Antiplatelet Aggregation Effect By Water Soluble Tomato Concentrate

PurposeTo investigate the antiplatelet aggregation effect of water soluble tomato concentrate(WSTC).BackgroundPlatelet activation at sites of vascular injury is essential for the arrest of bleeding;however,excessive platelet activation at regions of atherosclerotic plaque rupture or turbulent blood flow can result in the development of platelet thrombi,precipitating diseases such as acute myocardial infarction and ischemic stroke.Platelets in individuals with diabetes,sedentary life style,obesity and insulin resistance show hyperactivity at baseline and in response to agonists,ultimately leading to increased aggregation and plaque development.Hyperactive platelets are important mediators of atherogenesis in addition to their roles in thrombosis.However,antiplatelet drugs,such as aspirin or clopidogrel with prolong bleeding time,may not suitable for those with relatively low risk of cardiovascular events.It is of great importance to find more safe antiplatelet agents for those who has hyperactive platelets in order to prevent cardiovascular disease occurrence and development.It is recognized that some dietary components have the potential to reduce levels of specific risk factors for cardiovascular disease.There was compelling evidence for tomatoes to be considered as cardiovascular protective foods.Tomato(Lycopersicon esculentum)contains several components that are beneficial to overall health,including vitamin E,flavonoids,phytosterols,carotenoids,several water-soluble vitamins and minerals.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 compounds of WSTC were monitored from raw material to final product.The previous work reported that WSTC can influence platelet activity in vitro and ex vivo in human subjects.In healthy subjects,administration with tomato extract resulted in a reduction of 20.0±4.9%in platelet aggregation induced by ADP.In this study,we hypothesize that platelets PI3K p110β isoform might be the key signaling pathway for WSTC which helps maintain normal platelet aggregation contributing to healthy blood flow.Thereafter the following experiments were carried out to investigate the effect of WSTC on rat platelet aggregation and the possible signaling of WSTC on PDI and PI3K linked with blood flow.Methods1.Effects of WSTC on platelet aggregation in vitroRats underwent anesthesia by 3.0%sodium pentobarbital 60 mg·kg-1,and 8ml whole blood samples were withdrawn from abdominal aorta per rat with 109 mmol·L-1 sodium citrate(1:9)anti-coagulation.Platelet-rich plasma(PRP)was obtained following blood sample centrifuged at 200×g for 10 min.The PRP samples were again centrifuged at 200O×g for 10 min to obtain platelet-poor plasma(PPP).The mean platelets number in PRP was adjusted to 4.0× 1011 platelets/L by PPP for the aggregation assay.Platelets were counted by both microscope(Olympus BH-2)counting manually and Sysmex poch 100i counting automatically.All platelet preparations were conducted at room temperature.Aggregation was monitored by measuring light transmission via an aggregometer(LBY-NJ4,Tech link.Beijing China).PRP with 4.O×1011 platelets per liter were pre-incubated at 37℃ for 5 min with either WSTC or vehicle(saline),and stimulated by 2.5 μM ADP at 37 0C for 5 min.Three independent experiments were carried out and the same in the following.2.Ex vivo effects of WSTC on platelet aggregationPlatelet aggregation was examined as described above.Fifty-five male Sprague-Dawley rats,weight 210 ± 5 g were allotted into five groups.The control group rats were received distilled water.The positive control rats were given clopidogrel 7.5 mg·kg-1 once per day for 4 weeks.The WSTC group rats were orally administrated WSTC 25 mg·kg-1.75 mg·kg-1 and 150 mg·kg-1 in parallel.Blood samples were collected 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 rates were tested ex vivo3.Study on the effect of WSTC on the coagulation function of normal ratsFifty-five male Sprague-Dawley rats,weight 160±5 g were allotted into four treatment groups and an additional distilled water group randomly.WSTC were given to the rats orally 150 mg·kg-1,300 mg·kg-1 or 900 mg·kg-1 body weight once per day for 1 week.Blood was collected 120 min after administration of the last dose,and PPP were prepared as described above.Anti-coagulation activity of WSTC was evaluated by measuring plasma coagulation of PPP.The TT,FIB,APTT and PT were measured with a slight modification using a semi-automatic blood