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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 5  |  Issue : 4  |  Page : 260-268

Effects of water-soluble tomato concentrate on platelet aggregation


1 Center for Biomechanopharmacology, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
2 Science and Technology Center, ByHealth Co., Ltd., Guangzhou, China
3 Department of Cardiovascular, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China

Date of Submission25-Mar-2019
Date of Decision11-May-2019
Date of Acceptance16-May-2019
Date of Web Publication03-Dec-2019

Correspondence Address:
Prof. Yun You
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_35_19

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  Abstract 


Objective: To investigate the antiplatelet aggregation effect of water-soluble tomato concentrate (WSTC) and explore the underlying molecular mechanisms. Materials and Methods: Platelet aggregometry was used to quantify rat platelet aggregation with the maximum aggregation rate in vitro and ex vivo. Then, the fibrinogen (FIB) binding assay was employed to detect the effect of WSTC on the activation of platelet integrin αIIβ3 (GP IIb/IIIa). Furthermore, Western blot was performed to assess the platelet protein levels of phosphoinositide 3-kinase 110 β (PI3K110 β), protein disulfide isomerase (PDI), platelet endothelial cell adhesion molecule 1 (PECAM-1), and β1-Tubulin. Results: WSTC inhibited adenosine diphosphate (ADP) and collagen-induced platelet aggregation in a concentration-dependent manner in vitro, at IC50values of 3.05 g/L and 8.03 g/L, respectively. Significantly reduced ex vivo ADP induced platelet aggregation was observed after oral consumption of WSTC for 4 weeks in rats; average inhibition rates were 24.42%, 21.48%, and 20.87% for 25 mg/Kg, 75 mg/Kg, and 150 mg/Kg WSTC, respectively. It appeared that WSTC had no influence on coagulation function in rats. Incubation with WSTC decreased FIB binding to GP IIb/IIIa by 17.47% and 32.29% at the concentrations of 0.6 and 6 g/L, respectively. WSTC at 0.6 and 6 g/L markedly downregulated PI3K110 β, PDI, and PECAM-1 in platelets, and upregulated β1-Tubulin, in a concentration-dependent manner. Conclusion: WSTC inhibits platelet activation through modulation of platelet skeletal stability and suppresses GP IIb/IIIa receptor-mediated platelet aggregation, likely via the PI3K signaling pathway and PDI inhibition.

Keywords: GP IIb/IIIa, platelet aggregation, PDI, PI3K, PECAM-1, watersoluble tomato concentrate


How to cite this article:
Zhang Q, Zhang XG, Liu L, Zhang QL, Ding SL, Chen Y, Wang JY, Wang L, Liang RX, Liao FL, Wang YH, You Y. Effects of water-soluble tomato concentrate on platelet aggregation. World J Tradit Chin Med 2019;5:260-8

How to cite this URL:
Zhang Q, Zhang XG, Liu L, Zhang QL, Ding SL, Chen Y, Wang JY, Wang L, Liang RX, Liao FL, Wang YH, You Y. Effects of water-soluble tomato concentrate on platelet aggregation. World J Tradit Chin Med [serial online] 2019 [cited 2019 Dec 13];5:260-8. Available from: http://www.wjtcm.net/text.asp?2019/5/4/260/271969




  Introduction Top


Platelet activation at the site of vascular injury is essential to stop bleeding; however, excessive platelet activation in the region of atherosclerotic plaque rupture or turbulent blood flow can result in the development of platelet thrombi and coagulation diseases such as acute myocardial infarction and ischemic stroke. Platelets in individuals with diabetes, a sedentary lifestyle, obesity, and insulin resistance show hyperactivity at baseline and in response to agonists, ultimately leading to increased aggregation and plaque development.[1] Hyperactive platelets are important mediators of atherogenesis in addition to their roles in thrombosis. However, antiplatelet drugs, such as aspirin and clopidogrel with prolonged bleeding time may not be suitable for individuals with relatively low risk of cardiovascular events. It is of great importance to identify safer antiplatelet agents for patients with hyperactive platelets in order to prevent cardiovascular disease occurrence and development.

