A number of epidemiological studies have shown that tomato consumption was positively correlated with cardioprotection . Some studies suggested that the cardioprotective effects of tomatoes may arise in part due to lycopene . However, other studies have reported that dietary lycopene is not associated with the risk of cardiovascular disease (CVDs) . Therefore, there may be unidentified components in tomatoes that possess cardiovascular protective effects. In vitro studies have found that platelet aggregation was inhibited after the treatment of tomato juice, the main components of its anti-platelet function are water-soluble . In order to further clarify the protective effect of non-fat-soluble components in tomatoes on cardiovascular system, Fruitflow was obtained by physically removing fat-soluble components from tomatoes . Fruitflow was shown to possess the inhibitory effects on platelet aggregation and platelet glycoprotein IIb/IIIa expression in vitro and in vivo [22, 23]. However, the effects of Fruitflow on platelet granule secretion have not yet been investigated. Our present study demonstrated that Fruitflow inhibited the thrombin/collagen-induced platelet α-granule, dense granule, and lysosome secretion in human gel-filtered platelets in vitro. To the best of our knowledge, this study provides the first link between tomato extract Fruitflow and platelet granule secretion.
Platelet granule secretion was activated after the stimulation of different agonists, e.g., thrombin and ADP. Collagen is a common sub-endothelial matrix protein that is exposed to platelets when the vessel endothelium is damaged , thrombin is a physiological platelet agonist that exist in the circulation . Collagen and thrombin can bind to the platelet surface receptors GPVI and PARs, respectively, which initiates the activation of sarcoma tyrosine-protein kinase (Src) family members Lyn, Fyn and Src, and further phosphorylates Syk [14, 36]. Activation of Syk, in turn, promotes an intracellular signaling cascade that eventually leads to phosphorylation and activation of the LAT , resulting in the phosphorylation of PLCγ2 . PLCγ2 then acts as an adapter protein for PKC, which finally leading to the activation of downstream effectors and contribute to the platelet granule secretion . In order to clarify the mechanism of Fruitflow inhibiting platelet granule release, we investigated the activation of Src/PLCγ2/PKC signaling pathway. Our immunoblotting results showed that Fruitflow markedly inhibited Lyn expression and Src, Syk, LAT, PLCγ2, PKC phosphorylation. Moreover, we showed that GF109203X, a PKC inhibitor, has no significant additive effects on thrombin-induced platelet granule secretion when combined with Fruitflow, suggesting that PKC signaling pathway inhibition is probably one of the major mechanisms underlying the inhibition of Fruitflow on platelet granule secretion.
A lot of natural bioactive compounds exhibited platelet protection and each possesses multiple targets with pleiotropic and synergistic effects. For example, anthocyanins attenuated platelet activation and aggregation involving the signaling pathways of NF-κB, GPVI, PI3K/Akt, MAPK et al [40–42]; polyphenols improved the platelet function by suppressing ROS, PLCγ2/PKC, AMPK signaling pathways [43, 44]; Curcumin inhibited platelet reactivity through inhibition of thromboxane formation, Ca2+ and GPVI signaling [45, 46]. In our study, we only found Fruitflow may affect platelet granule secretion through platelet Src/PLCγ2/PKC signaling pathway, whether platelet granule release affected by Fruitflow through other signaling pathways remains unknown. We therefore need to explore multiple mechanisms that Fruitflow act on platelet granule release in our future study.
According to the HPLC results, Fruitflow contains three main active ingredients including nucleoside derivatives, phenolic conjugates, and flavonoid derivatives, the most representative among the three components are adenosine, chlorogenic acid, rutin respectively . The three substances can be used to quantify and evaluate the quality of Fruitflow and ensure that Fruitflow is as close to fresh tomatoes as possible. A large numerous of studies have already confirmed that nucleoside, polyphenols, flavonoid possessed the effects of platelet protection and improved platelet activation, adhesion, aggregation [47–49]. It is therefore reasonable to believe that Fruitflow’s effect on platelet granule secretion may be due to these three components, which components play the most important role remains to be further studied in the future.
Fruitflow is now authorized by EFSA for daily consumption with 150 mg in the format of powder, tablet or capsule. According to the results of pharmacokinetics, the maximum theoretical circulating concentration after ingestion of a single 150 mg dose of Fruitflow is 43 mg/L , we therefore used the dose of 20, 40, 80 mg/L in vitro experiment. These doses did not exert cytotoxic effects on platelets as measured by cytosolic LDH leakage (Fig. 4). As Fruitflow possessed a significant anti-platelet ability, care was taken to determine whether the intrinsic or extrinsic clotting pathways could be effected by Fruitflow alongside antiplatelet effects. Large numbers of trials proved that Fruitflow does not directly affect prothrombin time (PT) and thrombin clotting time (TCT) at any dose tested [50, 51]. Unlike many anti-platelet drugs, which irreversibly inhibit platelet function and affected the life span of circulating platelets, the antiplatelet effects of Fruitflow are not irreversible, or cumulative, and does not bring extra bleeding risks . In summary, Fruitflow is safe and reliable as a food supplement.