natural Science Foundation of China (No. 30973529, 81270278), Drug Innovative Program from Shanghai Municipal Science and Technology Commission (No. 11431920103), and Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning.
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Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
Salvianolic acid B inhibits platelets as a P2Y12 antagonist and PDE inhibitor: Evidence from clinic to laboratory Lei Liu a,1, Jian Li a, Yan Zhang b, Shenghui Zhang b, Jianqin Ye b, Zhichao Wen a, Jianping Ding c, Satya P. Kunapuli d, Xinping Luo a,⁎, Zhongren Ding b,⁎⁎ a
Department of Cardiology, Huashan Hospital, Fudan University Key Laboratory of Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai, China Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China d Department of Physiology, and Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, USA b c
a r t i c l e
i n f o
Article history: Received 4 April 2014 Received in revised form 5 June 2014 Accepted 13 July 2014 Available online 17 July 2014 Keywords: acute coronary syndrome antiplatelet P2Y12 receptor antagonist phosphodiesterase inhibitor salvianolate
a b s t r a c t Salviae miltiorrhiza (Danshen) has been used for thousands of years in China and some other Asian countries to treat atherothrombotic diseases. Salvianolate which consists of three water-soluble ingredients purified from Salviae miltiorrhiza, has been approved by Chinese SFDA to treat coronary artery disease. So far, there is no evidence clearly showing the clinical efficiency of salvianolate and the underlying mechanism. This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome and explore the underlying mechanism. We evaluated the effects of salvianolate on platelets in patients with acute coronary syndrome by measuring ADP-induced PAC-1 binding and P-selectin expression on platelets. Salvianolate significantly potentiated the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of salvianolatic acid B (Sal-B), the major component which composes 85% of salvianolate. Sal-B inhibits human platelet activation induced by multiple agonists in vitro by inhibiting phosphodiesterase (PDE) and antagonizing P2Y12 receptor. For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12 antagonism which may account for the clinical antiplatelet effects of salvianolate. Our results suggest that Sal-B may substitute salvianolate for clinical use. © 2014 Elsevier Ltd. All rights reserved.
Introduction Arterial thrombotic diseases, such as heart attack and stroke, are the leading cause of morbidity and mortality worldwide. Platelet activation triggered by atherosclerotic plaque disruption or endothelium injury caused by percutaneous coronary intervention (PCI) and the consequent intravascular arterial thrombogenesis is the common pathological basis
Abbreviations: Sal-B, salvianolatic acid B; VASP, vasodilator-stimulated phosphoprotein; AFM, atomic force microscopy; PDE, phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine; cAMP, 3,5-cyclic adenosine monophosphate; aspirin, acetylsalicylic acid; PRP, platelet-rich plasma; HPLC, high-performance liquid chromatography. ⁎ Correspondence to: X. Luo, Department of Cardiology, Huashan Hospital, Fudan University Shanghai Medical College, Shanghai 200040, China. Tel.: +86 21 52887165; fax: +86 21 64037268. ⁎⁎ Correspondence to: Z. Ding, Key Laboratory of Molecular Medicine, Ministry of Education, and Department of Biochemistry and Molecular Biology, Fudan University Shanghai Medical College, Shanghai 200032, China. Tel.: +86 21 54237896; fax: +86 21 64033738. E-mail addresses: [email protected] (X. Luo), [email protected] (Z. Ding). 1 Present address: Department of Cardiology, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai 201508, China.
http://dx.doi.org/10.1016/j.thromres.2014.07.019 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
of heart attack and stroke; therefore, antiplatelet drugs are effective for prevention and treatment of coronary artery disease (CAD) and stroke. Currently, cyclooxygenase inhibitor aspirin, thienopyridine P2Y12 receptor antagonists clopidogrel and prasugrel, fibrinogen receptor antagonists, and phosphodiesterase (PDE) inhibitor cilostazol, are the mainly used antiplatelet drugs. Among these antiplatelet drugs for arterial thrombotic diseases, aspirin and P2Y12 receptor antagonists are most successfully and most widely used for coronary heart disease and stroke [1], while the PDE inhibitor cilostazol is used mainly in peripheral arterial occlusion and is under clinical trials in patients undergoing PCI in combination with clopidogrel and aspirin [2–5]. Though the current antiplatelet drugs are proven to be beneficial to patients with coronary heart disease, stroke and peripheral arterial disease, morbidity and mortality are still high, and novel antiplatelet agents with improved efficacy and safety are still needed. Traditional Chinese medicine (TCM) Salviae miltiorrhiza (Dangshen) has been used clinically for the treatment of cardiovascular diseases in China and some other Asian countries for thousands of years. Salvianolate, also known as depsides salts, the active components extracted from Salviae miltiorrhiza, was approved by Chinese SFDA in 2005 and widely used in clinical practice to treat CAD. However, there are few evidences
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clearly showing its clinical efficiency as most clinically used TCMs, and the underlying mechanism is also not clear. Salvianolate is the mixture of salvianolic acid B (Sal-B, ≥ 85%), salvianolic acid A (Sal-A, ≥ 1.9%) and rosmarinic acid (RA, ≥ 10.1%) [6]. Huang et al have found that Sal-A inhibits human platelet activation in vitro via PI3K inhibition [7]. As the most abundant ingredient of salvianolate, Sal-B has also been reported to inhibit rat platelet activation by targeting integrin α2β1 [8]. Given the pivotal roles of platelets in CAD and the proven benefits of antiplatelet drugs in treating CAD, we hypothesize that salvianolate may exert its clinical effects via platelet inhibition in CAD patients. In this study, we evaluated the effects of salvianolate on platelets in acute coronary syndrome (ACS) patients and found that it potentiates the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of Sal-B, the major component of salvianolate and demonstrated that Sal-B inhibited platelet activation induced by multiple agonists by inhibiting PDE and antagonizing P2Y12 receptor.