It is admitted that dietary components have the potential to reduce the levels of specific risk factors for cardiovascular disease. There is compelling evidence for tomatoes to be considered a cardiovascular protective food. Tomato (Lycopersicon esculentum) contains several components that are beneficial to overall health, including vitamin E, flavonoids, phytosterols, carotenoids, and several water-soluble vitamins and minerals.[2] Water-soluble tomato concentrate (WSTC) is derived from ripe tomatoes, containing naturally occurring anti-platelet compounds such as nucleosides, simple phenolic derivatives and flavonoid derivatives, which have been shown to suppress blood platelet activity in healthy people after consumption. These representative WSTC compounds were monitored from raw material to final product.[3] WSTC has become the first product in Europe (Fruitflow®) approved with proprietary health claim under Article 13 (5) of the European Health Claims Regulation that “helps maintain normal platelet aggregation, which contributes to healthy blood flow.”[1]

A previous work reported that WSTC can influence platelet activityin vitro and ex vivo in human subjects.[3],[4] In healthy individuals, consumption of tomato extract results in a reduction by 20.0 ± 4.9% of platelet aggregation induced by adenosine diphosphate (ADP). Functionally, WSTC was observed to inhibit integrin αIIβ3 (GP IIb/IIIa) activation,[5] which is common to multiple aggregation pathways, indicating the wide range of WSTC effects.[3] Proteomic experiments carried out to examine the effects of WSTC on platelet signaling pathways showed that WSTC alters multiple platelet functions, including those regulating platelet structure, coagulation and redox status.[1] Protein disulfide isomerase (PDI), an oxidoreductase which catalyzes the formation and isomerization of disulfide bonds, is essential for normal thrombus formation. Severalin vivo thrombosis models have demonstrated that suppressing PDI with blocking antibodies inhibits a number of platelet activation processes, including aggregation, secretion, and fibrinogen (FIB) binding.[6],[7],[8] The common flavonoid quercetin-3-rutinoside (rutin) was identified as a natural inhibitor of PDI.[9] Rutin, a representative WSTC compound, prompts the interaction of WSTC with PDI,[1] but there is currently no direct evidence to demonstrate WSTC effects on PDI. Blood flow disturbances (high shear stress) play an important role in promoting arterial thrombosis by enhancing the adhesive and signaling functions of platelet integrin GP IIb-IIIa and GP Ib/V/IX. Several studies have indicated a key role for the phosphoinositide 3-kinase (PI3K) p110 β isoform in regulating the formation and stability of integrin αIIbβ3 adhesion bonds, which are necessary for shear activation of platelets.[10] PI3K p110 β isoform inhibitors eliminate occlusive thrombus formation in vivo. We hypothesized that platelet PI3K p110 β isoform might be the key WSTC ingredient which helps maintain normal platelet aggregation, contributing to healthy blood flow. Therefore, the following experiments were carried out to investigate the effect of WSTC on rat platelet aggregation and explore the possible underlying mechanisms.


  Materials and Methods Top


Materials

WSTC (Fruitflow®) was obtained from By-Health Co., Ltd (Guangzhou, China). Clopidogrel hydrogen sulfate was purchased from Salubris Pharmaceuticals Co., Ltd (Shenzhen, China). Aspirin was purchased from Sigma-Aldrich Co., Ltd (CA, USA). Activated partial thrombin time (APTT) assay kit, prothrombin time (PT) assay kit, thrombin time assay kit, and FIB content determination kit were from SunBio Pharmaceuticals Co., Ltd. (Shanghai, China).

The following antibodies were used: rabbit monoclonal antibody targeting PI3K 110 β (Abcam, MA, USA), mouse monoclonal antibody against PDI (Santa Cruz Biotechnology, CA, USA), mouse monoclonal antibody targeting platelet endothelial cell adhesion molecule 1 (PECAM-1) (Santa Cruz Biotechnology, CA, USA), rabbit monoclonal antibody against β1-Tubulin (Abcam, MA, USA) and mouse monoclonal antibody targeting GAPDH (Proteintech, Chicago, USA). All other chemicals were of reagent grade.

Animals

Male Sprague-Dawley rats (160–220 g) were obtained from Vital River Laboratory Animal Technology Co. (SCXK2016-0006, Beijing, China), and maintained in a standard laboratory animal facility with free access to feed and water, and acclimatized to these conditions for at least 1 week before use. All animals were handled according to the guidelines of the Animal Research Center Committee, China Academy of Chinese Medical Sciences. The experiments were carried out in accordance with internationally accepted guidelines on the use of laboratory animals, and the protocols were approved by the Institute of Chinese Materia Medica of China Academy of Chinese Medical Sciences.

