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Anti-platelet and anti-thrombogenic effects of shikimic acid in sedentary population

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Anti-platelet and anti-thrombogenic effects of shikimic acid in sedentary population

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Abstract

This ex vivo study was performed to evaluate the anti-platelet and anti-thrombogenic potential of shikimic acid (SA), a plant phenolic metabolite. Fasting blood samples were collected from 22 sedentary participants to analyse the effect of varying concentrations of SA (0.1 mM, 0.2 mM, 0.5 mM, 1 mM and 2 mM) on platelet surface-marker expression, platelet aggregation and biomarkers of thrombogenesis. Monocyte-platelet aggregates (CD14/CD42b) and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31), effective indicators of thrombus formation were evaluated. Procaspase-activating compound 1 (PAC-1) and P-selectin or CD62P were used to assess platelet activation-related thrombogenesis. Adenosine diphosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation to mimic the in vivo thrombogenic pathway. Platelet aggregation studies utilised both ADP and collagen as exogenous platelet agonists to target both P2Y1/P2Y12 and GPVI pathways of thrombus formation. It was observed with flow cytometry that SA produced a significant antiplatelet effect on PAC-1 (p = 0.03 at 2 mM) and CD62P (p = 0.017, p = 0.036 at 1 mM and 2 mM respectively) expression in addition to lowering monocyte-platelet aggregate formation (p = 0.013, p < 0.01 and p < 0.01 at 0.5 mM, 1 mM and 2 mM respectively). SA at 1 mM concentration reduced PECAM-1 expression (p = 0.035), signifying a reduction to endothelial leucocyte migration during thrombus growth. SA did not demonstrate a platelet aggregation inhibitory effect by targeting the GPVI collagen pathway but reduced ADP induced platelet aggregation at 2 mM concentration (p < 0.01 at 2 mM). The results suggest that SA, an active metabolite of polyphenol-rich food intake, could play an important role in reducing platelet activation, aggregation related thrombus formation and biomarkers of thrombogenesis in sedentary individuals.

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... Since shikimic acid is a natural compound isolated from the Japanese plant, Illicium verum [12], and seeds of Liquidambar styraciflua (sweetgum) abundant in North America [13] and Chinese star anise (Illicium verum), shikimic acid has been used as a base material for production of oseltamivir (Tamiflu) [14]. Also, though shikimic acid is known to have anti-diabetic [15], antibacterial [16], anti-inflammatory [17], analgesic [18], antioxidant [18], and anti-thrombogenic [19] effects, its hypolipogenic mechanism has never been reported. Thus, in the present study, hypolipogenic mechanism of shikimic acid was elucidated in HepG2 and Huh7 hepatocellular carcinoma HCC cells and 3T3-L1 adipocytes in association with AMPK/ACC and MID1IP1 signaling axis. ...
... Since shikimic acid is a natural compound isolated from the Japanese plant, Illicium verum [12], and seeds of Liquidambar styraciflua (sweetgum) abundant in North America [13] and Chinese star anise (Illicium verum), shikimic acid has been used as a base material for production of oseltamivir (Tamiflu) [14]. Also, though shikimic acid is known to have anti-diabetic [15], antibacterial [16], antiinflammatory [17], analgesic [18], antioxidant [18], and anti-thrombogenic [19] effects, its hypolipogenic mechanism has never been reported. Thus, in the present study, hypolipogenic mechanism of shikimic acid was elucidated in HepG2 and Huh7 hepatocellular carcinoma HCC cells and 3T3-L1 adipocytes in association with AMPK/ACC and MID1IP1 signaling axis. ...
Article
Full-text available
Although shikimic acid from Illicium verum has antioxidant, antibacterial, anti-inflammatory, and analgesic effects, the effect of shikimic acid on lipogenesis has not yet been explored. Thus, in the present study, hypolipogenic mechanism of shikimic acid was examined in HepG2, Huh7 and 3T3-L1 adipocyte cells. Shikimic acid showed weak cytotoxicity in HepG2, Huh7 and 3T3-L1 cells, but suppressed lipid accumulation in HepG2, Huh7 and 3T3-L1 cells by Oil Red O staining. Also, shikimic acid attenuated the mRNA expression of de novo lipogenesis related genes such as FAS, SREBP-1c, and LXR-α in HepG2 cells by RT-PCR analysis and suppressed the protein expression of SREBP-1c and LXR-α in HepG2 and 3T3-L1 cells. It should be noted that shikimic acid activated phosphorylation of AMP-activated protein kinase (AMPK)/Aacetyl-coenzyme A carboxylase (ACC) and reduced the expression of MID1 Interacting Protein 1 (MID1IP1) in HepG2, Huh7 and 3T3-L1 cells. Conversely, depletion of MID1IP1 activated phosphorylation of AMPK, while overexpression of MID1IP1 suppressed phosphorylation of AMPK in HepG2 cells. However, AMPK inhibitor compound c did not affect the expression of MID1IP1, indicating MID1IP1 as an upstream of AMPK. Taken together, our findings suggest that shikimic acid has hypolipogenic effect in HepG2 and 3T3-L1 cells via phosphorylation of AMPK/ACC and inhibition of MID1IP1 as a potent candidate for prevention or treatment of fatty liver and hyperlipidemia.
... Veach and co-workers [25] reported the anti-thrombogenic potential of SA through the inhibition of platelet activation and aggregation by targeting the P2Y1/P2Y12-ADP pathway using ex vivo human blood sample. This study demonstrated the reduction of PAC-1 and P-selectin/CD62P expression, where both are the biomarkers of the various platelet activation. ...
... in vivo Male Swiss Mice anti-hyperalgesic Inhibit nociceptive behavior, inhibit inflammatory nociception, attenuate mechanical hyperalgesia [25] ex vivo Human blood sample Anti-platelet and anti-thrombogenic ↓PAC-1, ↓P-selectin/CD62P , ↓monocyte-platelet aggregate formation, ↓platelet aggregation [26] in vivo ICR mice and SD rats fibrinolytic activity ↓fibrin clot solution turbidity, acidic pH Anti-thrombosis Inhibit mouse tail thrombus formation, ↑survival rate, ↓thrombosis [27] in [32] in vivo Wistar normal rats Anti-hyperglycemic ↓plasma glucose, ↑serum insulin, ↑liver glycogen [33] in vivo Wistar STZ-diabetic rats Anti-hyperglycemic ↓plasma glucose [34] in ...
Article
The oil palm tree (Elaeis guineensis) from the family Arecaceae is a high oil-producing agricultural crop. A significant amount of vegetation liquor is discarded during the palm oil milling process amounting to 90 million tons per year around the world. This water-soluble extract is rich in phenolic compounds known as Oil Palm Phenolics (OPP). Several phenolic acids including the three isomers of caffeoylshikimic acid (CFA), p-hydroxybenzoic acid (PHBA), protocatechuic acid (PCA) and hydroxytyrosol are among the primary active ingredients in the OPP. Previous investigations have reported several positive pharmacological potentials by OPP such as neuroprotective and atheroprotective effects, anti-tumor and reduction in Aβ deposition in Alzheimer's disease model. In the current review, the pharmacological potential for CFA, PHBA, PCA and hydroxytyrosol is carefully reviewed and evaluated.
... Monocyte-platelet aggregate formation was reduced by 29% (p < 0.05) post four-week ACN supplementation when compared to PBO supplementation. In an ex vivo study evaluating the potential anti-thrombogenic effects of shikimic acid (a plant phenolic metabolite), monocyte-platelet aggregate formation was also inhibited (Veach, Hosking, Thompson, & Santhakumar, 2016). Research shows that elevated monocyte-platelet aggregates are strongly associated with high-risk pro-thrombotic populations (Hui, Fuller, Erber, & Linden, 2015). ...