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and CD62P expression were subsequently measured using a FACS (FACSCalibur, Becton Dickinson). Results are expressed as percent of events designated positive for the marker of interest [10]. Preparation of Platelet Rich Plasma (PRP) and Washed Platelets from Normal Humans All experiments using human samples were performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board, Fudan University. The healthy volunteers (staff or students at Fudan University) without taking aspirin or other nonsteroidal anti-inflammatory drugs for at least two weeks were recruited after the informed consent was obtained. Blood (36 ml) was drawn into tubes containing 6 ml ACD (85 mM sodium citrate, 71.38 mM citric acid, and 27.78 mM glucose) solution. PRP and washed platelets were prepared as reported before [11–13].
Methods
Platelet Aggregation Assay
Study Design to Evaluate the Antiplatelet Effects of Salvianolate in ACS Patients
Aggregation of 0.5 ml human washed platelets in response to agonists or antagonists was analyzed using a lumi-aggregometer (Model 400VS; Chrono-Log, Havertown, PA, USA) under stirring condition (900 rpm) at 37 °C as reported before [11–14]. Platelet aggregation was initiated by addition of agonists with or without preincubating platelets with Sal-B (HPLC N 99%, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China) for 3 min. Each sample was allowed to aggregate for at least 3 min. The baseline was set using Tyrode’s buffer as blank.
Sixty three consecutive patients (Supplemental Table 1) were included after obtaining informed consent in abidance with the Declaration of Helsinki. Patients between 18 - 75 years old with chest pain suggestive of new-onset ACS were eligible, according to the 2007 ACC/ AHA guideline [9]. All patients enrolled were admitted to the Department of Cardiology in Huashan Hospital, Fudan University. Exclusion criteria were previous diagnosis of ACS before and relapse, long-term use of antiplatelet or anticoagulant therapy, receiving or scheduled use of platelet GP IIb/IIIa antagonist, and history of coagulopathy. Patients were randomly divided into two groups (Supplemental Table 1): the salvianolate group was given salvianolate (Shanghai Green Valley Pharmaceutical Co., Ltd, Shanghai, China) 200 mg/day intravenously in addition to standard treatment (according to the practice guideline of ACC/AHA) [9], and the control group was given standard treatment only (Fig. 1A). Salvianolate drip ended after one week. Current guidelines were followed for patient management and therapy, including percutaneous coronary interventions and thrombolysis. Drug therapy includes aspirin (Bay-aspirin) and clopidogrel (Plavix) (the loading dose of 300 mg, followed by a regimen therapy of 100 mg/d and 75 mg/d, respectively), statins (Lipitor 20 mg/d), LMWH (4000 IU twice daily, injected subcutaneously for 3 days) (Fig. 1A). To evaluate platelet activation status, PAC-1 binding and CD62P expression on platelet surface were determined by flow cytometry (FACS) using blood collected before and after antiplatelet treatment 7 days later, according to the protocol shown in Fig. 1A. PAC-1 Binding and CD62P Expression Assay in Whole Blood from ACS Patients Whole blood FACS analysis of PAC-1 binding and CD62P expression on platelet surface was taken before and 7 days after antiplatelet treatment. PAC-1 binding and CD62P expression were determined on resting and ADP-activated platelets from ACS patients, using monoclonal antibodies PAC-1 and anti-CD62P for activated αIIbβ3 and P-selectin, respectively, as reported before [10]. Blood was collected from an antecubital vein into Venous Blood Collection tubes containing 0.5 mL buffered sodium citrate (equiv. To 3.2% sodium citrate, Franklin Lakes, NJ, USA). The first 2 mL of blood were discarded. Isotonic HEPES Tyrode buffer was used to dilute the blood. Resting and platelets activated by ADP (10 μM) for 3 min were incubated with FITC-conjugated PAC-1 (Santa Cruz, San Diego, CA, USA) and PE-conjugated anti-CD62P (AbD Serotec, Oxford, UK) antibodies in the dark at room temperature for 15 min without stirring. PAC-1 binding