Water-soluble tomato concentrate preparation

The WSTC powder was dissolved in 0.9% saline. After 30 min of ultrasonication, the solution was centrifuged at 3500 r/min for 5 min. Then, the resulting supernatant was filtered with a 0.2 μm membrane, with the pH adjusted to 7.2–7.4.

Platelet preparation and aggregation

Rats underwent anesthesia by injection of 3.0% sodium pentobarbital at 60 mg/Kg, and 8 ml whole blood samples were withdrawn from the abdominal aorta per rat in tubes with 109 mmol/L sodium citrate (1:9) anticoagulation. Platelet-rich plasma (PRP) was obtained following blood sample centrifugation at 200 × g for 10 min. PRP samples were further centrifuged at 2000 × g for 10 min to obtain platelet-poor plasma (PPP). The mean platelets amounts in PRP were adjusted to 4.0 × 1011 platelets/L in PPP specimens for the aggregation assay. Platelets were counted both manually by microscopy (BH-2, Olympus, Japan) and automatically on a Sysmex poch 100i analyzer. All platelet preparations were conducted at room temperature. Aggregation was monitored by measuring light transmission on an aggregometer (LBY-NJ4, Techlink, Beijing, China). PRP samples with 4.0 × 1011 or 8.0 × 1011 platelets/l were preincubated at 37°C for 5 min with either WSTC or vehicle (saline) and stimulated with 2.5 μM ADP (Techlink, Beijing, China) or 4 mg/L collagen (Chrono-log, USA) separately at 37°C for 5 min. Three independent experiments were carried out.

Ex vivo platelet aggregation assay

Platelet aggregation was examined as described above. Fifty-five male Sprague-Dawley rats (210 ± 5 g) were randomized into five groups. Control rats received distilled water. The positive control group was administered clopidogrel hydrogen sulfate (Clopidogrel) at 7.5 mg/Kg once per day for 4 weeks. The WSTC groups were orally administered WSTC at 25 mg/Kg, 75 mg/Kg and 150 mg/Kg, respectively. Blood samples were collected for up to 2 h after the last dose, and platelets were prepared as described above. Platelet aggregation was induced by 2.5 μM ADP. WSTC effects on platelet aggregation were tested ex vivo.

Ex vivo coagulation assay

Fifty-five male Sprague-Dawley rats (160 ± 5 g) were randomized into four treatment groups and an additional distilled water group. WSTC was given to the animals orally at 150 mg, 300 mg, or 900 mg/Kg body weight once per day for 1 week. The positive control group was given aspirin (ASA) at 15 mg/Kg. Blood was collected 120 min after consumption of the last dose, and PPP samples were prepared as described above. The anticoagulation activity of WSTC was evaluated by measuring the plasma coagulation of PPP specimens. Then, TT, FIB, APTT, and PT were measured with slight modifications on a semi-automatic blood coagulation instrument (C2000-4, PRECIL, Beijing, China) using ELISA assay kits according to the manufacturer's instructions.

Platelet preparation and fibrinogen binding assay

Washed platelets were prepared as follows. PRP samples with 5 mM ACD (2.5% trisodium citrate, 2.0% glucose, and 1.5% citric acid) and 5 mM EDTA were further centrifuged at 400 × g for 10 min. Platelets were washed with Tyrode buffer (138 mM NaCl, 3.3 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 5.5 mM glucose, and 20 mM HEPES; pH 7.2) and centrifuged at 400 × g for 10 min. The platelets were adjusted to the proper number in Tyrode buffer. Washed platelets were initially incubated with Calcein AM (4 μM) for 1 h at room temperature in the dark. Calcein AM-platelets were pretreated with WSTC or vehicle (saline) for 10 min at 37°C in the dark. Then, the samples were transferred into a 96-well black plate preincubated with 250 μg/mL FIB (Thermo Fisher Scientific, MA, USA) overnight at 4°C. The plate was further blocked with 1% bovine serum albumin (BSA) for 1 h and washed with Ca2+-free HEPES/Tyrode buffer. Platelet binding to FIB was induced with ADP (10 μM) for 15 min and assessed by fluorescence detection on a spectrofluorometer (SpectraMax i3x, Molecular, USA). Unbound platelets were washed out by Ca2+-free HEPES/Tyrode buffer. The total platelets were only labeled with calcein AM without FIB addition. The binding rate was determined as the percentage of bound platelets among total platelets.

