Mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of rutin, a well-known flavonoid in platelet aggregation. In this study, rutin concentration-dependently (250 and 290 microM) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., collagen). Rutin (250 and 290 microM) did not significantly interfere with the binding of FITC-triflavin to the glycoprotein IIb/IIIa complex in human platelets. Rutin (250 and 290 microM) markedly inhibited intracellular Ca(2+) mobilization and thromboxane A(2) formation in human platelets stimulated by collagen. Rapid phosphorylation of a platelet protein of M(r) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by rutin (250 and 290 microM). On the other hand, rutin (250 and 290 microM) did not significantly increase the formations of cyclic AMP and nitric oxide/cyclic GMP in platelets. In conclusion, these results indicate that the antiplatelet activity of rutin may involve the following pathways: rutin inhibited the activation of phospholipase C, followed by inhibition of protein kinase C activity and thromboxane A(2) formation, thereby leading to inhibition of the phosphorylation of P47 and intracellular Ca(2+) mobilization, finally resulting in inhibition of platelet aggregation.     

Inhibition of malonaldehyde formation in oxidized calf thymus DNA with synthetic and natural antioxidants

Calf thymus DNA was oxidized by Fenton's reagent with or without synthetic antioxidants (Trolox and DMPO) and natural antioxidants-quercetin, apigenin, 2'-O-glycosylisovitexin (2'-O-GIV), (+)-catechin, cyanidin, pelargonidin, keracyanin, and callistephin. Malonaldehyde (MA) formed in oxidized DNA was analyzed using gas chromatography. MA formed from oxidized DNA without antioxidants was 4.0 +/- 0.53 nmol/mg of DNA in buffer solution, 3.7 +/- 0.34 nmol/mg of DNA in NaOH solution, and 4.6 +/- 0.19 nmol/mg of DNA in HCl solution. MA formed from DNA with antioxidants (at the level of 0.1 micromol/mL) ranged from 1.90 +/- 0.18 (catechin) to 4.10 +/- 0.18 nmol/mg of DNA (cyanidin). Trolox and DMPO inhibited MA formation from DNA by 41.2% and 18.6%, respectively, at the level of 0.1 micromol/mL. Trolox (water-soluble vitamin E) exhibited dose-dependent inhibition. The decreasing order of inhibitory effect by flavonoids at the level of 0.1 micromol/mL was catechin (48.5%) > quercetin (47.1%) > 2'-O-GIV (40.5%) > apigenin (29.9%) and by the anthocyanins at the level of 0.1 micromol/mL was callistephin (45%) > keracyanin (33.2%) > pelargonidin (25.1%) > cyanidin (10.2%).     

Exploration of the antiplatelet activity profile of betulinic acid on human platelets

Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including antiretroviral, antibacterial, antimalarial, and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated, and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-14, and arachidonic acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR-derived structure of betulinic acid and prostacyclin agonists (PGI2), and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that deserves further investigation.     

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, β/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.     

Dietary apigenin suppresses IgE and inflammatory cytokines production in C57BL/6N mice

Flavonoids ubiquitously exist in plants, vegetables, fruits, and teas. We evaluated the effect of dietary apigenin, one of the well-known flavonoids, on the immune system in C57BL/6N mice. Mice were fed experimental diets containing apigenin for 2 weeks. After the experimental period, there was no significant difference in body and organ weights between the control and the apigenin group. The total immunoglobulin (Ig) E levels in mice fed apigenin were significantly suppressed, whereas levels of IgG, IgM, and IgA were not affected. We also examined the effect of the apigenin diet on cytokine expression in mice sera using a cytokine array. The production of regulated upon activation normal T cell expressed and secreted (RANTES) and soluble tumor necrosis factor receptor I (sTNFRI) in mice sera was down-regulated by the apigenin diet. These results suggest that a diet containing apigenin can reduce serum IgE and inflammatory cytokines such as RANTES and sTNFRI in mice.     