... P-selectin expression was reduced by 9% (p < 0.05) post ACN supplementation in this study demonstrating a reduction in leucocyte recruitment and platelet activation dependent agranule release. In vitro studies have been performed demonstrating the effect of ACNs and its active in vivo metabolites in the inhibition of P-selectin expression hence alleviating platelet hyperactivity (Veach et al., 2016). In a dietary intervention trial performed on sedentary population, Santhakumar et al. also noted a reduction in P-selectin expression after 28-day supplementation with antioxidant richQueen Garnet plum juice (QGPJ) consequently reducing thrombotic risk by inhibiting a-granule release (Santhakumar, Kundur, Fanning, et al., 2015). ...
Article
The aim was to evaluate the effect of anthocyanin (ACN) supplementation in reducing thrombogenesis and maintaining hemostasis in pro-thrombotic overweight and obese individuals. Twenty-six (M = 9, F = 17) overweight/obese (BMI > 25) individuals participated in this randomized, double-blind, placebo-controlled, crossover design dietary intervention trial. Volunteers consumed ACN (320 mg/day) or placebo capsules for 28-days followed by a two-week wash-out period. ACN supplementation inhibited adenosine diphosphate (ADP)-induced platelet activation-related conformational change and degranulation by reducing PAC-1 expression by 12% and P-selectin expression by 9% respectively. ACN supplementation also alleviated thrombogenic progression by reducing monocyte-platelet aggregate formation by 29% and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression by 21%. Platelet aggregation induced by ADP, collagen and arachidonic acid was reduced by 36%, 17%, and 24% respectively. ACN supplementation has the potential to reduce the risk of thrombosis in overweight/obese population by targeting specific pathways of platelet activation/aggregation and endothelial dysfunction associated leucocyte migration.
... The compound lowered PAC-1 expression (> 1 mM); reduction of monocyte-platelet aggregate formation and whole-blood platelet aggregation stimulated by ADP but not by collagen (1-2 mM). [79] ( Table 3) Gardenia (Gardenia jasminoides) extract; geniposide (main constituent) was acquired from commercial sources and tested isolated. ...
... Veach et al. [79] performed an ex vivo study in sedentary patients to investigate the anticoagulant effects of shikimic acid (47), a phenolic metabolite. The acid reduced platelet aggregation induced by ADP and lowered procaspase-activating compound 1 (PAC-1) expression. ...
... The anthocyanin Cy-3-g is a member of the polyphenol family, which includes flavonoids, coumarin, shikimic acid, resveratrol, quercetin, and catechin (58)(59)(60)(61)(62)(63). Multiple studies have suggested that a diet high in polyphenol compounds may inhibit platelet aggregation (64,65). ...
Article
Background: Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Glycoprotein VI (GPVI) is a major platelet receptor that interacts with exposed collagen on injured vessel walls. Our previous studies have shown that anthocyanins (a type of natural plant pigment) attenuate platelet function; however, whether anthocyanins affect collagen-induced GPVI signaling remains unknown. Objective: The objective of this study was to explore the effects of cyanidin-3-glucoside (Cy-3-g, one of the major bioactive compounds in anthocyanins) on platelet activation and thrombosis and the GPVI signaling pathway. Methods: Platelets from healthy men and women were isolated and incubated with different concentrations (0, 0.5, 5, and 50 μM) of Cy-3-g. The expression of activated integrin αIIbβ3, P-selectin, CD63, and CD40L, fibrinogen binding to platelets, and platelet aggregation were evaluated in vitro. Platelet adhesion and aggregation in whole blood under flow conditions were assessed in collagen-coated perfusion chambers. Thrombosis and hemostasis were assessed in 3–4-wk-old male C57BL/6J mice through FeCl3-induced intravital microscopy and tail bleeding time. The effect of Cy-3-g on collagen-induced human platelet GPVI signaling was explored with Western blot. Results: Cy-3-g attenuated platelet function in a dose-dependent manner. The 0.5-μM dose of Cy-3-g inhibited (P < 0.05) human platelet adhesion and aggregation to collagen at both venous (−54.02%) and arterial (−22.90%) shear stresses. The 5-μM dose inhibited (P < 0.05) collagen-induced human platelet activation (PAC-1: −48.21%, P-selectin: −50.63%), secretion (CD63: −73.89%, CD40L: −43.70%), fibrinogen binding (−56.79%), and aggregation (−17.81%). The 5-μM dose attenuated (P < 0.01) thrombus growth (−66.67%) without prolonging bleeding time in mice. The 50-μM dose downregulated (P < 0.05) collagen-induced GPVI signaling in human platelets and significantly decreased phosphorylation of Syk–linker for activation of T cells (LAT)–SLP76 (Syk: −39.08%, LAT: −32.25%, SLP76: −40.00%) and the expression of Lyn (−31.89%), Fyn (−36.27%), and phospholipase C-γ2 (−39.08%). Conclusions: Cy-3-g inhibits human platelet activation, aggregation, secretion, and thrombus formation, and downregulates the collagen-GPVI signaling pathway. Supplementation of Cy-3-g may have protective effects against atherothrombosis.
... Another important aspect to consider in the pathophysiology of cardiovascular diseases is platelet hyper-activation associated thrombus development. An optimal platelet function, via the reduction of diet-based platelet hyperactivity, can be considered a viable alternative for the maintenance of cardiovascular health Erlund et al., 2008;Franzini et al., 2012;Santhakumar et al., 2014Santhakumar et al., , 2015aSanthakumar et al., , 2015bSanthakumar et al., , 2015cVeach et al., 2016;Yu et al., 2011). ...
Article
Epidemiological studies have demonstrated that nutritional habits, like those based on high consumption of fruits and vegetables, have been associated with a longer life expectancy and a significant decrease in the incidence and prevalence of several chronic diseases with inflammatory basis, such as cardiovascular diseases (CVD). This beneficial activity has been related to the content of several bioactive compounds in fruit and vegetables, such as polyphenols. The cardioprotective effects of polyphenols have been linked mainly to its antioxidant properties; however, recent findings attribute its anti-atherosclerotic potential to modulate simultaneous signaling and mechanistic pathways. Emerging data suggest that polyphenols can regulate cellular lipid metabolism; vascular and endothelial function; haemostasis; as well as platelet function; which represent primary conditions for atherosclerotic plaque formation and development. This review presents the results of a selection of experimental studies and clinical trials regarding the atheroprotective effects of the most common dietary polyphenols.
... 49 Shikimic acid (12), a key intermediate involved in most of biosynthesis of phenolic compounds of plant is also a promising antiplatelet and antithrombotic agent as confirmed by research performed with human blood of sedentary population. 50 Syringic acid (13), a product from the shikimate pathway, was found to attenuate the development of thrombosis and thromboembolism by inhibiting fibrin clot formation, coagulant factors, and platelet stimulation through DEP-1/PTP1B/aIIbb3/kinases. 51 A number of studies carried out with plant resources without fractionation or isolation of active components have demonstrated that polyphenol contents of materials are strongly correlated with their potential for antiplatelet and antithrombotic activity. ...
Article
Cardiovascular disease (CVD)-associated thrombosis are the most threatening cause of death worldwide. To resolve this crisis, efforts must be made to discover antithrombotic compounds for development of therapeutic agents with novel mechanisms of action, higher efficacy, and less adverse effects. It might also impact on the development of nutraceuticals to ameliorate vascular disease, and the development of appropriate diets to prevent CVDs. Recently, the research on antithrombotic compounds from edible natural sources increased swiftly by demand, and there has been remarkable progress. This review article covers bioactive components with antithrombotic activity that have been investigated in recent 5 years, more focused on the chemical structure classification (phenolics, polysaccharides, peptides, terpenes, and so on) and their original natural sources (traditional medicines, plants, animals, and foods).
... 53,54 The polyphenolic acid derivative shikimic acid at 10 À3 M moderately affected P2Y 12 -, but not GPVI-dependent platelet responses. 55 Stilbenoids that are regularly studied are the analogs isorhapontigenin and resveratrol, both of which are panassay interference compounds, hence linking to a wide variety of signaling pathways. This also holds for platelet research. ...