Western blot

Washed platelets from rats were incubated with saline, WSTC (0.06 g/L, 0.6 g/L, or 6 g/L), aspirin (2 mM), rutin (6 mM), or LY294002 (25 μM) for 10 min, and stimulated with 10 μM ADP for 10 min at 37°C. Then, platelets were obtained by centrifugation for 10 min at 1200 × g, followed by lysis in RIPA buffer containing 1 mM PMSF. After centrifugation for 5 min at 12 000 r/min and 4°C, protein amounts in the supernatants were determined with a BCA assay kit (Thermo, USA) according to the manufacturer's instructions. Equivalent amounts of protein (30–40 μg/lane) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes. Each membrane was blocked with 5% skim milk in Tris Buffered Saline containing Tween-20 (TBST) (20 mM Tris-HCl, pH 7.5, 137 Mm, NaCl, and 0.1% Tween 20) at room temperature for 2 h and incubated with anti-PI3K 110 β (1:1,000), anti-PDI (1:100), anti-PECAM-1 (1:1,000), anti-β1-Tubulin (1:1,000), and anti-GAPDH antibody (1:5,000) primary antibodies, respectively, at 4°C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 2 h at room temperature. Bands were visualized by enhanced chemiluminescence (ECL) (Millipore, USA) and analyzed on an ECL detection system (Syngene, Cambridge, UK). The mean density of each band was analyzed with the Image J software. Each experiment was carried out for at least three times.


  Results Top


Effects of water-soluble tomato concentrate on platelet aggregation in vitro and ex vivo

The effects of WSTC on ADP or collagen induced platelet aggregation were assessed by the light transmission method. As shown in [Figure 1]a, ADP (2.5 μM) mediated platelet aggregation was obviously inhibited by WSTC in a concentration-dependent manner, with average inhibitory rates of 12.74%, 20.63%, 54.98% and 74.07% at WSTC concentrations of 1.0, 2.0, 4.0 and 6.0 g/L, respectively; the 50% inhibitory concentration (IC50) of WSTC was 3.05 g/L. Collagen at a concentration of 4 mg/L induced the maximum platelet aggregation with a rate of 71.04 ± 3.16%, and WSTC showed marked inhibitory effects on platelet aggregation [Figure 1]b, with an IC50 of 8.03 g/L. Significant differences were observed at a concentration of 6 g/L under both ADP and collagen inductions.
Figure 1: Effects of water-soluble tomato concentrate on platelet aggregation in vitro. Platelets were preincubated at 37°C for 5 min with either water-soluble tomato concentrate or vehicle (saline) and stimulated with 2.5 μM adenosine diphosphate or 4 mg/L collagen at 37°C for 5 min. Three independent experiments were carried out. (a) Water-soluble tomato concentrate inhibited platelet aggregation induced by 2.5 μM adenosine diphosphate; platelet-rich plasma was 4 × 1011 platelets/l (n = 3). (b) water-soluble tomato concentrate inhibited platelet aggregation induced by 4 mg/L collagen; platelet-rich plasma was 8 × 1011 platelets/l (n = 4). Data are mean ± standard deviation *P < 0.05, **P < 0.01 versus adenosine diphosphate/Col group,△△P < 0.01 group comparisons

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Ex vivo effects of water-soluble tomato concentrate on platelet aggregation and blood coagulation

The effects of WSTC on ADP induced platelet aggregation after 4 weeks of oral consumption in rats are shown in [Figure 2]. WSTC significantly inhibited ADP-induced platelet aggregation ex vivo compared with the model group (P < 0.01). The average inhibitory rates were 24.42%, 21.48% and 20.87%, respectively, under the doses of 25, 75 and 150 mg/Kg in rats. Clopidogrel at a dose of 7.5 mg/Kg inhibited ADP-induced platelet aggregation by 95.47%. The effects of WSTC on coagulation function in rats were also evaluated by FIB, TT, APTT and PT assays. The coagulation function was not influenced by WSTC at doses ranging from 150 to 900 mg/Kg [Table 1].
Figure 2: Effects of water-soluble tomato concentrate on platelet aggregation ex vivo. Fifty-five male standard deviation rats were randomized into four treatment groups, and an additional distilled water group. Water-soluble tomato concentrate was given to the animals orally at 25 mg/Kg, 75 mg/Kg, and 150 mg/Kg body weight, respectively, once per day for 4 weeks; clopidogrel hydrogen sulfate was administered at 7.5 mg/Kg as a positive control. Blood was collected 120 min after administration of the final dose, and platelets were prepared as described above. Platelet aggregation was induced by 2.5 μM adenosine diphosphate. Water-soluble tomato concentrate effects on aggregation were tested ex vivo. Data are mean ± standard deviation (n = 11). *P < 0.05, **P < 0.01 versus adenosine diphosphate group