C-phycocyanin, a very potent and novel platelet aggregation inhibitor from Spirulina platensis

The aim of this study was to systematically examine the inhibitory mechanisms of C-phycocyanin (C-PC), one of the major phycobiliproteins of Spirulina platensis (a blue-green alga), in platelet activation. In this study, C-PC concentration-dependently (0.5-10 nM) inhibited platelet aggregation stimulated by agonists. C-PC (4 and 8 nM) inhibited intracellular Ca2+ mobilization and thromboxane A2 formation but not phosphoinositide breakdown stimulated by collagen (1 microg/mL) in human platelets. In addition, C-PC (4 and 8 nM) markedly increased levels of cyclic GMP and cyclic GMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation. Rapid phosphorylation of a platelet protein of Mw 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (150 nM). This phosphorylation was markedly inhibited by C-PC (4 and 8 nM). In addition, C-PC (4 and 8 nM) markedly reduced the electron spin resonance (ESR) signal intensity of hydroxyl radicals in collagen (1 microg/mL)-activated platelets. The present study reports on a novel and very potent (in nanomolar concentrations) antiplatelet agent, C-PC, which is involved in the following inhibitory pathways: (1) C-phycocyanin increases cyclic GMP/VASP Ser157 phosphorylation and subsequently inhibits protein kinase C activity, resulting in inhibition of both P47 phosphorylation and intracellular Ca2+ mobilization, and (2) C-PC may inhibit free radicals (such as hydroxyl radicals) released from activated platelets, which ultimately inhibits platelet aggregation. These results strongly indicate that C-PC appears to represent a novel and potential antiplatelet agent for treatment of arterial thromboembolism.     

Differential inhibition of human platelet aggregation by selected Allium thiosulfinates

Thiosulfinates (TSs) have been implicated as a principle source of the antiplatelet property of raw onion and garlic juice. The in vitro responses of human platelets to dosages of four TSs were measured using whole blood aggregometry and compared by regression analysis. Of the compounds evaluated, methyl methane-TS (MMTS), propyl propane-TS (PPTS), and 2-propenyl 2-propene-TS (allicin) are present in freshly cut Allium vegetables, whereas ethyl ethane-TS (EETS) has not been detected. All TSs were synthesized using a model reaction system. PPTS and allicin had the strongest antiplatelet activity at 0.4 mM, inhibiting aggregation by 90 and 89%, respectively. At the same concentration, EETS and MMTS were significantly weaker, inhibiting 74 and 26%, respectively. Combinations of TSs were not additive in their inhibition of aggregation, indicating that the antiplatelet potential of Allium extracts cannot be easily predicted by quantifying organosulfur components. EETS, PPTS, and allicin were significantly more potent platelet inhibitors than aspirin at nearly equivalent concentrations.     

Absorption and excretion of luteolin and apigenin in rats after oral administration of Chrysanthemum morifolium extract

Chrysanthemum morifolium extract (CME) has the protective effect on cardiovascular diseases. Luteolin and apigenin are two major bioactive components in vivo when CME is orally administrated to experimental animal. The present paper shows the study of the absorption and excretion of luteolin and apigenin in rats after a single oral dose of CME (200 mg/kg). The levels of luteolin and apigenin in plasma, urine, feces, and bile were measured by HPLC after deconjugation with hydrochloric acid or beta-glucuronidase/sulfatase. The results showed that the plasma concentrations of luteolin and apigenin reached the highest peak level at 1.1 and 3.9 h after dosing, respectively. The area under the concentration-time curves (AUC) for luteolin and apigenin were 23.03 and 237.6 microg h mL-1, respectively. The total recovery of the dose was 37.9% (6.6% in urine; 31.3% in feces) for luteolin and 45.2% (16.6% in urine; 28.6% in feces) for apigenin. The cumulative luteolin and apigenin excreted in the bile was 2.05% and 6.34% of the dose, respectively. All of the results suggest apigenin may be absorbed more efficiently than luteolin in CME in rats, and both luteolin and apigenin have a slow elimination phase, with a quick absorption, so a possible accumulation of the two flavonoids in the body can be hypothesized.     

New C-deoxyhexosyl flavones and antioxidant properties of Passiflora edulis leaf extract

The flavonoids present in passion fruit (Passiflora edulis) leaves were identified by a high-performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) method. Sixteen apigenin or luteolin derivatives were characterized, which included four mono-C-glycosyl, eight O-glycosyl- C-glycosyl, and four O-glycosyl derivatives. With the exceptions of C-hexosyl luteolin and C-hexosyl apigenin, all the compounds exhibited a deoxyhexose moiety. Moreover, the uncommon C-deoxyhexosyl derivatives of luteolin and apigenin have been identified for first time in P. edulis by HPLC-DAD-MS/MS. The antioxidative capacity of passion fruit leaves was checked against DPPH radical and several reactive oxygen species (superoxide radical, hydroxyl radical, and hypochlorous acid), revealing it to be concentration-dependent, although a pro-oxidant effect was noticed for hydroxyl radical.     