Assay of PAC-1 Binding on Platelets from Normal Humans Whole blood from normal humans was incubated with Sal-B at different concentrations. PAC-1 binding on the surface of resting or ADP activated platelets was determined by FACS as described above. [Ca2+]i Measurements Human PRP was prepared as described above. 1 mg Fluo-3/AM (Biotium, Hayward, CA, USA) was dissolved in DMSO with 20% F-127 as 1 mM stock solution [15]. Platelets pelleted from PRP were resuspended in Ca2+-free Tyrode’s solution, and then incubated with 2 μM Fluo-3/AM for 30 min at 37 °C. After washing twice, the fluo-3-loaded platelets were finally suspended in Tyrode’s buffer containing 0.5 mM EGTA, at a density of 3 × 108 platelets/ml. The fluo-3-loaded platelets were preincubated with Sal-B at 37 °C for 3 min before the addition of the platelet agonists. Fluorescence (excitation 505 nm, emission 530 nm) was measured with a fluorescence spectrophotometer (Model F4500; Hitachi, Tokyo, Japan) [15]. Western Blotting Analysis of VASP (Vasodilator-Stimulated Phosphoprotein) Phosphorylation in Normal Human Platelets Washed platelets was prepared as described above and treated or untreated with 5 μM prostaglandin E1 (PGE1) at 37 °C for 30 min [16], followed by incubation with Sal-B (70, 140, 280 μM) or AR-C69931MX (100 nM, a gift from AstraZeneca, Loughborough, UK) under stirring condition (900 rpm) for 3 min. After stimulation with 10 μM ADP for 3 min the reaction was stopped by addition of 5 x sample buffer and boiled for 5 min. Platelet lysates were further subjected to western blot analysis of VASP phosphorylation using anti-pSer157-VASP (Cell signalling, Beverly, MA, USA)[17,18]. The optical density of the bands was measured using Image J (National Institutes of Health, Bethesda, MD).
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Fig. 1. Study protocol for (A) blood sampling and (B) FACS assay of PAC-1 binding and CD62P expression before and after antiplatelet therapy in ACS patients. Panel B is a typical dot figure showing antiplatelet treatment significantly inhibits CD62P expression and PAC-1 binding on both resting and ADP-activated platelets from patients, while salvianolate group having more significant inhibition on CD62P expression and PAC-1 binding on ADP-activated platelets in salvianolate group (anti-CD62P: 46.2/36.2; PAC-1: 53.2/39.3) compared with control group.
Measurement of cAMP in Platelets Intracellular cAMP of stimulated platelets was measured using chromatography as described before [11,14]. After incubation with Sal-B or AR-C69931MX for 30 s, ADP 10 μM was added to activate platelets for 2.5 min. Levels of cAMP were determined and cAMP conversion from ATP was calculated using the following formula: cAMP conversion from ATP = [3H]cAMP/([3H]ATP + [3H]cAMP) x 103. We also assayed cAMP in resting platelets using commercially available cAMP 125I radioimmunoassay kits (Isotype Laboratory of Shanghai University of Traditional Chinese Medicine, Shanghai, China) as reported previously [11,19]. Assay of the Activity of Phosphodiesterase (PDE) Extracted from Human Platelets Human platelet PDE extracts were prepared as previously reported [11,12]. The assay for cAMP-PDE activity was performed using the
method previously reported [12,20]. The inhibition of PDE activity was calculated using the following formula: % inhibition of PDE activity = (1 - converted cAMP in enzyme reaction system treated by different agents/converted cAMP in untreated enzyme reaction system) x 100%.
Interaction Between ADP and P2Y12 Receptor with Atomic Force Microscopy (AFM) Effects of Sal-B on the interaction between ADP and P2Y12 receptors stably expressed in Chinese hamster ovary (CHO-K1) cells were measured using a MFP-3D-BIO atomic force microscope (Asylum Research, Santa Barbara, CA, USA), integrated with an IX71 inverted microscope (Olympus, Tokyo, Japan) as previously reported [11,12]. P2Y12 receptor antagonist AR-C69931MX (100 nM) was used as positive control while P2Y1 receptor antagonist MRS2179 (100 μM, Tocris Bioscience, Ellisville, MO, USA) was used as negative control.
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Platelet Spreading Experiment
Sal-B Inhibits Platelet Aggregation and GPIIb/IIIa Activation Induced by ADP
Glass coverslips were coated with 10 μg/mL fibrinogen in 0.1 M NaHCO3 (pH 8.3) at 4 °C overnight. Human washed platelets (2 × 107/ mL) preincubated with or without inhibitors were allowed to adhere and spread on the fibrinogen-coated glass coverslips at 37 °C for 90 min. After washing with PBS, attached platelets were fixed with freshly prepared 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 min, stained with fluorescein-labeled phalloidin (Molecular Probes, Eugene, OR, USA), and viewed as previously described [21,22].
