Lynn Crosbie

Effects of tomato extract on human platelet aggregation in vitro

Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 w l of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 2 g supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 microl of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 xg supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.

A residential study comparing the effects of diets rich in stearic acid, oleic acid, and linoleic acid on fasting blood lipids, hemostatic variables and platelets in young healthy men.

Dietary fat is known to influence the variables of blood coagulation and fibrinolysis associated with vascular disease. However, the role of fat content and/or fat composition of the diet in this regard is still not well understood. In the present study, we investigated the effects of three isoenergic diets of differing fat composition in nine healthy young men in a strictly controlled residential study. Subjects consumed the three experimental diets for periods of 2 weeks each, separated by a washout period of at least 5 weeks in a randomized crossover design. The diets provided 38% of total energy intake as fat, 45% as carbohydrate, and 17% as protein, and differed only with respect to the fatty acid composition (stearic acid-rich diet: 34.1% stearic acid, 36.6% oleic acid; oleic acid-rich diet: 65.8% oleic acid; linoleic acid-rich diet: 36.5% linoleic acid, 38% oleic acid). Blood samples were collected at the beginning and at the end of each dietary period from fasted subjects for determination of factor VII coagulant activity (FVIIc), activated factor VII (FVIIa), factor VII antigen (FVIIag), tissue plasminogen activator (tPA) activity, plasminogen activator inhibitor type 1 (PAI-1) activity, fibrinogen, prothrombin fragment 1+2 (F(1+2)), and plasma lipids. There were no significant differences between diets in fasting plasma concentrations of FVIIc, FVIIa, FVIIag, fibrinogen, F(1+2), PAI-1 activity, and tPA activity. Plasma concentrations of lipids (high density lipoproteins, low density lipoproteins, triacylglycerols, and total cholesterol) were also unaffected. Although there were no changes in platelet aggregation response and membrane fluidity observed in any of the diets, increased anti-aggregatory prostaglandin E(1) binding to platelet membranes was observed only in the case of linoleic acid-rich diet. In conclusion, diets with very different fatty acid compositions, at 38% of energy as fat intake, did not significantly influence blood coagulation, fibrinolysis, or blood lipids in the fasting state in young healthy men.

Inhibitory effect of Ginkgo biloba extract on human platelet aggregation

The effect of pure flavonoids and Gingko biloba extract (GBE) on human platelet aggregation was investigated. Most of the flavonoids and vitamin E did not affect platelet aggregation in platelet-rich plasma (PRP); however some of these flavonoids inhibited platelet aggregation in gel-filtered platelets (GFP). GBE inhibited both ADP- and collagen-induced platelet aggregation in PRP, GFP and in whole blood in a dose-dependent manner. GBE at very low concentrations inhibited whole blood aggregation induced by ADP compared with those used for PRP or GFP. Flavonoids and GBE decreased the production of TxA(2) induced by collagen and ADP in PRP. However, no correlation was observed between the inhibition of platelet aggregation and the decrease of TxA(2) synthesis. GBE and flavonoids did not affect platelet membrane fluidity. However, the incubation of PRP with GBE increased cAMP levels in platelets, which is known to inhibit platelet activation by lowering intracellular Ca2+ levels. GBE is a mixture of many compounds, including flavonoids and gingkoglides, which affect metabolism of cAMP, TxA(2) and Ca2+ in platelets. It is effective in the inhibition of platelet aggregation, both in PRP and whole blood, and thus may be potentially used as an effective oral anti-platelet therapeutic agent.

Inhibitory effect of aqueous extracts of some herbs on human platelet aggregation in vitro

Effect of aqueous extract of several herbs on human platelet aggregation in vitro was investigated. Out of 28 herbs/nutriceuticals investigated, camomile, nettle alfalfa, garlic and onion exhibited most significant anti-platelet activity (≥45% inhibition). Aqueous extracts of alfalfa, fresh nettle, and camomile inhibited ADP induced-platelet aggregation by 73, 65 and 60%, respectively, compared with control (P < 0.05). Camomile and alfalfa inhibited collagen-induced platelet aggregation by 84 and 65%, respectively, but nettle could not inhibit collagen-induced aggregation. In contrast, nettle was the most potent inhibitor (66%) of whole blood aggregation induced by collagen, followed by alfalfa (52%), and camomile (30%) compared with control (P < 0.05). None of these three herbs however could inhibit arachidonic acid or thrombin induced platelet aggregation. Camomile and alfalfa strongly inhibited thromboxane B2 synthesis induced by ADP or collagen, but nettle had no effect. Alfalfa and nettle increased cGMP levels in platelets by 50 and 35%, respectively, compared with the control (1.85 ± 0.23 nM) (P < 0.005). All these data indicate that camomile, nettle and alfalfa have potent anti-platelet properties, and their inhibitory actions are mediated via different mechanisms.