Article
Cardiovascular disease, in particular due to arterial thrombosis, is a leading cause of mortality and morbidity, with crucial roles of platelets in thrombus formation. For multiple plant-derived phytochemicals found in common dietary components, claims have been made regarding cardiovascular health and antiplatelet activities. Here we present a systematic overview of the published effects of common phytochemicals, applied in vitro or in nutritional intervention studies, on agonist-induced platelet activation properties and platelet signaling pathways. Comparing the phytochemical effects per structural class, we included general phenols: curcuminoids (e.g., curcumin), lignans (honokiol, silybin), phenolic acids (caffeic and chlorogenic acid), derivatives of these (shikimic acid) and stilbenoids (isorhapontigenin, resveratrol). Furthermore, we evaluated the flavonoid polyphenols, including anthocyanidins (delphinidin, malvidin), flavan-3-ols (catechins), flavanones (hesperidin), flavones (apigenin, nobiletin), flavonols (kaempferol, myricetin, quercetin), isoflavones (daidzein, genistein); and terpenoids including carotenes and limonene; and finally miscellaneous compounds like betalains, indoles, organosulfides (diallyl trisulfide) and phytosterols. We furthermore discuss the implications for selected phytochemicals to interfere in thrombosis and hemostasis, indicating their possible clinical relevance. Lastly, we provide guidance on which compounds are of interest for further platelet-related research.
... f. (Chinese star anise) have been traditionally used to treat skin inflammation and stomach aches (Rabelo et al. 2015). Shikimic acid is a plant polyphenolic compound known to protect against oxidative stress (Rabelo et al. 2015) and has antiplatelet and anti-thrombogenic effects (Veach et al. 2016). Other studies have shown that shikimate can cause a dose-dependent activation of the aryl hydrocarbon receptor, a ligand-activated transcription factor with important roles in multiple tissues, including the mucosal immune system (Sridharan et al. 2014). ...
Article
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BACKGROUND: There is intense debate on whether glyphosate can inhibit the shikimate pathway of gastrointestinal microorganisms, with potential health implications. OBJECTIVES: We tested whether glyphosate or its representative EU herbicide formulation Roundup MON 52276 affects the rat gut microbiome. METHODS: We combined cecal microbiome shotgun metagenomics with serum and cecum metabolomics to assess the effects of glyphosate [0.5, 50, 175 mg=kg body weight ðBWÞ per day] or MON 52276 at the same glyphosate-equivalent doses, in a 90-d toxicity test in rats. RESULTS: Glyphosate and MON 52276 treatment resulted in ceca accumulation of shikimic acid and 3-dehydroshikimic acid, suggesting inhibition of 5-enolpyruvylshikimate-3-phosphate synthase of the shikimate pathway in the gut microbiome. Cysteinylglycine, c-glutamylglutamine, and valylgly-cine levels were elevated in the cecal microbiome following glyphosate and MON 52276 treatments. Altered cecum metabolites were not differentially expressed in serum, suggesting that the glyphosate and MON 52276 impact on gut microbial metabolism had limited consequences on physiological biochemistry. Serum metabolites differentially expressed with glyphosate treatment were associated with nicotinamide, branched-chain amino acid, methionine, cysteine, and taurine metabolism, indicative of a response to oxidative stress. MON 52276 had similar, but more pronounced, effects than glyphosate on the serum metabolome. Shotgun metagenomics of the cecum showed that treatment with glyphosate and MON 52276 resulted in higher levels of Eggerthella spp., Shinella zoogleoides, Acinetobacter johnsonii, and Akkermansia muciniphila. Shinella zoogleoides was higher only with MON 52276 exposure. In vitro culture assays with Lacticaseibacillus rhamnosus strains showed that Roundup GT plus inhibited growth at concentrations at which MON 52276 and glyphosate had no effect. DISCUSSION: Our study highlights the power of multi-omics approaches to investigate the toxic effects of pesticides. Multi-omics revealed that glypho-sate and MON 52276 inhibited the shikimate pathway in the rat gut microbiome. Our findings could be used to develop biomarkers for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on humans. https://doi.org/10.1289/EHP6990
... The copyright holder for this preprint . is a plant polyphenolic compound known to protect against oxidative stress (Rabelo et al. 2015), and has anti-platelet and anti-thrombogenic effects (Veach et al. 2016). ...
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There is intense debate as to whether glyphosate can interfere with aromatic amino acid biosynthesis in microorganisms inhabiting the gastrointestinal tract, which could potentially lead to negative health outcomes. We have addressed this major gap in glyphosate toxicology by using a multi-omics strategy combining shotgun metagenomics and metabolomics. We tested whether glyphosate (0.5, 50, 175 mg/kg bw/day), or its representative EU commercial herbicide formulation MON 52276 at the same glyphosate equivalent doses, has an effect on the rat gut microbiome in a 90-day subchronic toxicity test. Clinical biochemistry measurements in blood and histopathological evaluations showed that MON 52276 but not glyphosate was associated with statistically significant increase in hepatic steatosis and necrosis. Similar lesions were also present in the liver of glyphosate-treated groups but not in the control group. Caecum metabolomics revealed that glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the shikimate pathway as evidenced by an accumulation of shikimic acid and 3-dehydroshikimic acid. Levels of caecal microbiome dipeptides involved in the regulation of redox balance (γ-glutamylglutamine, cysteinylglycine, valylglycine) had their levels significantly increased. Shotgun metagenomics showed that glyphosate affected caecum microbial community structure and increased levels of Eggerthella spp . and Homeothermacea spp.. MON 52276, but not glyphosate, increased the relative abundance of Shinella zoogleoides . Since Shinella spp. are known to degrade alkaloids, its increased abundance may explain the decrease in solanidine levels measured with MON 52776 but not glyphosate. Other glyphosate formulations may have different effects since Roundup® GT Plus inhibited bacterial growth in vitro at concentrations at which MON 52276 did not present any visible effect. Our study highlights the power of a multiomics approach to investigate effects of pesticides on the gut microbiome. This revealed the first biomarker of glyphosate effects on rat gut microbiome. Although more studies will be needed to ascertain if there are health implications arising from glyphosate inhibition of the shikimate pathway in the gut microbiome, our findings can be used in environmental epidemiological studies to understand if glyphosate can have biological effects in human populations. Graphical Abstract
... [7] Consequently, the use of novel plant-derived compounds as potential adjuvants for the treatment of these diseases, as proposed by numerous authors, [8][9][10][11] is experiencing increased research interest. [12,13] Previous reviews have highlighted the potential of naturally derived phenolic and antioxidant compounds for the treatment or prevention of various non-communicative diseases, [3,14,15] including age-related macular degeneration, [16][17][18] Alzheimer's disease, [19][20][21] Huntington's disease, [22,23] Parkinson's disease, [21,[23][24][25] atherosclerosis, [26] cardiovascular disease, [27][28][29][30] diabetes, [31,32] cancers [33][34][35][36][37] and some autoimmune disorders. [38][39][40][41] In addition, certain phenolic compounds show promise for the treatment of certain communicable diseases, including viral, [42,43] bacterial, [44][45][46] fungal [44,47] and protozoal infections. ...