Sal-B is the major component of salvianolate, composes 85% of salvianolate. After showing the antiplatelet effects of salvianolate in ACS patients, we sought to investigate the role of Sal-B on human platelet activation. A plasma concentration ranging from 8 to 280 μM has been reported in rat [23], therefore the antiplatelet role of Sal-B was tested in this concentration range. In the range of 8 to 280 μM, Sal-B concentration-dependently inhibited platelet aggregation induced by 10 μM ADP in aspirintreated human washed platelets with IC50 of 55.5 μM (Fig. 2B and C). Interestingly, we have shown that Sal-B and salvianolate equivalently inhibit platelet aggregation induced by ADP (Supplemental Fig. 1). The inhibitory effect of Sal-B on ADP-induced platelet activation was further confirmed in whole blood from normal humans by FACS analysis of PAC-1 binding (Fig. 2D). At higher concentrations (140 and 280 μM), the antiplatelet effects of Sal-B are even more profound than 100 nM AR-C69931MX, a P2Y12 receptor antagonist under clinical trials as antiplatelet drugs.
Statistics For ex vivo measurements, baseline platelet glycoprotein data were analyzed for differences by analysis of variance (ANOVA) with the use of treatment and sex as factors. Data were expressed as the mean ± SD for continuous variables and as frequencies and percentages for categorical variables. Wilcoxon test was used to compare continuous variables, and a Chi square test or Fisher’s exact test was used to compare the categorical variables. For in vitro measurements, data were expressed as mean ± SEM. Differences between the groups were analyzed by one-way ANOVA followed by a Newman-Keuls test using GraphPad Prism version 5.0 unless otherwise stated. P b 0.05 was considered to be statistically significant. Results Patient Characteristics Sixty seven patients with chest pain between November 1, 2010 and April 31, 2011 were consecutively enrolled, among which 4 patients died of non-cardiac reasons were excluded. A total of 63 subjects fulfilled the study (Supplemental Table 1), the average age in salvianolate group (n = 32) and control group (n = 31) is 66 ± 9 and 66 ± 11 years old, respectively. There were no differences in demographic characteristics and baseline values between 2 groups (Supplemental Table 1). Salvianolate Potentiates the Antiplatelet Effects of Aspirin Plus Clopidogrel in ACS Patients There were no significant differences in PAC-1 binding and CD62P expression on the surface of resting and ADP-activated platelets between the 2 groups before antiplatelet treatment (Fig. 1B and Table 1). Antiplatelet treatment significantly inhibits both PAC-1 binding and CD62P expression on the surface of resting and ADP-activated platelets in both groups (Fig. 1B and Table 1). Compared with control group, addition of salvianolate attenuated PAC-1 binding on ADP-activated platelets from ACS patients more significantly (47.0 ± 10.0 % vs 52.1 ± 6.2 %, P b 0.05). CD62P expression on ADP-activated platelets was also attenuated more significantly in salvianolate group compared with control group (39.5 ± 8.3 % vs 45.0 ± 6.7%, P b 0.01) (Table 1). These results indicate that addition of salvianolate to the standard dual antiplatelet therapy further enhances the antiplatelet effects in ACS patients.
Sal-B Inhibits Platelet Aggregation Induced by Thrombin, Arachidonic Acid, Collagen and U46619 Platelet activation involves complex signal transduction cascades mediated by multiple agonists including ADP, thrombin, collagen, and thromboxane A2. ADP receptor P2Y12 contributes to platelet activation induced by other platelet agonists [24]. Hence, after showing excellent antiplatelet efficacy of Sal-B on human platelets activated by ADP, we further explored the antiplatelet effects of Sal-B on platelet activation induced by thrombin, arachidonic acid, collagen, and U46619. As shown in Fig. 2E, in the range of 8 - 280 μM, Sal-B also abolished or drastically inhibited human platelet aggregation induced by thrombin, arachidonic acid, collagen, and U46619. Sal-B Inhibits Platelet Spreading Platelet aggregation is the result of fibrinogen binding to the activated integrin GPIIb/IIIa mediated by inside-out signal pathways. Upon GPIIb/IIIa binding to fibrinogen, it triggers outside-in signaling, causing platelet spreading and clot retraction which play a crucial role in thrombosis. We have shown that Sal-B inhibits platelet aggregation induced by multiple agonists and integrin GPIIb/IIIa activation induced by ADP, the typical inside-out signaling events. We next explored whether SalB also influence platelet outside-in signaling. As shown in Fig. 3, Sal-B 280 μM dramatically inhibited platelet spreading on immobilized fibrinogen, similarly to LY294002 (Calbiochem, San Diego, CA, USA), a PI3K inhibitor, which has been reported to inhibit platelet spreading [7,22]. Sal-B did not inhibit platelet spreading at 140 μM, a concentration that dramatically inhibited platelet aggregation and PAC-1 binding (Fig. 3). Sal-B Increases cAMP Level in Resting and ADP-Stimulated Platelets Sal-B inhibits platelet aggregation induced by multiple agonists including ADP, thrombin, collagen, and U46619 with more potent