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Table 1: Effects of water-soluble tomato concentrate on coagulation in rats ex vivo (mean}standrad deviation, n=11)

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Water-soluble tomato concentrate inhibited GP IIb/IIIa activation of platelet

Next, we performed the FIB binding assay to detect the effect of WSTC on GP IIb/IIIa activation. As shown in [Figure 3], ADP (10 μM) significantly increased platelet GP IIb/IIIa activation by 52.65% (P < 0.01). After incubation for 10 min with WSTC, the FIB binding rates decreased obviously, and the average inhibitory rates by WSTC were 11.15%, 17.47%, and 32.29% at concentrations of 0.06, 0.6, and 6 g/L, separately; a significant difference was observed at a concentration of 6 g/L [Figure 3]. These results indicated that WSTC partially inhibited the activation of GP IIb/IIIa. In addition, LY294002 at a concentration of 25 μM showed a significant inhibitory effect on GP IIb/IIIa activation by 38.19%.
Figure 3: Water-soluble tomato concentrate inhibited GP IIb/IIIa activity. Washed platelets were initially incubated with calcein AM (4 μM) for 1 h at room temperature in the dark. Calcein AM-platelets were pretreated with water-soluble tomato concentrate or vehicle (saline) for 10 min at 37°C in the dark. Then, the samples were transferred into a 96-well black plate preincubated with 250 μg/mL fibrinogen overnight at 4°C. The plate was further blocked with 1% bovine serum albumin for 1 h and washed with Ca2+-free HEPES/Tyrode buffer. Platelet binding to fibrinogen was induced by adenosine diphosphate (10 μM) for 15 min and assessed by fluorescence detection. Unbound platelets were washed out with Ca2+-free HEPES/Tyrode buffer. The total platelets were only labeled with calcein AM without fibrinogen addition. The binding rate was determined as the percentage of bound platelets among total platelets. Data are mean ± standard deviation (n = 3).##P < 0.01 versus control; *P < 0.05, **P < 0.01 versus adenosine diphosphate group,△△P < 0.01 group comparisons

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Effects of water-soluble tomato concentrate on platelet aggregation-related proteins

Western blot results demonstrated that WSTC treatment decreased platelet protein levels of PI3K110 β, PDI, and PECAM-1 [Figure 4]a-c]. WSTC at concentrations of 0.6 and 6 g/L showed overt inhibitory effects on the protein level of these proteins. Treatment with WSTC at a concentration of 0. 6 g/L decreased PI3K110 β levels by 61.09% [Figure 4]a; WSTC at 6 g/L reduced PDI levels by 58.75% accordingly, and LY294002 showed overt inhibitory effects on PDI levels as well [Figure 4]b. The PECAM-1 protein was obviously down-regulated by WSTC treatment at concentrations of 0.06 g/L (P < 0.01) and 6 g/L (P < 0.05).
Figure 4: Inhibitory effects of water-soluble tomato concentrate on PI3K 110 β and protein disulfide isomerase. Washed platelets were pretreated with water-soluble tomato concentrate or saline for 10 min at 37°C. Fibrinogen binding to platelets was induced by adenosine diphosphate (10 μM) for 15 min. The protein levels of PI3K 110 β, protein disulfide isomerase, platelet endothelial cell adhesion molecule 1, and β1-tubulin were measured by Western blot. (a) The expression of PI3K 110 β, n = 3. (b) The expression of protein disulfide isomerase, n = 3. (c) The expression of platelet endothelial cell adhesion molecule 1, n = 4. (d) The expression of platelet endothelial cell adhesion molecule 1, n = 4. The data were given as the means ± standard deviation Data are mean ± standard deviation *P < 0.05, **P < 0.01 versus model group

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To elucidate the mechanism by which WSTC affects platelet aggregation, the β1-Tubulin protein was analyzed. The results [Figure 4]d showed that β1-Tubulin protein levels were greatly increased after pre-treatment with WSTC at 0.6 g/L (P < 0.05) and 6 g/L (P < 0.01). These results indicated that WSTC inhibited platelet aggregation by increasing cytoskeleton stability. No differences were found among the various WSTC concentrations for their effects on PI3K110 β, PDI, PECAM-1 and β1-Tubulin levels.