Article
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Chloromethylated cross-linked polystyrene (CMCPS) microsphere is hydroxylated, obtaining hydroxylated cross-linked polystyrene microsphere (CPS-OH). Methacrylic acid (MAA) is grafted on the surface of CPS-OH microspheres by cerium salt-hydroxyl group redox initiation system, obtaining graft-polymerization functional microsphere CPS-g-PMAA. The chemical structure and physicochemistry characters of CPS-g-PMAA are characterized with infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The adsorption performance of the grafted microspheres CPS-g-PMAA for shikimic acid was mainly investigated and the adsorption mechanism was explored. The experiment results show that for the graft-polymerization of MAA, the suitable concentration of MAA in solution is 3.2 mol/L, the optimal concentration of initiator cerium salt and catalyzer Sulfuric acid is 1.3 × 10⁻² and 0.3 1 mol/L, respectively, the appropriate temperature is 55 °C, and reaction time is 6 h, the grafting degree of PMAA can reach 43.3 g/100 g. The kinetics demonstrating adsorption kinetic of SA by CPS-g-PMAA is fitted PFO model in the first 50 min and is then fitted with the PSO model. The CPS-g-PMAA microsphere adsorbs SA via hydrogen bond interaction, the adsorption capacity reaches 92 mg/g in ethanol. The adsorption amount decreases with the increase of temperature, Freundlich isothermal model is fitted the adsorption experimental data.
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Several in vitro studies have proposed syringic acid as a benzoic acid derivative found in plant foods, which can reduces collagen-stimulated platelet aggregation. Nonetheless, to date, there are no reports elucidating the role of syringic acid in platelet activation and thrombosis. Syringic acid inhibited clot formation and procoagulant protease activity and induced blood clot degradation. Syringic acid decreased the expression of density-enhanced phosphatase-1 (DEP-1)/protein tyrosine phosphatase-1B (PTP1B)/αIIbβ3, as well as the phosphorylation of kinases in collagen/epinephrine-stimulated platelets both in vitro and in vivo. Moreover, syringic acid inhibited the secretion of granule constituents, clot retraction, and FeCl3-induced vascular occlusion of the carotid artery. Syringic acid (up to 100 μM) did not show any effect on the viability of RAW 264.7 cells. Our findings demonstrate that syringic acid attenuate the development of thrombosis and thromboembolism by inhibiting fibrin clot formation, coagulant factors, and platelet stimulation through DEP-1/PTP1B/αIIbβ3/kinases.
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The anti-thrombotic properties of anthocyanin (ACN) supplementation was evaluated in this randomised, double-blind, placebo (PBO) controlled, cross-over design, dietary intervention trial in sedentary population. In all, sixteen participants (three males and thirteen females) consumed ACN (320 mg/d) or PBO capsules for 28 d followed by a 2-week wash-out period. Biomarkers of thrombogenesis and platelet activation induced by ADP; platelet aggregation induced by ADP, collagen and arachidonic acid; biochemical, lipid, inflammatory and coagulation profile were evaluated before and after supplementation. ACN supplementation reduced monocyte-platelet aggregate formation by 39 %; inhibited platelet endothelial cell adhesion molecule-1 expression by 14 %; reduced platelet activation-dependant conformational change and degranulation by reducing procaspase activating compound-1 (PAC-1) (↓10 %) and P-selectin expression (↓14 %), respectively; and reduced ADP-induced whole blood platelet aggregation by 29 %. Arachidonic acid and collagen-induced platelet aggregation; biochemical, lipid, inflammatory and coagulation parameters did not change post-ACN supplementation. PBO treatment did not have an effect on the parameters tested. The findings suggest that dietary ACN supplementation has the potential to alleviate biomarkers of thrombogenesis, platelet hyperactivation and hyper-aggregation in sedentary population.
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Thrombosis is considered to be closely related to several diseases such as atherosclerosis, ischemic heart disease and stroke, as well as rheumatoid arthritis, hyperuricemia, and various inflammatory conditions. More and more studies have been focused on understanding the mechanism of molecular and cellular basis of thrombus formation as well as preventing thrombosis for the treatment of thrombotic diseases. In reality, there is considerable interest in the role of natural products and their bioactive components in the prevention and treatment of thrombosis related disorders. This paper briefly describes the mechanisms of thrombus formation on three aspects, including coagulation system, platelet activation, and aggregation, and change of blood flow conditions. Furthermore, the natural products for antithrombosis by anticoagulation, antiplatelet aggregation, and fibrinolysis were summarized, respectively.
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Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/ feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid ‘‘surge’’ capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the b2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.
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Platelet flow cytometry is an emerging tool in diagnostic and therapeutic hematology. It is eminently suited to study the expression of platelet surface receptors both qualitatively as well as quantitatively. It can serve as a useful marker for the documentation of in vivo platelet activation, and thus, fore-warn the risk of thromboembolism in patients with diabetes mellitus, coronary syndromes, peripheral vascular diseases, and pre-eclampsia. This technique can also be extended to study and compare the effect of various antiplatelet drugs on the level of activation of platelets and to establish any dose-effect relationship of these drugs. Topographical localization of platelet granules and study of platelet-platelet and platelet-leukocyte interaction is also possible by this procedure. All these parameters serve as pointers towards the presence of activated platelets in the circulation with its thromboembolic consequences. This is a simple reliable and cost effective technique which has a wide application in the diagnosis of various inherited and acquired platelet disorders. Study of platelet cluster of differentiation (CD) markers in various inherited disorders i.e. Bernard Soulier's disease, von Willebrand disease, Glanzman's disease, and Grey platelet syndrome may help categories the molecular lesions in these oft under-studied disorders.
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Epidemiological studies indicate that the higher intake of fruits and vegetables may reduce the risks of many degenrative diseases like cancer, cardiovascular disease, cataract etc. This is attributed mainly to the intake of dietary polyphenols as seen in Mediterranean diets. However, the bioavailability of polyphenols is reported to be low due to poor absorption in the gut, intestine and colon and depends on the type of compounds, chemical structure, food matrix, extent of conjugation and individual colon microflora. In general, flavonoids, aglycones and pure compounds are absorbed more when compared to the glycosides. Diversity in intestinal mocroflora also contributes to a great extent for the variation in absorption of polyphenols as seen in a few studies for the absorption of isoflavones. Among the polyphenols, isoflavones are known to be more bioavailable followed by phenolic acids, flavanols, flavanones, flavonols and lowest bioavailability was seen for anthocyanins and proanthocyanidins. Many human and animal studies have shown that dietary polyphenols reduce the cardiovascular diseases by inhibiting LDL oxidation, promoting vasodilation and by antiplatelet properties. However, the relationship between the level of polyphenols in plasma and their in vivo cardioprotective effects are poor. It is clear that more studies with improved methods are needed to understand the involvement of polyphenols in reducing the risks of degenerative diseases. In order to translate the in vitro results to in vivo, bioavailability of dietary polyphenols have to be increased significantly. One way to achieve this would be to study food preparation methods that can increase the bioavailability of these compounds through the use of different additives, cooking methods, enzymes and microorganisms. Microorganisms appear to play an important role in increasing the bioavailability of polyphenols by removing the conjugation and by breaking polyphenols into simpler absorbable phenols. We feel more bacteria and fungi should be used in food preparations such as yeasts for bread and wine making; lactic acid bacteria for fermenting idlies, dosas, curds; and Bacillus strains for soy fermentation products to increase bioavalability of polyphenols. Enzyme treated or microbial digested food may become the future of food industry. This article was written with a view to supplement Dr. R. B. Singh's life long ambition to prevent cardio-vascular diseases through the use of diet and discuss the above points in greater detail.
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The goal of this review is to briefly summarize the two primary pathways of hemostasis, primary hemostasis and secondary hemostasis, as well as to summarize anticoagulant mechanisms and fibrinolysis. In addition, this review will discuss pathologies of hemostasis and the mechanisms of the various drugs that are available to impact these pathways to prevent either thrombosis or bleeding. While many of the main drugs that are used to treat disorders of hemostasis have been used for decades, greater understanding of hemostasis has led to development of various new drugs that have come onto the market recently or are close to coming onto the market. Thus, improved understanding of hemostasis continues to lead to benefits for patients.