Table 1 Antiplatelet treatment inhibits platelet function in ACS patients as evaluated by FACS analysis of PAC-1 binding and CD62P expression on platelet surface. PAC-1 positive (%) before treatment control group (n = 31) salvianolate group (n = 32)
resting ADP-activated resting ADP-activated
9.7 69.9 10.2 71.8
± ± ± ±
3.9 8.0 6.4 12.7
anti-CD62P positive (%) after treatment 5.5 52.1 6.2 47.0
± ± ± ±
#
1.4 6.2## 2.8# 10.0#⁎
before treatment
after treatment
7.8 60.0 7.0 59.0
4.7 45.0 4.5 39.5
± ± ± ±
2.8 10.5 3.5 13.5
± ± ± ±
1.7# 6.7# 2.3# 8.3#⁎⁎
Antiplatelet treatment for control group is standard of care, salvianolate treatment group received standard of care plus salvianolate. #P b 0.05, ##P b 0.01 compared with before treatment; ⁎P b 0.05 (47.0 ± 10.0 in salvianolate group compared with 52.1 ± 6.2 in control group), ⁎⁎P b 0.01 (39.5 ± 8.3 in salvianolate group compared with 45.0 ± 6.7 in control group).
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Fig. 2. Sal-B concentration-dependently inhibits human platelet aggregation induced by ADP, thrombin, arachidonic acid (AA), collagen and U46619. A, Structure of Sal-B and Sal-A. B, Sal-B concentration-dependently inhibits ADP (10 μM) induced platelet aggregation in aspirin-treated human washed platelets. C, Concentration-response curve of Sal-B inhibition on platelet aggregation induced by ADP 10 μM in aspirin-treated washed human platelets. Platelets were preincubated with saline, Sal-B (10 μM) or AR-C69931 MX (100 nM) followed by stimulation with ADP (10 μM). Tracings shown are representative of at least three experiments using platelets from different donors. 0.9% saline was used as a vehicle control. D, Sal-B concentrationdependently inhibits PAC-1 binding on platelets in human blood measured by FACS analysis. Data were expressed as mean ± SEM representing three separate experiments measured in duplicate. E, Sal-B was preincubated with human washed platelets for 3 min before stimulation with thrombin (0.05 U/ml), AA (0.5 mM), collagen (2 μg/ml) and U46619 (1 μM). Tracings shown are representative of at least 3 experiments using platelets from different donors. 0.9% saline was used as vehicle control.
inhibition on ADP-induced aggregation (Fig. 2), suggesting that Sal-B may target ADP receptors to exert its antiplatelet effects. Sal-B inhibits ADP-induced platelet aggregation without influence on platelet shape change (Fig. 2B), indicating that Sal-B may target P2Y12 receptor rather than P2Y1 [25]. To elucidate the antiplatelet mechanism of Sal-B, we first measured the effects of Sal-B on cAMP level in platelets stimulated with ADP to explore its possible P2Y12 antagonizing role. As shown in Fig. 4A, in the antiplatelet concentration range (35 – 280 μM), Sal-B concentration-dependently inhibited ADP-induced decrease in cAMP levels, consistent with its putative P2Y12 antagonizing role. At 280 μM, Sal-B exhibited similar effects as AR-C69931MX, a P2Y12 receptor
antagonist. Higher concentrations of Sal-B (140 and 280 μM) also significantly elevated intracellular cAMP levels of resting platelets as forskolin did (Fig. 4B), indicating that Sal-B may also exert its antiplatelet effects via mechanisms besides P2Y12 antagonism. Sal-B Enhances VASP Phosphorylation in Platelets Stimulated with ADP VASP is the major substrate of PKA (cAMP-dependent protein kinase), which prefers to phosphorylate Ser157 of the 3 phosphorylation sites on VASP [17]. Similar to cAMP decrease, P2Y12- dependent inhibition by ADP of phosphorylation of VASP is another specific assay to
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Fig. 3. Sal-B inhibits platelet spreading on fibrinogen-coated surface. Human washed platelets were seeded on fibrinogen-coated cover slips in the presence of Sal-B 280 μM, PI3-kinase inhibitor LY294002 (100 nM) or vehicle control. Platelets were stained with FITC-phalloidin and observed under a 40 x objective with magnification of 5 (upper panel). Further amplified single platelets in the squares from upper panel are shown in bottom panel. Results shown are representative of 3 experiments using platelets from different donors run in duplicate.
reflect P2Y12 receptor activation status [26,27] and is widely used to measure the effects of antiplatelet drugs targeting P2Y12 receptor [28]. Similar to AR-C69931, at 140 and 280 μM Sal-B concentrationdependently inhibited ADP-induced reduction of VASP phosphorylation on Ser157 (Fig. 4C), in line with its ability to inhibit platelet aggregation and cAMP decrease induced by ADP. Taken together, these results suggest that P2Y12 receptor antagonism is involved in the antiplatelet roles of Sal-B.