As shown in [Figure 5], ADP is considered one of the key agonists that trigger P2Y12 receptor, initiating a complex signaling cascade that ultimately results in platelet activation and aggregation. The activation of platelets is accompanied by a conformational change in GP IIb/IIIa, exposing the FIB binding site. WSTC inhibited GP IIb/IIIa activation. PDI could regulate the rearrangement of disulfide bonds into the ligand-competent state via its thiol isomerase activity to make GP IIb/IIIa switch to a high affinity form from a low affinity state. When the spatial configuration changes, GP IIb/IIIa binds to FIB and promotes aggregation. WSTC could downregulate the PDI protein and was greatly inhibited by LY294002 (PI3K inhibitor). PI3K is involved in both promoting and maintaining GP IIb/IIIa activation, necessary for stable thrombus growth, and PI3Kβ110 appears to play an important role in regulating GP IIb/IIIa function. Meanwhile, PECAM-1 is involved in the process of PI3K activation, and both PI3K and PECAM-1 are associated with the blood flow shear stress mechanosensory complex.[11] WSTC also reduced the protein levels of PI3K and PECAM-1. Thus, WSTC could inhibit ADP-mediated platelet aggregation and GP IIb/IIIa activation, likely by downregulating platelet PDI, PI3K, and PECAM-1.
Figure 5: The mechanism underlying water-soluble tomato concentrate's inhibitory effects on platelet aggregation. Water-soluble tomato concentrate inhibits adenosine diphosphate-mediated platelet aggregation and GP IIb/IIIa activation, likely via downregulation of platelet protein disulfide isomerase, phosphoinositide 3-kinase and platelet endothelial cell adhesion molecule 1

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  Discussion Top


Platelet activation and aggregation play a crucial role in physiological hemostasis and the process of thrombosis formation.[12] Platelet aggregation represents an important parameter which reflects the physiological function and pathological damage of platelets. Hyperactive platelets and increased platelet aggregation can be seen in patients with cardiovascular diseases and may induce thrombosis. Aspirin and clopidogrel, commonly used antiplatelet drugs, can significantly reduce the risk of cardiovascular events. However, because of “aspirin and/or clopidogrel resistance,” thrombotic events still occur in some patients undergoing antiplatelet therapy. Therefore it is important to identify alternative safe antiplatelet agents for vulnerable individuals with hyperactive platelets to reduce the risk of cardiovascular disease.[1]

Diets and nutrients play a potential role in altering platelet function. For instance, dark chocolate, garlic, red grape juice, omega-3 polyunsaturated fatty acids, ginger, onion, tomato, and wine reduce platelet aggregation in human subjects.[13],[14] Specifically, the antiplatelet effects of tomato have attracted increasing attention. Tomatoes, as an agricultural product, represent an important commodity worldwide and constitute a significant part of the human diet. Regular consumption of tomatoes and tomato products has been correlated with reduced risk of cardiovascular diseases and other positive effects.[15],[16] A previous study showed that tomato juice inhibits platelet aggregationin vitro significantly, possibly via the interruption of the interaction between platelet FIB receptor and FIB.[5] Tomato extracts include lipid-soluble and water-soluble extracts. A representative constituent of the lipid-soluble tomato extract is lycopene, which has a variety of biological functions beneflcial to humans such as antioxidation, antitumor, protection of cardiac vessels, and immunity enhancement.[17] WSTC containing bioactive components was developed and marketed globally as Fruitflow®. It was demonstrated that WSTC could effectively inhibit platelet aggregation in human subjects bothin vitro and in vivo,[3],[4],[5],[18] but the molecular mechanisms of WSTC effects on platelets have not been studied directly and clearly. Therefore, this study focused on the mechanisms of WSTC's antiplatelet activity in an animal model.