Human gut microbial degradation of flavonoids: structure-function relationships

The relationship between chemical structure and gut microbial degradation rates of 14 flavonoids, flavone, apigenin, chrysin, naringenin, kaempferol, genistein, daidzein, daidzin, puerarin, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, 5,3'-dihydroxyflavone, and 4'-hydroxyflavone, was investigated by anaerobically fermenting the flavonoids with human gut microflora (n = 11 subjects). Degradation rates for the 5,7,4'-trihydroxyl flavonoids, apigenin, genistein, naringenin, and kaempferol, were significantly faster than the other structural motifs. Puerarin was resistant to degradation by the gut microflora. Extensive degradation of flavonoids by gut microflora may result in lower overall bioavailability than those flavonoids that are slowly degraded because rapidly degrading flavonoids are less likely to be absorbed intact.     

Involvement of Ca2+ in the inhibition by crocetin of platelet activity and thrombosis formation

Crocetin, a unique carotenoid with potent antioxidative and anti-inflammatory activities, is a major ingredient of saffron used as an important spice and food colorant in various parts of the world. In the present study, the effects of crocetin on platelet activity and thrombosis formation were systematically investigated. Crocetin showed a dose-dependent inhibition of platelet aggregation induced by ADP, collagen, but not by arachidonic acid (AA). Crocetin significantly attenuated dense granule release, while neither platelets adhesion to collagen nor cyclic AMP level was altered by crocetin. Pretreatment with crocetin was confirmed to partially inhibit Ca (2+) mobilization via reducing both intracellular Ca (2+) release and extracellular Ca (2+) influx. Besides that, crocetin prolonged the occlusive time in electrical stimulation-induced carotid arterial thrombosis. These findings suggest that the favorable impacts of crocetin on platelet activity and thrombosis formation may be related to the inhibition of Ca (2+) elevation in stimulated platelets.     

Antiplatelet activity of soy sauce as functional seasoning.

In seeking the functionality of foodstuffs applicable to medicine, soy sauce was found to show antiplatelet activity. Therefore, the active components in soy sauce were purified, structurally identified, and studied for their inhibitory effects on the aggregation of human platelets. Aqueous 2-fold diluents of soy sauce inhibited platelet aggregation induced by collagen and epinephrine depending on the dilution factor. Since a basic extract with diethyl ether completely inhibited collagen-induced aggregation, it was subjected to serial extractions and multistep HPLC fractionations for purifying antiplatelet components. The finally obtained isolates were identified as 1-methyl-1,2,3,4-tetrahydro-beta-carboline and 1-methyl-beta-carboline on the basis of EI-MS, (1)H NMR, diode array, and fluorescence spectra. Their spectral data and chromatographic behaviors were the same as those of synthetic ones. 1-Methyl-1,2,3, 4-tetrahydro-beta-carboline showed mean concentrations (n = 5-6) of 4.6, 4.2, 28.6, 11.6, and 65.8 microgram/mL to produce 50% inhibition of the maximal aggregation response induced by epinephrine, platelet-activating factor, collagen, adenosine 5'-diphosphate, and thrombin, respectively. Its inhibitory effect was much greater than that of 1-methyl-beta-carboline on platelet aggregation by all the tested inducers. The quantitative HPLC analysis revealed that the significant amounts of both antiplatelet compounds were uniformly contained in commercially available soy sauce. From these results, soy sauce may be referred to as functional seasoning containing alkaloidal components with the potent preventive effect on thrombus formation.     

Baicalein induction of hydroxyl radical formation via 12-lipoxygenase in human platelets: an ESR study

The pro-oxidant activities of baicalein, morin, myricetin, quercetin, and rutin were examined in various cell-containing systems including human platelets, rat vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs), human THP-1 cells, and fibroblast cells. Electron spin resonance (ESR) results showed that only baicalein generated hydroxyl radicals in a resting human platelet suspension, whereas the other flavonoids showed no effects on any of the resting cell systems. A low concentration of arachidonic acid (AA) increased the intensity of hydroxyl radicals, but a high concentration inhibited it. Collagen and thrombin, platelet aggregatory agents that can cause the release of AA by platelets, enhanced baicalein-induced hydroxyl radical formation, whereas ADP and U44619 showed no significant effects. Quinacrine and 5,8,11,14-eicosatetraenoic trifluoromethyl ketone, both PLA2 inhibitors, significantly attenuated baicalein-induced hydroxyl radical formation. These results suggest that baicalein-induced hydroxyl radical formation is associated with AA metabolite enzymes in human platelets. The formation of hydroxyl radicals was significantly inhibited by lipoxygenase inhibitors including nordihydroguaiaretic acid, (-)-epicatechin, (-)-epicatechin gallate, and hinokitiol, but was not affected by desferroxamine or the heme protein inhibitors KCN and NaN3. On the other hand, semiquinone free radicals were generated when baicalein was incubated with horseradish peroxidase/H2O2 or platelets/AA. The semiquinone radicals formed in the platelets/AA system could be extensively inhibited by desferroxamine, diethylenetriaminepentaacetic acid, KCN, and NaN3, indicating that prostaglandin H synthase (PGHS)-peroxidase may be involved. The results of this study led to the proposal that baicalein induces hydroxyl radical formation via 12-lipoxygenase and induces semiquinone radical formation via PGHS-peroxidase in human platelets.     

Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity

Significantly, much of the activity of Citrus flavonoids appears to impact blood and microvascular endothelial cells, and it is not surprising that the two main areas of research on the biological actions of Citrus flavonoids have been inflammation and cancer. Epidemiological and animal studies point to a possible protective effect of flavonoids against cardiovascular diseases and some types of cancer. Although flavonoids have been studied for about 50 years, the cellular mechanisms involved in their biological action are still not completely known. Many of the pharmacological properties of Citrus flavonoids can be linked to the abilities of these compounds to inhibit enzymes involved in cell activation. Attempts to control cancer involve a variety of means, including the use of suppressing, blocking, and transforming agents. Suppressing agents prevent the formation of new cancers from procarcinogens, and blocking agents prevent carcinogenic compounds from reaching critical initiation sites, while transformation agents act to facilitate the metabolism of carcinogenic components into less toxic materials or prevent their biological actions. Flavonoids can act as all three types of agent. Many epidemiological studies have shown that regular flavonoid intake is associated with a reduced risk of cardiovascular diseases. In coronary heart disease, the protective effects of flavonoids include mainly antithrombotic, anti-ischemic, anti-oxidant, and vasorelaxant. It is suggested that flavonoids decrease the risk of coronary heart disease by three major actions: improving coronary vasodilatation, decreasing the ability of platelets in the blood to clot, and preventing low-density lipoproteins (LDLs) from oxidizing. The anti-inflammatory properties of the Citrus flavonoids have also been studied. Several key studies have shown that the anti-inflammatory properties of Citrus flavonoids are due to its inhibition of the synthesis and biological activities of different pro-inflammatory mediators, mainly the arachidonic acid derivatives, prostaglandins E 2, F 2, and thromboxane A 2. The anti-oxidant and anti-inflammatory properties of Citrus flavonoids can play a key role in their activity against several degenerative diseases and particularly brain diseases. The most abundant Citrus flavonoids are flavanones, such as hesperidin, naringin, or neohesperidin. However, generally, the flavones, such as diosmin, apigenin, or luteolin, exhibit higher biological activity, even though they occur in much lower concentrations. Diosmin and rutin have a demonstrated activity as a venotonic agent and are present in several pharmaceutical products. Apigenin and their glucosides have been shown a good anti-inflammatory activity without the side effects of other anti-inflammatory products. In this paper, we discuss the relation between each structural factor of Citrus flavonoids and the anticancer, anti-inflammatory, and cardiovascular protection activity of Citrus flavonoids and their role in degenerative diseases.     

HPLC-PDA-MS and NMR characterization of C-glycosyl flavones in a hydroalcoholic extract of Citrus aurantifolia leaves with antiplatelet activity

A hydroalcoholic extract of lime ( Citrus aurantifolia) leaves has been developed in Cuba to be used as a nutritional supplement and phytomedicine in the form of tincture (TLL). A HPLC-PDA-ESI/MS/MS method has been used for the comprehensive analysis of C-glycosyl flavones in TLL. Six C-glycosyl flavones were characterized and, to confirm the proposed structures and to elucidate the nature of the sugar units, a preparative procedure was applied, and isolated compounds were characterized by NMR. Apigenin-6,8-di-C-beta-D-glucopyranoside (vicenin II) (1), diosmetin-6,8-di- C-beta- d-glucopyranoside (2), apigenin-8-C-beta-D-glucopyranoside (vitexin) (3), apigenin-8-C-[alpha-L-arabinopyranosyl-(1-->6)]-O-beta-D-glucopyranoside (4), apigenin-6-C-[alpha-l-arabinopyranosyl-(1-->6)]-O-beta-D-glucopyranoside (5). and apigenin-6-C-beta-D-glucopyranoside (isovitexin) (6) were identified in TLL and quantified by HPLC-PDA. Compounds 4 and 5 were two new arabinosyl derivatives of vitexin and isovitexin. Inhibitor effect of TLL on platelet aggregation induced by physiological agonists of platelets was evaluated in human plasma. TLL inhibited significantly ADP and epinephrine-induced platelet aggregation in a concentration-dependent manner (IC 50=0.40 and 0.32 mg/mL, respectively).     