coagulation instrument(C2000-4,PRECIL).4.Effect of WSTC on the activation of platelet GP Ⅱb/ⅢaWashed platelets were initially incubated with Calcein AM(4 μM)for 1h at room temperature in the dark.Calcein AM-platelets were pre-treated with the WSTC or vehicle(saline)for 10 min at 37℃ in the dark.Then,the samples were transferred into a 96-well black plate which was pre-incubated with 250 μg·mL-1 fibrinogen(Thermo)overnight at 4℃with blocking of nonspecific binding by 1%BSA for 1 h at 37℃ after washing once time with 0.1 mL Ca2+-free Hepes/Tyrode buffer.Fibrinogen binding to platelets induced by ADP(10 μM)for 15 min was determined by fluorescence detection using a spectrofluorometer(SpectraMax i3x,Molecular,USA).5.Western Blot assayWashed platelets of rats were incubated with saline,WSTC(0.06 g·L-1,0.60g L-1,6.00 g·L-1),asprin(2 mM),rutin(6 mM)or LY294002(25 μM)for 10 minutes and stimulated by 10 μM ADP for 10 minutes both at 37℃ then platelets were obtained by centrifuging for 10 min at 1200×g,followed lysed in RIPA buffer containing 1 mM PMSF.After centrifugation for 5 min at 12 000 r·min-1 at 4℃,the protein concentration in the supernatant was determined with a BCA assay kit(Thermo,USA)according to the manufacturer’s instruction.The expression of PI3K p110β,PDI,PEC AM-1 and β1-tubulin was detected by western blot.Results1.Effects of WSTC on platelet aggregation in vitro and ex vivo.The effects of WSTC on ADP induced platelet aggregation were assessed using a light transmission method.Results shown in Figure 1,ADP of 2.5μM mediated platelet aggregation was obviously inhibited by WSTC with the average inhibitory rate of 64.02%,concentration of 6.00 g·L-1,and the 50%inhibitory concentrations(IC50)of WSTC was 3.46 g·L-1.2.Ex vivo effects of WSTC on platelet aggregation and blood coagulationThe effects of WSTC on ADP induced platelet aggregation after four weeks oral administration in rats are shown in Figure2.WSTC significantly inhibited the ADP-induced platelet aggregation ex vivo compared with the model group(P<0.01).The average inhibitory rate was 24.42%,21.48%and 20.87%respectively,under doses of 25,75 and 150mg·Kg-1 in rats.Clopidogrel at dose of 7.5mg·kg-1 inhibited ADP induced platelet aggregation by 95.47%.The influences of WSTC on coagulation system in rats were also evaluated by FIB,TT,APTT and PT assay.The coagulation system was not influenced by WSTC at concentrations from 150 to 900 mg·kg-1.3.Effects of WSTC on GPⅡb/Ⅲa activation of plateletFurthermore,we used fibrinogen binding assay to detect the effect of WSTC on the activation of GP Ⅱb/Ⅲa.Results showed shown ADP(10μM)significantly stimulated platelets GP Ⅱb/Ⅲa activation by 52.65%(P<0.01).After incubation 15min with WSTC,the fibrinogen binding rates decreased obviously,and the average inhibitory rate by WSTC was 11.15%,17.47%and 32.29%at concentrations of 0.06 g·L-1,0.60 g·L-1 and 6.00 g·L-separately.The results indicated that the WSTC partially inhibited the activation of GPⅡb/Ⅲa.In addition,LY294002 at concentration of 25μM showed significantly inhibitory effect on GP Ⅱb/Ⅲa activation by 38.19%.4.Effects of WSTC on platelet aggregation related protein expressionThe western blot results demonstrated that WSTC treatment decreased platelets protein expression of PI3K p110β,PDI and PECAM-1.WSTC at concentrations of 0.60and 6.00 g·L-1 showed obvious inhibitory effect on the expression of these proteins.Treatment with WSTC at concentration of 0.60 g·L-1 decreased PI3K p110β expression by 61.09%;WSTC at concentration of 6.00 g·L-1 reduced PDI expression by 58.75%accordingly,and LY294002 showed the visibly inhibitory effect on the PDI expression either.PECAM-1 protein was obviously down-regulated by WSTC treatment at concentration of 0.06 g·L-1(P<0.01)and 6.00 g· L-1(P<0.05).In order to elucidate of WSTC on the defonmation of platelet,β1-tubulin protein was analyzed.The result showed that β1-tubulin protein levels were greatly increased under the pre-treatment of WSTC in 0.60 g·L-1(P<0.05)and 6.00 g·L-1(P<0.01).These results indicated that WSTC inhibited platelet aggregation by increasing cytoskeleton stability.Conclusion1.WSTC inhibited ADP induced platelet aggregation in vitro and significantly inhibited platelet aggregation ex vivo without affecting the coagulation system in rats.2.WSTC inhibited platelet activation and aggregation through modulation platelet skeletal stability and signalling downstream of the integrin GP Ⅱb/Ⅲa,which might be via PI3K p-110β/PDI/PECAM-1 signaling pathway.