Platelets could be activated and aggregated through various physiological stimuli, such as ADP, collagen, thrombin, and shear stress.[19] Many traditional Chinese medicine (TCM) preparations show antiplatelet effects, such as Andrographolide (active component of Andrographis Paniculata), Bupleurumin (aerial parts of Bupleurum falcatum), Tanshinone IIA, and crude aqueous extract of parsley.[20] Psm2, one of the pyrrolidinoindoline alkaloids isolated from whole Selaginella moellendorffi plants, dose-dependently inhibits human platelet aggregation induced by ADP and collagen with IC50 values of 0.64 and 0.87 g/L, respectively.[21] A previous study showed that tomato has the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple display little or no activity. Tomato extract (20–50 mL of 100% juice) inhibits both ADP-and collagen-induced aggregation by up to 70%.[5] Another study also demonstrated that tomato aqueous extract shows significantly inhibition of ADP (10 μM) and collagen (4 mg/L)-induced aggregation of human platelets in vitro, with IC50 values of 2.0 g/L and 2.7 g/L, respectively.[3] Our results [Figure 1] showed that WSTC inhibited ADP and collagen-induced platelet aggregation significantlyin vitro in a concentration-dependent manner, with IC50 values of 3.05 g/L and 8.03 g/L, respectively. After oral consumption of WSTC for 4 weeks, ADP-induced platelet aggregation ex vivo was reduced markedly, and average inhibition rates were 24.42%, 21.48%, and 20.87%, respectively, under the doses of 25 mg/Kg, 75 mg/Kg, and 150 mg/Kg; Clopidogrel at 7.5 mg/Kg also showed a significant reduction, with an average inhibition rate of 95.48% [Figure 2]. WSTC had no influence on coagulation function in rats after oral consumption for one week at the highest dose of 900 mg/Kg, which is approximately 180 times the clinical dose [Table 1].

The platelet GP IIb/IIIa complex mediates aggregation and is a member of the cytoadhesin family of receptors that bind adhesive proteins such as FIB, fibronectin, and von Willebrand factor.[22] Platelet aggregation and thrombus formation start with platelet–platelet cross-linking of FIB bound to activated GP IIb/IIIa with a high-affinity.[23] Then, the conformational change in the GP IIb/IIIa complex leading to FIB exposure indicates GP IIb/IIIa activation. Thus, we evaluated GP IIb/IIIa activation in rat platelets by the FIB binding assay using fluorescence. Previous studies have shown that blockade of platelet GP IIb/IIIa is a promising antiplatelet strategy.[24],[25],[26],[27],[28],[29],[30],[31],[32] Many natural products exert antiplatelet effects by inhibiting FIB binding to GP IIb/IIIa, such as 95% ethanol extract of Spatholobus suberectus,[33] indole-3-carbinol of cruciferous vegetables,[34] 15%–20% ethanol extract of aged garlic,[35] aqueous extract of Agrimonia Pilosa and so on.[36] Meanwhile, Ginseng (Korean Red Ginseng), the root of Panax ginseng Meyer, frequently used in traditional Oriental medicine, was found to inhibit FIB binding to GP IIb/IIIa via dephosphorylation of PI3K and Akt.[37] Our results showed that WSTC decreased FIB binding to GP IIb/IIIa by 17.47% and 32.29% at concentrations of 0.6 and 6 g/L, respectively, indicating the inhibitory effects of WSTC on GP IIb/IIIa activation [Figure 3]. It is particularly worth mentioning that stimulation of PI3-Kinases mediates IIb/IIIa activation,[28] and our results showed that FIB binding was greatly inhibited by LY294002 (PI3K inhibitor) at a concentration of 25 μM, with an average inhibition rate of 38.19%. PI3K is involved in both promoting and maintaining GP IIb/IIIa activation, necessary for stable thrombus growth, and PI3K signaling appears to play an important role in regulating GP IIb/IIIa function.[29] We thus preliminary suggest that WSTC efficacy in inhibiting ADP-stimulated platelet activation occurs via PI3K pathway modulation.

PI3Kα, β, and γ isoforms regulate platelet function,[31] and PI3Kβ that is generally studied in platelets appears to play an important role downstream of the GP IIb/IIIa receptor.[30],[31],[32] Tangeretin, a fl‚avonoid abundant in the peel of citrus fruits incorporated in traditional medicines in India and China, was shown to inhibit PI3K mediated signaling to suppress agonist-induced human platelet activation and aggregation.[38] This study demonstrated that WSTC inhibited PI3K110 β expression [Figure 4]a, corroborating previous reports showing that isoform-selective PI3K 110 β inhibitors prevent GP IIb/IIIa activation and adhesion, indicating PI3K 110 β is an important new target for antithrombotic therapy.[10] The above results suggested that WSTC functions by inhibiting ADP-stimulated platelet GP IIb/IIIa activation via PI3K 110 β suppression. A previous study showed that a redox-dependent mechanism is involved in PDI on endothelial cells and PI3K/Akt pathway activation.[39] This suggests a possible connection between PI3K and PDI in WSTC anti-platelet effects, which deserves further investigation.