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Epidemiological evidence suggests that polyphenols may, in part, explain the cardioprotective properties of fruits. This review aims to summarise the evidence for the effects of fruit polyphenols on four risk factors of CVD: platelet function, blood pressure, vascular function and blood lipids. This review includes human dietary intervention studies investigating fruits and their polyphenols. There was some evidence to suggest that fruits containing relatively high concentrations of flavonols, anthocyanins and procyanindins, such as pomegranate, purple grapes and berries, were effective at reducing CVD risk factors, particularly with respect to anti-hypertensive effects, inhibition of platelet aggregation and increasing endothelial-dependent vasodilation than other fruits investigated. Flavanone-rich fruits, such as oranges and grapefruits, were reported to have hypocholesterolaemic effects, with little impact on other risk factors being examined. However, the evidence was limited, inconsistent and often inconclusive. This is in part due to the heterogeneity in the design of studies, the lack of controls, the relatively short intervention periods and low power in several studies. Details of the polyphenol content of the fruits investigated were also omitted in some studies, negating comparison of data. It is recommended that large, well-powered, long-term human dietary intervention studies investigating a wider range of fruits are required to confirm these observations. Investigations into the potential synergistic effects of polyphenols on a combination of CVD risk markers, dose-response relationships and standardisation in methodology would facilitate the comparison of studies and also provide valuable information on the types of fruits which could confer protection against CVD.
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Platelets are central mediators of haemostasis at sites of vascular injury, but they also mediate pathologic thrombosis. Activated platelets stimulate thrombus formation in response to rupture of an atherosclerotic plaque or endothelial cell erosion, promoting atherothrombotic disease. They also interact with endothelial cells and leukocytes to promote inflammation, which contributes to atherosclerosis. Multiple pathways contribute to platelet activation, and current oral antiplatelet therapy with aspirin and a P2Y(12) adenosine diphosphate (ADP) receptor antagonist target the thromboxane A(2) and ADP pathways, respectively. Both can diminish activation by other factors, but the extent of their effects depends upon the agonist, agonist strength, and platelet reactivity status. Although these agents have demonstrated significant clinical benefit, residual morbidity and mortality remain high. Neither agent is effective in inhibiting thrombin, the most potent platelet activator. This lack of comprehensive inhibition of platelet function allows continued thrombus formation and exposes patients to risk for recurrent thrombotic events. Moreover, bleeding risk is a substantial limitation of antiplatelet therapy, because these agents target platelet activation pathways critical for both protective haemostasis and pathologic thrombosis. Novel antiplatelet therapies that provide more complete inhibition of platelet activation without increasing bleeding risk could considerably decrease residual risk for ischemic events. Inhibition of the protease-activated receptor (PAR)-1 platelet activation pathway stimulated by thrombin is a novel, emerging approach to achieve more comprehensive inhibition of platelet activation when used in combination with current oral antiplatelet agents. PAR-1 inhibition is not expected to increase bleeding risk, as this pathway does not interfere with haemostasis.
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Hemostasis and pathological thrombus formation are dynamic processes that require a co-ordinated series of events involving platelet membrane receptors, bidirectional intracellular signals, and release of platelet proteins and inflammatory substances. This review aims to summarize current knowledge in the key steps in the dynamics of thrombus formation, with special emphasis on the crucial participation of platelet receptors and signaling in this process. Initial tethering and firm adhesion of platelets to the exposed subendothelium is mediated by glycoprotein (GP) Ib/IX/V complex and collagen receptors, GP VI and alpha(2)beta(1) integrin, in the platelet surface, and by VWF and fibrillar collagen in the vascular site. Interactions between these elements are largely influenced by flow and trigger signaling events that reinforce adhesion and promote platelet activation. Thereafter, soluble agonists, ADP, thrombin, TxA(2), produced/released at the site of vascular injury act in autocrine and paracrine mode to amplify platelet activation and to recruit circulating platelets to the developing thrombus. Specific interactions of these agonists with their G-protein coupled receptors generate inside-out signaling leading to conformational activation of integrins, in particular alpha(IIb)beta(3), increasing their ligand affinity. Binding of alpha(IIb)beta(3) to its ligands, mainly fibrinogen, supports processes such as clot retraction and platelet aggregation. Stabilization of thrombi is supported by the late wave of signaling events promoted by close contact between aggregated platelets. The best known contact-dependent signaling is outside-in signaling through alphaIb beta(3), but new ones are being clarified such as those mediated by interaction of Eph receptors with ephrins, or by Sema 4D and Gas-6 binding to their receptors. Finally, newly identified mechanisms appear to control thrombus growth, including back-shifting of activated integrins and actuation of compensatory molecules such as ESAM or PECAM-1. The expanding knowledge of thrombotic disease is expected to translate into the development of new drugs to help management and prevention of thrombosis.
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Because shikimic acid is the key intermediate in the shikimate pathway in plants and microorganisms, shikimic acid and its derivatives have been described as herbicides and anti-microbial agents. Triacetylshikimic acid (TSA) is an acetylate derivative of shikimic acid. The possible anti-platelet activity and anti-thrombotic efficacy of TSA were evaluated and its effect on arachidonic acid (AA) metabolism and second messengers including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) was evaluated. After oral pretreatment with TSA, adenosine diphosphate (ADP)-, collagen-, and AA-induced rat platelet aggregation was inhibited ex vivo in a dose-dependent manner. In an arteriovenous-shunt thrombosis model, oral administration of TSA resulted in a dose-dependent inhibition of thrombus growth. TSA markedly increased the cAMP level and showed no effect on the cGMP level in rat platelets. Also, no significant changes in ADP-induced thromboxane B2 formation in rat platelets or 6-keto-prostaglandin F 1alpha production from the abdominal aorta were observed after oral administration of low and medium doses of TSA (12.5 and 50 mg/kg). Additionally, prothrombin time, activated partial thromboplastin time, and thrombin time were unchanged at effective anti-platelet doses of TSA. These results demonstrate that TSA exerts oral anti-platelet and anti-thrombotic efficacy without perturbation of systemic hemostasis in rats, which was partially concerned with the elevation of cAMP in platelets.
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Polyphenols are abundant micronutrients in our diet, and evidence for their role in the prevention of degenerative diseases such as cancer and cardiovascular diseases is emerging. The health effects of polyphenols depend on the amount consumed and on their bioavailability. In this article, the nature and contents of the various polyphenols present in food sources and the influence of agricultural practices and industrial processes are reviewed. Estimates of dietary intakes are given for each class of polyphenols. The bioavailability of polyphenols is also reviewed, with particular focus on intestinal absorption and the influence of chemical structure (eg, glycosylation, esterification, and polymerization), food matrix, and excretion back into the intestinal lumen. Information on the role of microflora in the catabolism of polyphenols and the production of some active metabolites is presented. Mechanisms of intestinal and hepatic conjugation (methylation, glucuronidation, sulfation), plasma transport, and elimination in bile and urine are also described. Pharmacokinetic data for the various polyphenols are compared. Studies on the identification of circulating metabolites, cellular uptake, intracellular metabolism with possible deconjugation, biological properties of the conjugated metabolites, and specific accumulation in some target tissues are discussed. Finally, bioavailability appears to differ greatly between the various polyphenols, and the most abundant polyphenols in our diet are not necessarily those that have the best bioavailability profile. A thorough knowledge of the bioavailability of the hundreds of dietary polyphenols will help us to identify those that are most likely to exert protective health effects.
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Increasing evidence suggests that polyphenols from fruits, vegetables and beverages such as wine and tea may exert protective effects on the cardiovascular system. Indeed, research in the field of polyphenols points out their antioxidant and free radical scavenging properties, leading to lower low-density lipoprotein (LDL) oxidation and platelet aggregation. These compounds are also able to modulate the generation of nitric oxide (NO) from vascular endothelium and to interfere with the mechanisms leading to inflammation and endothelial apoptosis, contributing to the prevention of the endothelial dysfunction, known to play a central role in the pathogenesis of cardiovascular diseases. This article reviews the potential targets of polyphenols involved in the complex pathophysiological events occurring in cardiovascular diseases, such as hypertension, atherosclerosis and stroke.