Even at 280 μM, Sal-B only slightly inhibited ADP-evoked Ca2+ rise. In contrast, P2Y1 receptor antagonist 100 μM MRS2179 totally abolished Ca2+ rise. P2Y12 receptor was reported to sustain the increased Ca2+ level [29], in agreement with this, Sal-B accelerated Ca2 + decay (Fig. 6B and C), consistent with its P2Y12 antagonizing role. These results indicate that Sal-B antagonize ADP receptor P2Y12 rather than P2Y1.
Sal-B Inhibits the Interaction Between ADP and P2Y12 Receptor Measured by AFM
The ability of Sal-B to enhance cAMP in resting platelets implicates another mechanism in addition to P2Y12 antagonism that might contribute to its antiplatelet effects. We extracted PDE from human platelets and investigated the effects of Sal-B on PDE activity by measuring residual cAMP in a reaction mixture containing Sal-B, PDE extracts and PDE substrate cAMP using HPLC. As shown in Fig. 7, at concentrations ranging from 70 to 280 μM, Sal-B concentration-dependently inhibited the activity of platelet PDE (Fig. 7), in line with its cAMP-enhancing effect in resting platelets (Fig. 4B). Compared with IBMX, a non-specific PDE inhibitor, the PDE inhibition activity is weak even at a high concentration of 280 μM. Furthermore, in the range of 70 - 280 μM, Sal-B increased VASP phosphorylation in human platelets (Fig. 7C), further confirming that Sal-B is a PDE inhibitor.
Sal-B reverses ADP-induced decrease of intracellular cAMP and VASP phosphorylation in platelets (Fig. 4A and C) strongly suggests that Sal-B may work as a P2Y12 antagonist to exert its antiplatelet roles. To provide direct evidence that Sal-B blocks ADP binding to P2Y12 receptor, the interaction between ADP and single P2Y12-expressing CHO-K cell was assayed in the presence of Sal-B by direct force measurement using AFM. As shown in Fig. 5, similar to P2Y12 receptor antagonist ARC69931MX, 280 μM Sal-B remarkably suppressed the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells. In contrast, MRS2179, a P2Y1 antagonist of ADP receptors, did not affect the interaction between ADP and P2Y12 receptor. Sal-B inhibits the interaction between ADP and P2Y12 receptor confirmed its P2Y12 antagonist activity as an antiplatelet drug. Effects of Sal-B on Platelet Intracellular Ca2+ Mobilization We investigated whether Sal-B affects P2Y1-Gq pathway by assaying ADP-induced Ca2+ mobilization. As shown in Fig. 6, in the absence of extracellular Ca2 +, 10 μM ADP elicited rapid Ca2 + rise, which then returned to baseline as a result of Ca2 + re-uptake or extrusion. After preincubating with Sal-B 140 μM, ADP elicited similar rapid Ca2+ rise.
Sal-B Inhibits the Activity of PDE from Human Platelets
Discussion In this study, we first investigated the antiplatelet effects of salvianolate in ACS patients. We found that addition of salvianolate to the standard antiplatelet therapy could further enhance platelet inhibition in ACS patients. To our knowledge, this is the first report to show that salvianolate potentiates the antiplatelet effects of the standard antiplatelet therapy in ACS patients ex vivo. We further investigated the antiplatelet effects and the underlying mechanism of Sal-B, the major component of salvianolate. We found that Sal-B inhibited platelet
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Fig. 4. Effects of Sal-B on platelet cAMP and VASP phosphorylation in washed human platelets. A & B, Sal-B concentration-dependently increases intracellular cAMP in ADP-stimulated (A) and resting (B) human platelets. Data are expressed as mean ± SEM representing three separate experiments measured in duplicate. C, Sal-B concentration-dependently increases VASP phosphorylation in washed human platelets stimulated with ADP. After preincubation with 5 μM prostaglandin E1, platelets were incubated with vehicle, AR-C69931MX (ARC) or Sal-B and further stimulated for 5 min with 10 μM ADP. Equal amounts of proteins were separated by SDS-PAGE, western blotted, and probed for anti-phospho-VASP or antiGAPDH antibody. The results shown are representative of 3 experiments using platelets from different donors.
activation induced by multiple agonists, antagonized ADP binding to P2Y12 receptor, and inhibited platelet PDE activity at the antiplatelet concentrations. Clinical trials have demonstrated that antiplatelet drugs including aspirin, clopidogrel and GP IIb/IIIa inhibitors provide substantial therapeutic benefits in patients with ACS [30,31]. After platelet stimulation, GP IIb/IIIa serves as an anchoring site for soluble fibrinogen and von Willebrand factor (vWF), and P-selectin released from platelet granules to the surface membrane permits platelet binding to white blood cells and endothelium [32]. Therefore, ACS patients may benefit from salvianolate given in combination with the standard antiplatelet therapy, which warrants further study.