PDI is a multifunctional protein of the thioredoxin superfamily. PDI mediates proper protein folding by oxidation or isomerization, and disrupts disulfide bonds by reduction; it also has chaperone and antichaperone activities. Although PDI localizes primarily to the endoplasmic reticulum, it is secreted and expressed on the cell surface.[7] Recent studies emphasized that the PDI protein is involved in the process of platelet activation, including adhesion, aggregation, and release.[8],[9],[40],[41] PDI could regulate disulfide bond formation into the ligand-competent state via its thiol isomerase activity to make GP IIb/IIIa switch to high from low affinity state. When the spatial configuration changes, GP IIb/IIIa could bind to FIB and promote thrombosis.[23],[24],[25],[26],[27],[28] Both endothelial αVβ3 and platelet β3 integrins are important for initial and sustained PDI accumulation, as well as platelet thrombus development after vascular injury.[41] Previous findings confirm PDI as a new antithrombotic agent, and quercetin-3-rutinoside (rutin), a key component of WSTC, inhibits thrombus formation by blocking PDI.[9] HPW-RX40, a chemical derivative of β-nitrostyrene, was found to prevent human platelet activation by attenuating cell surface PDI[42] and is considered a potential molecule for the development of novel antithrombotic agents. Moreover, a novel ERp57 (a member of PDI family) inhibitor considered an antiplatelet agent with potential roles in platelet aggregation was isolated from Danshen (Salvia miltiorrhiza), a well-known herb in TCM for treating cardiovascular diseases.[43] The current results first showed that PDI expression was downregulated directly by WSTC. An interesting finding of this study is that PDI was significantly downregulated by LY294002 [Figure 4]b. This indicates that WSTC might inhibit ADP-stimulated platelet GP IIb/IIIa activation also via PDI inhibition.

PECAM-1; CD31, a member of the immunoglobulin superfamily, is highly expressed in endothelial cells and platelets.[44] PECAM-1 is the key factor in platelet adhesion and aggregation. Each quiescent platelet expresses about 8000 PECAM-1 molecules, and activated platelets in thrombosis express twice as much PECAM-1.[45] Schwartz MA reported that PECAM-1, vascular endothelial (VE)-cadherin, and VE growth factor receptor 2 constitute a mechanosensory complex in endothelial cells, with PECAM-1−/− endothelial cells showing no PI3K activation.[11] This suggests a correlation between PECAM-1 and PI3K activation, and PI3K most likely leads to GP IIb/IIIa activation through a conserved pathway.[10] In a word, the current results indicated that WSTC inhibits ADP mediated platelet aggregation and GP IIb/IIIa activation by downregulating PDI/PI3K and controlling PECAM-1 [Figures 4c and 5].

The cytoskeletal network defines the cell shape and local membrane architecture, and provides strength and stability for cell/cell and cell/matrix interactions. In the cardiovascular system, it is clear that cytoskeleton membrane proteins are essential for cellular function.[46] β1-tubulin is one of the platelet cytoskeleton proteins, and serves as a potential target of curdione, which attenuates thrombin-induced human platelet activation.[47] The present data showed that WSTC greatly enhanced β1-tubulin expression, with increased platelet skeletal stability [Figure 4]d, providing further evidence to support the emerging notion of antiplatelet effects of WSTC.


  Conclusion Top


The above findings showed that WSTC inhibits ADP and collagen-induced platelet aggregation in vitro, in a concentration-dependent manner, and significantly suppresses ADP-induced platelet aggregation ex vivo without affecting the coagulation system in rats. In addition, we found that WSTC inhibits platelet activation and aggregation through modulation of platelet skeletal stability and signaling downstream of the integrin GP IIb/IIIa, likely via the PDI/PI3Kβ110/PECAM-1 signaling pathway.

Statistical analysis

Data are mean ± standard deviation All statistical analyses were performed with the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and assessed by one-way ANOVA to test for synergism; a least significant difference post hoc test was used to evaluate differences for each pair of factor levels. P < 0.05 was considered as statistically significant.

Acknowledgments

This work was financially supported by the China Academy of Chinese Medical Sciences (ZZ11-044).

Financial support and sponsorship

This work was financially supported by the China Academy of Chinese Medical Sciences (ZZ11-044).

Conflicts of interest

There are no conflicts of interest.



 
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