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Aim of this study was to evaluate the biotransformation of simple phenols after ingestion of edible fruits and mixed food. It was analyzed hippuric acid in urine as biomarker of conjugation in the liver cells of glycine with aromatic phenolic acids such benzoic and salicylic acid from ingested food. Measurement of hippuric acid in urine samples of 10 healthy individuals: 5 female and 5 male with a mean age 51,5 years were recruited to participate in this study. Urine samples were collected for 24 hours. The additional meals 300 g of fruits: blueberry, cherry, raspberry, melon, blackberry and mixed food were given immediately before the 24 hr urine sampling. Otherwise, the meals given during 24 hr was a usually food. Biotransformation of phenols in edible fruits, that are together with liver glycins precursors of hippuric acid biosynthesis, was evaluated by direct spectrophotometric measurement of excreted hippuric acid in urine at 410 nm. It was established that the highest quantity of hippuric acid was after ingestion of 300 g of bilberry fruits (p< 0,003), and same quantity of cherries (p< 0,003). Concentration of excreted hippuric acid was twice higher after ingestion of these fruits in comparison with hippuric acid concentrations in urine after ingestion of common - mixed food. Quantity of biosynthesised hippuric acid was in direct correlation with the concentrations of its precursors, primarily phenol acids and other simple aromatic acids ingested with food.
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We assessed the effects of guaraná (Paullinia cupana) consumption on plasma catechins, erythrocyte antioxidant enzyme activity (superoxide dismutase, catalase and glutathione peroxidase) and biomarkers of oxidative stress (ex vivo LDL oxidation, plasma total antioxidant status and ORAC, and lymphocyte single cell gel electrophoresis) in healthy overweight subjects. Twelve participants completed a 15-day run-in period followed by a 15-day intervention with daily intake of 3 g guaraná seed powder containing 90 mg (+)-catechin and 60 mg (-)-epicatechin. Blood samples were taken on the first and last day of the intervention period, at fasting and 1h post-dose. The administration of guaraná increased plasma ORAC, while reducing ex vivo LDL oxidation (only in the first study day) and hydrogen peroxide-induced DNA damage in lymphocytes, at 1h post-dose. Plasma catechin (0.38 ± 0.12 and 0.44 ± 0.18 nmol/mL), epicatechin (0.59 ± 0.18 and 0.64 ± 0.25 nmol/mL) and their methylated metabolites were observed at 1h post-dose but were almost negligible after overnight fasting. The activities of catalase (in both study days) and glutathione peroxidase (in the last intervention day) increased at 1h post-dose. Furthermore, the activity of both enzymes remained higher than basal levels in overnight-fasting individuals in the last intervention day, suggesting a prolonged effect of guaraná that continues even after plasma catechin clearance. In conclusion, guaraná catechins are bioavailable and contribute to reduce the oxidative stress of clinically healthy individuals, by direct antioxidant action of the absorbed phytochemicals and up-regulation of antioxidant/detoxifying enzymes.
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Platelet dysfunction, oxidative stress and dyslipidemia are important contributors to pro-thrombotic progression particularly in obese and hyper-cholesterolemic populations. Becoming an increasingly widespread endemic, obesity causes a dysfunction in the metabolic system by initiating endothelial dysfunction; increasing free radical production; lipid peroxidation; platelet hyperactivity and aggregation; thereby accelerating thrombogenesis. In the event of increased free radical generation under pro-thrombotic conditions, antioxidants act as scavengers in reducing physiological oxidative stress; free radical-mediated thrombosis and hemostatic function. Anthocyanin, a subclass of the polyphenol family flavonoids has been shown to exhibit anti-dyslipidemic and anti-thrombotic properties by virtue of its antioxidant activity. Current anti-platelet/coagulant therapeutics target specific receptor pathways to relieve the extent of dysfunction and plaque acceleration in pro-thrombotic individuals. Though effective, they have been associated with high bleeding risk and increased response variability. The following review focuses on the potential role of natural dietary anthocyanins in targeting simultaneous mechanistic pathways in alleviating platelet activation, dyslipidemia, and oxidative stress-associated thrombus acceleration in obese pro-thrombotic populations.
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Previous evidence suggested an active role for cocoa products and flavanols in modulating platelet aggregation. However, cocoa flavanols are characterized by a low bioavailability that can deeply affect their presence in biological fluids and raise questions on their biological effect in humans. We performed a systematic search on MEDLINE, EMBASE, COCHRANE and ProQuest Databases, until April 2015, on the effect of cocoa products on platelet aggregation in human intervention studies. We identified 13 interventions, of which only five involved repeated administration. Different effects were observed on the basis of the platelet aggregation test used, whereas neither a longer duration of treatment nor a higher dose were associated with a higher inhibition of platelet aggregation. In conclusion, the reviewed results suggest that consumption of cocoa products in bolus administration positively affects platelet aggregation in both healthy subjects and disease patients. On the other hand, more evidence is required in order to assess the effect of long-term cocoa products ingestion and to identify the bioactive components involved.
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The anti-thrombotic properties of an anthocyanin-rich Queen Garnet plum juice (QGPJ) and anthocyanin-free prune juice (PJ) were studied in this randomised, double-blind, crossover trial. Twenty-one healthy subjects (M = 10, F = 11) consumed QGPJ, PJ or placebo, 200 mL/day for 28-days followed by a 2-week wash-out period. Only QGPJ supplementation inhibited platelet aggregation induced by ADP (<5%, P = 0.02), collagen (<2.7%, P < 0.001) and arachidonic acid (<4%, P < 0.001); reduced platelet activation-dependent surface-marker P-selectin expression of activated de-granulated platelets (<17.2%, P = 0.04); prolonged activated-partial thromboplastin clotting time (>2.1 s, P = 0.03); reduced plasma-fibrinogen (<7.5%, P = 0.02) and malondialdehyde levels, a plasma biomarker of oxidative stress (P = 0.016). PJ supplementation increased plasma hippuric acid content (P = 0.018). QGPJ or PJ supplementation did not affect blood cell counts, lipid profile, or inflammation markers. Our findings suggest that QGPJ but not PJ has the potential to significantly attenuate thrombosis by reducing platelet activation/hyper-coagulability and oxidative stress.
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Dietary sources of polyphenols, which are derivatives and/or isomers of flavones, isoflavones, flavonols, catechins and phenolic acids, possess antioxidant properties and therefore might be important in preventing oxidative‐stress‐induced platelet activation and attenuating adverse haemostatic function. Free radicals, including reactive oxygen and nitrogen species, promote oxidative stress, leading to platelet hyperactivation and the risk of thrombosis. The consumption of antioxidant/polyphenol rich foods might therefore impart anti‐thrombotic and cardiovascular protective effects via their inhibition of platelet hyperactivation or aggregation. Most commonly‐used anti‐platelet drugs such as aspirin block the cyclooxygenase (COX)‐1 pathway of platelet activation, similar to the action of antioxidants with respect to neutralising hydrogen peroxide (H2O2), with a similar effect on thromboxane production via the COX‐1 pathway. Polyphenols also target various additional platelet activation pathways (e.g. by blocking platelet‐ADP, collagen receptors); thus alleviating fibrinogen binding to platelet surface (GPIIb‐IIIa) receptors, reducing further platelet recruitment for aggregation and inhibiting platelet degranulation. As a result of the ability of polyphenols to target additional pathways of platelet activation, they may have the potential to substitute or complement currently used anti‐platelet drugs in sedentary, obese, pre‐diabetic or diabetic populations who can be resistant or sensitive to pharmacological anti‐platelet therapy.