Effect of cooking on garlic (Allium sativum L.) antiplatelet activity and thiosulfinates content

The raw form of garlic and some of its preparations are widely recognized as antiplatelet agents that may contribute to the prevention of cardiovascular disease. Herein, we examined the in-vitro antiaggregatory activity (IVAA) of human blood platelets induced by extracts of garlic samples that were previously heated (in the form of crushed versus uncrushed cloves) using different cooking methods and intensities. The concentrations of allicin and pyruvate, two predictors of antiplatelet strength, were also monitored. Oven-heating at 200 degrees C or immersing in boiling water for 3 min or less did not affect the ability of garlic to inhibit platelet aggregation (as compared to raw garlic), whereas heating for 6 min completely suppressed IVAA in uncrushed, but not in previously crushed, samples. The latter samples had reduced, yet significant, antiplatelet activity. Prolonged incubation (more than 10 min) at these temperatures completely suppressed IVAA. Microwaved garlic had no effect on platelet aggregation. However, increasing the concentration of garlic juice in the aggregation reaction had a positive IVAA dose response in crushed, but not in uncrushed, microwaved samples. The addition of raw garlic juice to microwaved uncrushed garlic restored a full complement of antiplatelet activity that was completely lost without the garlic addition. Garlic-induced IVAA was always associated with allicin and pyruvate levels. Our results suggest that (1) allicin and thiosulfinates are responsible for the IVAA response, (2) crushing garlic before moderate cooking can reduce the loss of activity, and (3) the partial loss of antithrombotic effect in crushed-cooked garlic may be compensated by increasing the amount consumed.     

Isolation and identification of antiplatelet aggregatory principles from the leaves of Piper lolot

The methanolic extract of Piper lolot, having shown potent inhibitory activity on platelet aggregation induced by arachidonic acid (AA) and platelet activating factor (PAF), was subjected to activity-guided isolation to yield twelve new amide alkaloids, piperlotine A-L (1-12), along with twenty-nine known compounds. Their structures were elucidated on the basis of spectroscopic analysis. The isolated compounds were tested for their inhibitory activity on the rabbit platelet aggregation. The compounds piperlotine A (1), piperlotine C (3), piperlotine D (4), piperlotine E (5), 3-phenyl-1-(2,4,6-trihydroxyphenyl)propan-1-one (21), 3-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one (22), 1-trans-cinnamoylpyrrolidine (24), sarmentine (26), pellitorine (27), methyl 3-phenylpropionate (32), and (10S)-10-hydroxypheophorbide a methyl ester (40) showed potent antiplatelet aggregation activity.     

alpha-Naphthoflavone, a potent antiplatelet flavonoid, is mediated through inhibition of phospholipase C activity and stimulation of cyclic GMP formation

The aim of this study was to systematically examine the inhibitory mechanisms of the flavonoid alpha-naphthoflavone (alpha-NF) in platelet activation. In this study, alpha-NF concentration dependently (5-20 microM) inhibited platelet aggregation stimulated by agonists. alpha-NF (5 and 10 microM) inhibited intracellular Ca(2+) mobilization, phosphoinositide breakdown, and thromboxane A(2) formation stimulated by collagen (1 microg/mL) in human platelets. In addition, alpha-NF (5 and 10 microM) markedly increased levels of cyclic GMP and cyclic GMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (60 nM). This phosphorylation was markedly inhibited by alpha-NF (5 and 10 microM). However, alpha-NF (5 and 10 microM) did not reduce the electron spin resonance (ESR) signal intensity of hydroxyl radicals in collagen (1 microg/mL)-activated platelets. These results indicate that the antiplatelet activity of alpha-NF may be involved in the following pathways. (1) alpha-NF may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A(2) formation, thereby leading to inhibition of intracellular Ca(2+) mobilization. (2) alpha-NF also activated the formation of cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that alpha-NF appears to represent a novel and potent antiplatelet agent for treatment of arterial thromboembolism.     

Apigenin-induced suicidal erythrocyte death

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 μM), increased ceramide formation (15 μM), decreased ATP concentration (15 μM), decreased forward scatter (≥ 1 μM), and increased annexin V binding (≥ 5 μM) but did not significantly modify hemolysis. The effect of 15 μM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.