As the major component of salvianolate, Sal-B constitutes 85% of salvianolate. Previously Sal-B has been reported to inhibit platelet adhesion to collagen [8,33] and platelet aggregation induced by collagen and ADP in rat platelets [8,34]. Consistent with these results, in the present study, we demonstrated that Sal-B effectively inhibited platelet activation elicited by multiple agonists including collagen, ADP, thrombin, arachidonic acid, and U46619 in human platelets. Importantly, we demonstrated that salvianolate and Sal-B exhibited the almost equivalent inhibitory effects on platelet aggregation induced by ADP (Supplemental Fig. 2), indicating that salvianolate inhibits platelet aggregation mainly through Sal-B. We also found that Sal-B inhibited platelet spreading on immobilized fibrinogen and ADP-induced PAC-1 binding
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Fig. 5. Sal-B inhibits interaction between ADP and P2Y12 receptor. A, Typical atomic force microscopy force-displacement measurements of the interaction between ADP and P2Y12 receptors expressed in CHO-K1 cells in the presence of different ADP receptor antagonists, Sal-B or vehicle control. The measurements were required with 100 pN indentation force, 0.2 s contact time, and a cantilever retraction speed of 3 μm/s. B, Histograms of individual unbinding forces of ADP and P2Y12 in the presence of different ADP receptor antagonists, Sal-B or vehicle control from force displacement measurements. The y-axis plots the number of force transitions detected and x-axis is the unbinding force (pN). C, Statistical analysis of unbinding forces of ADP and P2Y12 in the presence of different ADP receptor antagonists, Sal-B (filled bars) or vehicle control (blank bars). AR-C69931MX (100 nM), MRS2179 (100 μM) and Sal-B (280 μM) were used. Data are expressed as mean ± SEM of 100 measurements in panel A.
in human whole blood, which further confirmed the antiplatelet role of Sal-B on human platelets. The effective concentrations of Sal-B range from 8 to 280 μM, which is in line with antiplatelet concentrations previously reported in rats [8,34] and is within the blood drug concentration range of in rats [35]. Platelet activation is a complex process involving multiple agonists and signaling pathways. Among the numerous signaling molecules regulating platelet activation, haemostasis and thrombosis, P2Y12 receptor plays a central role [24] and has been one of the most successful antiplatelet targets. cAMP is also an important signaling molecule in platelet activation, and increased platelet cAMP level negatively regulates platelet function. PDE inhibitors, which increase cAMP by inhibiting cAMP degrading, inhibit platelet activation elicited by all agonists. In this
study, we found that Sal-B inhibits platelet activation induced by a broad range of agonists, consistent with the central role of P2Y12 receptor in platelet activation [24] and the broad spectrum antiplatelet activity of PDE inhibitors. Our results confirm and further extend the previous finding that Sal-B inhibits rat platelet aggregation induced by ADP [34]. Concomitant activation of Gq-coupled P2Y1 and Gi-coupled P2Y12 is essential for ADP induced platelet aggregation [25]. P2Y1 induces Ca2+ mobilization, platelet shape change, and reversible platelet aggregation, while P2Y12 inhibits adenylyl cyclase, decreases cAMP, amplifies P2Y1induced platelet aggregation, leading to irreversible aggregation. P2Y12 also amplifies platelet activation induced by other agonists, plays a central role in platelet activation [24], and is one of the most successful
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Fig. 6. Effects of Sal-B on ADP-induced Ca2+ mobilization in platelets. Sal-B accelerates Ca2+ decay at 140 and 280 μM and only slightly inhibits Ca2+ rise in human platelets stimulated with ADP 10 μM. Intracellular Ca2+ mobilization was assayed as the Ca2+ fluorescence in Fluo-3-loaded human platelets in the absence of extracellular Ca2+. Results shown are representative of three experiments using platelets from different donors.