Article
Dietary sources of polyphenols, which are derivatives and/or isomers of flavones, isoflavones, flavonols, catechins and phenolic acids, possess antioxidant properties and therefore might be important in preventing oxidative-stress-induced platelet activation and attenuating adverse haemostatic function. Free radicals, including reactive oxygen and nitrogen species, promote oxidative stress, leading to platelet hyperactivation and the risk of thrombosis. The consumption of antioxidant/polyphenol rich foods might therefore impart anti-thrombotic and cardiovascular protective effects via their inhibition of platelet hyperactivation or aggregation. Most commonly-used anti-platelet drugs such as aspirin block the cyclooxygenase (COX)-1 pathway of platelet activation, similar to the action of antioxidants with respect to neutralising hydrogen peroxide (H2 O2 ), with a similar effect on thromboxane production via the COX-1 pathway. Polyphenols also target various additional platelet activation pathways (e.g. by blocking platelet-ADP, collagen receptors); thus alleviating fibrinogen binding to platelet surface (GPIIb-IIIa) receptors, reducing further platelet recruitment for aggregation and inhibiting platelet degranulation. As a result of the ability of polyphenols to target additional pathways of platelet activation, they may have the potential to substitute or complement currently used anti-platelet drugs in sedentary, obese, pre-diabetic or diabetic populations who can be resistant or sensitive to pharmacological anti-platelet therapy.
Article
The complex relationship between flavonoid-based nutrition and cardiovascular disease may be dissected by understanding the activities of these compounds in biological systems. The aim of the present study was to explore a hierarchy for the importance of dietary flavonoids on cardiovascular health by examining the structural basis for inhibitory effects of common, dietary flavonoids (quercetin, apigenin, and naringenin) and the plasma metabolite, tamarixetin. Understanding flavonoid effects on platelets in vivo can be informed by investigations of the ability of these compounds to attenuate the function of these cells. Inhibition of platelet function in whole blood and plasma was structure-dependent. The order of potency was apigenin > tamarixetin > quercetin = naringenin indicating that in vivo, important functional groups are potentially a methylated B ring, and a non-hydroxylated, planar C ring. Apigenin and the methylated metabolite of quercetin, tamarixetin significantly reduced thrombus volume at concentrations (5 μM) that suggested their reported physiological levels (0.1-1 μM) may exert low levels of inhibition. Flavonoid interactions with erythrocytes, leukocytes and human serum albumin in whole blood reduce their inhibitory activities against platelet function. The diminished inhibitory activity of flavonoids that we observed in whole blood and plasma indicated that these interactions do not overcome the attenuating effects of these compounds. Furthermore, inhibition of platelet aggregation by flavonoids was enhanced with increases in exposure time, indicating the potential for measurable inhibitory effects during resident plasma times. We conclude that flavonoid structures may be a major influence of their activities in vivo with methylated metabolites and those of flavones being more potent than those of flavonols and flavanones.
Article
Junctional adhesion molecule (JAM)-A expressed in endothelial, epithelial and blood cells can regulate permeability and leukocyte extravasation. Atherosclerosis develops at sites of disturbed flow in large arteries but the mechanisms guiding inflammatory cells into these predilection sites remain unknown. To characterize cell-specific functions of JAM-A in atherosclerosis, we employed apolipoprotein E-deficient mice with a somatic or endothelium-specific deficiency in JAM-A and bone marrow chimeras with JAM-A-deficient leukocytes. We show that impaired JAM-A expression in endothelial cells reduced mononuclear cell recruitment into the arterial wall and limited atherosclerotic lesion formation in hyperlipidemic mice. In contrast, JAM-A deficiency in bone marrow cells impeded monocyte de-adhesion, thereby increasing vascular permeability and lesion formation, whereas somatic JAM-A deletion revealed no significant effects. Regions with disturbed flow displayed a focal enrichment and luminal redistribution of endothelial JAM-A and were preferentially protected by its deficiency. The functional expression and redistribution of endothelial JAM-A was increased by oxidized low-density lipoprotein, but confined by athero¬protective laminar flow through an up-regulation of miR-145, which repressed JAM-A. Our data identify endothelial JAM-A as an important effector molecule integrating atherogenic conditions to direct inflammatory cell entry at predilection sites of atherosclerosis.
Article
EGC was prepared from green tea polyphenols through column chromatography of a polyamide (3.6 × 40 cm). Three dosages of EGC (0.25, 0.5, 1.0 g kg(-1) d(-1)) were ingested respectively by ICR mice via gavage. Compared with the control group, group EGC0.5 (dosage, 0. 5 g kg(-1) d(-1)) and group EGC1.0 (dosage, 1.0 g kg(-1) d(-1)) presented significant inhibition on platelet aggregation in mice accompanied by 18.4 and 25.6% of inhibition ratio, respectively. The bleeding times (BT) of mice in group EGC0.5 and group EGC1.0 were significantly prolonged (P < 0.01) as well as blood clotting time (BCT) in group EGC1.0 (P < 0.05). All three dosages of EGC prolonged activated partial thromboplastin time (APTT) significantly (P < 0.01), but had no prominent effect on prothrombin time (PT) and fibrinogen level which indicated that the anticoagulation of EGC could not be attributed to the level decrease of coagulation factor such as fibrinogen. The results demonstrated that EGC had prominent antiplatelet activity and blood anticoagulation in a dose-dependent manner.
Article
Platelets are a remarkable mammalian adaptation that are required for human survival by virtue of their ability to prevent and arrest bleeding. Ironically, however, in the past century, the platelets' hemostatic activity became maladaptive for the increasingly large percentage of individuals who develop age-dependent progressive atherosclerosis. As a result, platelets also make a major contribution to ischemic thrombotic vascular disease, the leading cause of death worldwide. In this brief review, I provide historical descriptions of a highly selected group of topics to provide a framework for understanding our current knowledge and the trends that are likely to continue into the future of platelet research. For convenience, I separate the eras of platelet research into the "Descriptive Period" extending from ~1880-1960 and the "Mechanistic Period" encompassing the past ~50 years since 1960. We currently are reaching yet another inflection point, as there is a major shift from a focus on traditional biochemistry and cell and molecular biology to an era of single molecule biophysics, single cell biology, single cell molecular biology, structural biology, computational simulations, and the high-throughput, data-dense techniques collectively named with the "omics postfix". Given the progress made in understanding, diagnosing, and treating many rare and common platelet disorders during the past 50 years, I think it appropriate to consider it a Golden Age of Platelet Research and to recognize all of the investigators who have made important contributions to this remarkable achievement..
Article
Antiplatelet drugs are important components in the management of atherothrombotic vascular disease. However, several limitations restrict the safety and efficacy of current antiplatelet therapy in clinical practice. Interpatient variability and resistance to aspirin and/or clopidogrel has spurred efforts for the development of novel agents. Indeed, several antiplatelet drugs are at various stages of evaluation; those at advanced stage of development are the focus of this review.
Article
Hippuric acid has been a major human metabolite for years. However, there is no well-known documented health benefit associated with it except for excretion of environmental-toxic exposures of aromatic compounds such as toluene, or from dietary protein degradation and re-synthesis by intestinal microflora metabolism of quinic acid via the shikimate pathway. Thus hippuric acid can appear in humans as an excretory product from natural or unnatural sources. It has been believed over the years that the major source of urinary hippuric acid levels in humans has come from environmental toxic solvent exposures. However, more recently it was been shown that approximately 1-2 mM hippuric acid is excreted daily in the urine, even in the absence of organic solvent exposure, signalling abundant metabolic dietary sources of hippuric acid are also apparent. One of these has been dietary proteins. The other is from the well-documented presence of quinic acid in healthy colored foodstuffs. Quinic acid is a key metabolite associated with the shikimate pathway existing only in plants, and it is responsible for essential amino acid biosynthesis such as tryptophan, phenylalanine and tyrosine. Here we review the evidence that the human gastrointestinal tract microflora are responsible for quinic acid metabolism not only to hippuric acid, but more importantly to efficacious antioxidant amino acids and vitamins.