Arachidonic acid uptake by human platelets is mediated by CD36

The involvement of glycoprotein (GP) IV (CD36) in arachidonic acid uptake by human platelets was investigated using an anti-CD36 monoclonal antibody (MAB). The binding of [(14)C]arachidonic acid to MAB-treated platelets was significantly reduced compared with untreated platelets. The MAB also inhibited arachidonic acid-induced platelet aggregation and thromboxane A(2) synthesis in a dose-dependent manner. Pre-incubation of gel-filtered platelets with the MAB (10mg/I) inhibited arachidonic acid-induced platelet aggregation by 50% and collagen-induced platelet aggregation by 7-8% and the lag time was increased by 200%. Although the mechanism of platelet aggregation is not fully understood yet, the inhibition of arachidonic acid-induced platelet aggregation by the MAB could be the result of a reduced uptake of exogeneously added arachidonic acid by the MAB-treated platelets. Our data clearly indicate that arachidonic acid uptake by platelets is mediated, at least in part, by CD36.

The effects of structurally defined triglycerides of differing fatty acid composition on postprandial haemostasis in young, healthy men.

The aim of this study was to investigate whether a number of key haemostatic factors were altered when healthy young individuals were challenged with a fat load of physiological size contained within a meal composed of normal ingredients and whether this response was modified when the fatty acid composition of the meal was altered radically. Eight healthy male volunteers each randomly consumed four meals which were identical in terms of gross nutritional content (41% of energy provided as fat, 17% as protein and 42% as carbohydrate) but which differed in fatty acid composition. To reduce the possible influence of fatty acid position within the triglyceride molecule on lipid absorption and subsequent metabolic effects, the structural integrity of 91% of fat (test triglycerides such as 1,3 distearoyl-2-oleoyl glycerol (S-O-S), trioleine (O-O-O), and 1,3 dilinoleoyl-2-oleoyl glycerol, (L-O-L)) in the meals was controlled so that the principal fatty acid in the sn-2 position was oleic acid (18:1n-9). Meals rich in either a test triglyceride or a control oil provided 44+/-6 g of fat. No significant alterations from fasted values of elevated plasma factor VII coagulant activity (FVIIc) or F1 + 2 were observed. FVIIA varied significantly over the postprandial time course; however, when expressed as a percentage of the fasting value, the FVIIa responses to O-O-O and L-O-L differed significantly but this was not evident when the absolute values were analysed. Similarly, no difference in plasma fibrinopeptide A (FPA) concentrations were evident. After all four meals, chylomicron contained proportionately more palmitic acid and generally less oleic acid than the ingested lipids. This study clearly demonstrates that postprandial haemostatic responses of young healthy individuals to a physiological fat load are minimal, (irrespective of triglyceride structure).

A randomised controlled trial comparing a dietary antiplatelet, the water-soluble tomato extract Fruitflow, with 75 mg aspirin in healthy subjects

Background/Objectives:Increasing numbers of food ingredients are gaining acknowledgement, via regulated health claims, of benefits to human health. One such is a water-soluble tomato extract, Fruitflow (FF), a dietary antiplatelet. We examined relative platelet responses to FF and to 75 mg aspirin (ASA) in healthy subjects.Subjects/Methods:A total of 47 healthy subjects completed a double-blinded randomised controlled trial following a crossover design. Acute and 7-day treatments with 75 mg ASA were compared with control with and without concomitant FF, over a 5-h timecourse. Platelet aggregation response agonist, platelet thromboxane A2 release, plasma clotting times and time to form a primary haemostatic clot (PFA-100 closure time, TTC) were measured.Results:Administration of all treatments lowered platelet function and thromboxane A2 generation, and extended the TTC, relative to baseline (P<0.001) and to control (P<0.001). Plasma clotting times were not affected. A single 75 mg dose of ASA showed approximately equal efficacy to a dose of FF, whereas daily 75 mg ASA was approximately three times as effective after 7 days (P=0.002). Platelet responses were heterogenous with distinct weak and strong responder groups. Weak ASA responders retained a functional platelet response to collagen agonist and were responsive to FF. Concomitant FF and ASA did not lead to significant additive effects.Conclusions:The suppression of platelet function observed after consuming FF is approximately one-third that of daily 75 mg ASA. The reversible action of FF renders it less likely to overextend the time to form a primary haemostatic clot than ASA, an important safety consideration for primary prevention.