antiplatelet drug targets. In this study we found that Sal-B inhibits platelet activation via P2Y12 antagonism as evidenced by: 1) it concentrationdependently inhibited ADP-induced cAMP decrease in platelets; 2) it concentration-dependently inhibited ADP-induced VASP phosphorylation decrease in platelets; 3) it did not affect Ca2+ mobilization amplitude, but accelerated the decay of the elevated Ca2 + in platelets stimulated with ADP, consistent with the previous report that P2Y12 sustains the increased Ca2+ level [29] while P2Y1 elicits Ca2+ mobilization; 4) it inhibited the interaction between ADP and P2Y12. The fact that Sal-B does not influence ADP-induced platelet shape change and Ca2+ mobilization amplitude rules out P2Y1 as its antiplatelet target. Sal-B increased cAMP in resting platelets cannot be explained by P2Y12 antagonism, suggesting the involvement of an alternative mechanism underlying the antiplatelet effects of Sal-B, which could be adenylyl cyclase activation or PDE inhibition. Direct assay of the activity of PDE extracted from human platelets confirmed that Sal-B inhibits the activity of PDE concentration-dependently in its antiplatelet concentration range (Fig. 7). Sal-B inhibiting platelets as a PDE inhibitor is in agreement with the previous report that Injectio Salvia Miltiorrhizae inhibits human platelet activation by increasing platelet cAMP [36]. However, even at 280 μM, the concentration Sal-B remarkably inhibited platelet activation, PDE is only partly inhibited (42% inhibition), indicating that PDE inhibition only partly contributes to the antiplatelet effects of Sal-B with the rest effects attributed to P2Y12 antagonism. Taken together, our results indicate that Sal-B exerts its antiplatelet effects via P2Y12 antagonism and PDE inhibition, similarly to the BF compounds
previously reported in our lab [11,12]. Interestingly, the two BF compounds share similar structure, while Sal-B is structurely distinctive. Sal-B has been reported to target α2β1 [8,33]. We found that Sal-B did not affect anti-α2β1 antibody binding to integrin α2β1 of human platelets at 280 μM, the concentration that dramatically inhibits platelet activation (Supplemental Fig. 2). Therefore, we think that α2β1antagonism does not contribute the antiplatelet and antithrombotic effects of Sal-B. Multiple signaling pathways contribute to platelet activation and thrombosis, therefore combined antiplatelet therapy targeting different pathways is recommended. Currently dual antiplatelet therapy with aspirin plus thienopyridine P2Y12 receptor antagonists is widely used, while triple antiplatelet therapy (dual antiplatelet therapy plus PDE inhibitor cilostazol) is under intensive evaluation with improved efficacy and safety reported recently [2,37,38]. Consistently, in this study we demonstrated that addition of salvianolate to the standard dual antiplatelet therapy (aspirin plus clopidogrel) enhanced platelet inhibition without affecting hemostasis in ACS patients, which can be attributed to the dual antiplatelet activity (targeting P2Y12 and PDE) of Sal-B. Platelet outside-in signaling pathway is mainly involved in thrombosis rather than hemostasis of platelet function. Targeting platelet outside-in signaling pathway represents a more attractive approach to develop effective and safe antiplatelet agents [21]. In this study, we found that Sal-B dramatically inhibited platelet spreading on immobilized fibrinogen at high concentration (280 μM) (Fig. 3). However, this inhibitory effect was not observed at the lower concentrations profoundly inhibiting platelet aggregation. In concert with our findings,
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Fig. 7. Sal-B inhibits cAMP-phosphodiesterase activity in human platelet extracts and increases VASP phosphorylation in washed human platelets. A, Sal-B concentration-dependently inhibits the activity of PDE extracted from human platelets. Data are expressed as mean ± SEM representing three separate experiments. B, Representative HPLC tracings of residual cAMP in the presence of PDE and various inhibitors. C, Sal-B increases VASP phosphorylation in washed human platelets. Platelets were incubated with vehicle, Sal-B or IBMX for 5 min. Equal amounts of proteins were separated by SDS-PAGE, western blotted, and probed for anti-phospho-VASP or anti-GAPDH antibody. All concentrations of Sal-B increase VASP phosphorylation compared with the vehicle control using t-test. The results shown are representative of 4 experiments using platelets from different donors.
salvianolate acid A, another antiplatelet component in salvianolate, also inhibits platelet spreading at high concentration (202 μM) [7]. We have shown that salvianolate potentiated platelet inhibition in ACS patients receiving dual antiplatelet therapy without affecting hemostasis in the present study. Whether the inhibition of Sal-B on platelet spreading contributes to the antiplatelet and antithrombotic effects of salvianolate without affecting hemostasis remains to be elucidated. In conclusion, for the first time, we show that salvianolate potentiates the antiplatelet effects of the standard antiplatelet therapy without affecting hemostasis in ACS patients. We also find that Sal-B, which constitutes 85% of salvianolate, inhibits platelet activation via P2Y12 antagonism and PDE inhibition, which may account for the clinical antiplatelet effects of salvianolate. Whether Sal-B can substitute salvianolate for clinical use deserves further investigation. Conflict of Interest Statement None of the authors have any conflict of interests. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.thromres.2014.07.019.
Acknowledgements This work was partially supported by National Natural Science Foundation of China (No. 30973529, 81270278), Drug Innovative Program from Shanghai Municipal Science and Technology Commission (No. 11431920103), and Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning.
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