Article
Currently, clopidogrel is recommended for treatment of patients with acute coronary syndrome and/or percutaneous coronary intervention. However, the delayed onset of the effect and the occurrence of poor platelet inhibition responders with clopidogrel as well as non-compliance to dual antiplatelet treatment are associated with a raised risk of stent thrombosis. The molecular target of the active metabolite of clopidogrel and several emerging antiplatelet treatments is the P2Y(12) receptor, which is the main platelet receptor responsible for ADP-induced platelet aggregation. Active metabolites of the thienopyridine prodrugs (ticlopidine, clopidogrel, and prasugrel) covalently bind to the P2Y(12) receptor and are irreversible, indirect platelet inhibitors. The newer, direct-acting P2Y(12) inhibitors (cangrelor and ticagrelor) change the conformation of the P2Y(12) receptor, resulting in reversible, concentration dependent inhibition of the receptor. An understanding of the similarities and differences in the properties and mechanisms of action of these new inhibitors compared with clopidogrel is needed in order to optimize the development and use of these agents in clinical practice. The objectives of this systematic review are to summarize the pharmacokinetics, pharmacodynamics, and pharmacogenetics of the different P2Y(12) inhibitors and to discuss the clinical implications for treatment of patients.
Article
The monopalmityloxy shikimic acids have been synthesized from shikimic acid and palmitic acid catalyzed by Novozym 435 in 2-methyl-2-butanol. The anticoagulation activity in vivo via oral administration of monopalmityloxy shikimic acid has been evaluated through arteriovenous shunt model of rats and through the determination of thrombin time, prothrombin time, and activated partial thromboplastin time via rats. After reaction, the solid shikimic acid has been observed to dissolve in the reaction system completely. The subsequent high-performance liquid chromatography-mass spectroscopy analysis showed that the monopalmityloxy shikimic acids, as the only products, had been formed and the overall conversion rate was over 70%. The result showed that it had anti-thrombosis activity, could prolong the coagulating time and bleeding time in vivo, and lengthen the coagulating time in vitro. Compared with control group, the differences of the treatment group and aspirin group of rats are significant (P < 0.05) for prothrombin time and thrombin time, and very significant (P < 0.01) for activated partial thromboplastin time. It suggested that the product had the anticoagulation activity. The mechanism might be the co-action of the inhibition of intrinsic coagulation and the inhibition of extrinsic coagulation, and the inhibiting effect on intrinsic pathway is stronger than that on extrinsic pathway.
Article
For patients with inborn errors of urea synthesis, oral administration of sodium benzoate is the usual treatment to increase the nitrogen excretion. Thus, monitoring hippuric acid and benzoic acid simultaneously in human biological fluids is considered to be clinically important. We developed a simple and accurate high-performance liquid chromatographic method for the simultaneous determination of hippuric acid and benzoic acid in human plasma and urine. This method requires no extraction step. Aliquots of urine and plasma are added to a solution of internal standard (o-chlorobenzoic acid) in acetonitrile and directly injected onto a reversed-phase column using an acidic (pH 2.7) eluent and ultraviolet detection at 235 nm. The preliminary plasma concentration-time and urinary excretion rate-time profiles of hippuric acid and benzoic acid from a healthy subject receiving small, medium and large doses of sodium benzoate are reported.
Article
To investigate the effect of triacetylshikimic acid (TSA) on the platelet adhesion to neutrophils and P-selectin expression on activated platelet membrane induced by thrombin and reperfusion after focal cerebral ischemia. The platelet adhesion to neutrophils was evaluated by rosette assay, and P-selectin expression on platelet membrane was determined by flow cytometry. TSA 10 - 1000 micromol/L markedly inhibited thrombin(0.4 kU/L)-induced platelet adhesion to neutrophils. The platelet adhesion to neutrophils induced by a 21-h reperfusion after middle cerebral artery occlusion for 3 h was also inhibited in a dose-dependent manner by TSA 50 - 200 mg/kg given by ig immediately and at 60 min again after the onset of cerebral ischemia. TSA was also shown to decrease the P-selectin expression on platelet surface induced by thrombin in washed platelet and by adenosine diphosphate (ADP) 5 micromol/L in whole blood. Reperfusion after cerebral ischemia and thrombin induced platelet adhesion to neutrophils, which could be reduced by TSA probably due to its inhibition of P-selectin expression on activated platelets.
The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites. All pathway intermediates can also be considered branch point compounds that may serve as substrates for other metabolic pathways. The shikimate pathway is found only in microorganisms and plants, never in animals. All enzymes of this pathway have been obtained in pure form from prokaryotic and eukaryotic sources and their respective DNAs have been characterized from several organisms. The cDNAs of higher plants encode proteins with amino terminal signal sequences for plastid import, suggesting that plastids are the exclusive locale for chorismate biosynthesis. In microorganisms, the shikimate pathway is regulated by feedback inhibition and by repression of the first enzyme. In higher plants, no physiological feedback inhibitor has been identified, suggesting that pathway regulation may occur exclusively at the genetic level. This difference between microorganisms and plants is reflected in the unusually large variation in the primary structures of the respective first enzymes. Several of the pathway enzymes occur in isoenzymic forms whose expression varies with changing environmental conditions and, within the plant, from organ to organ. The penultimate enzyme of the pathway is the sole target for the herbicide glyphosate. Glyphosate-tolerant transgenic plants are at the core of novel weed control systems for several crop plants.
Article
Clopidogrel is a potent antithrombotic drug that inhibits ADP-induced platelet aggregation. The results of large clinical trials have demonstrated an overall benefit of clopidogrel over aspirin in the prevention of vascular ischemic events (myocardial infarction, stroke, vascular death) in patients with a history of symptomatic atherosclerotic disease. The antiaggregating effect of clopidogrel is attributed to an irreversible inhibition of ADP binding to a purinergic receptor present at the platelet surface. Clopidogrel is not active in vitro and can be considered a precursor of an active metabolite formed in the liver. The chemical structure of this active metabolite and its biological activity have been described recently. Several purinergic receptors have been described on platelets; P2X (1), a calcium channel, and P2Y1 a Gq-coupled seven-transmembrane domain receptor, have been found not to be antagonized by clopidogrel. Another Gi (2)-coupled receptor (named P2Y12) has been recently cloned and stably expressed in CHO cells. These cells displayed a strong affinity for (33)P-2MeS-ADP, a stable analogue of ADP, the binding characteristics of which corresponded in all points to those observed on platelets. The binding of (33)P-2MeS-ADP to these cells was strongly inhibited by the active metabolite of clopidogrel with a potency that was consistent with that observed for this compound on platelets. In these transfected CHO cells, as in platelets, ADP and 2MeS-ADP induced adenylyl cyclase downregulation, an effect that was inhibited by the active metabolite of clopidogrel. These results demonstrate that this receptor corresponds to the previously called "P2t" platelet receptor and show that the active metabolite of clopidogrel binds in a covalent manner to this receptor, thus explaining how it blocks the aggregating effect of ADP on platelets.
Article
Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a molecule expressed on all cells within the vascular compartment, being expressed to different degrees on most leukocyte sub-types, platelets, and on endothelial cells where its expression is largely concentrated at junctions between adjacent cells. As well as exhibiting adhesive properties, PECAM-1 is an efficient signaling molecule and is now known to have diverse roles in vascular biology including roles in angiogenesis, platelet function, and thrombosis, mechanosensing of endothelial cell response to fluid shear stress, and regulation of multiple stages of leukocyte migration through venular walls. This review will focus on some new developments with respect to the role of PECAM-1 in inflammation and vascular biology, highlighting the emerging complexities associated with the functions of this unique molecule.
Article
Platelet-dependent thrombus formation may be influenced by alteration of platelet or vascular redox state, the presence of endogenous or exogenous antioxidants, as well as the formation of reactive oxygen and nitrogen species. Specifically, settings and pathways that influence the formation of superoxide and nitric oxide, as well as their metabolism, may influence platelet function and thrombus formation. Although some antioxidant regimens have been associated with bleeding and hemorrhagic stroke, the therapeutic value of antioxidants in clinical syndromes that lead to platelet-dependent thrombosis is not clear, as supplemental antioxidants have not been generally associated with better cardiovascular outcome.