Inhibition of platelet aggregation has been well documented by numerous authors studying the in vitro and ex vivo effect of various garlic preparations (1-11). Some in vivo studies examined the effect of a garlic preparation on arterial thrombus formation in dogs (12) and of ajoene on platelet adhesion in pigs (13). In humans the results of platelet-aggregation studies have been variable, however, with some showing reduction in platelet aggregability (14) but others not (15-17). This is not totally unexpected, as some of the most inhibitory organosulfur compounds associated with garlic are also very unstable, may not be absorbed from the intestinal tract or only very incompletely and are extremely rapidly inactivated in the circulation (18,19). Furthermore, the amounts of the active principles in garlic preparations may be so low that their overall effect becomes almost negligible. There are as yet no data available on the effect of garlic on platelet adhesion in humans.
Beneficial effects of garlic or garlic extracts on total and low-density lipoprotein (LDL) cholesterol as well as blood pressure have been described by a number of investigators both in normal and in hypercholesterolemic individuals (20,21). The additional benefit of reducing platelet function when taking these dietary supplements would be extremely important in the prevention of thromboembolic events associated with atherosclerotic lesions in the arterial circulation. The possible role of oxidative modification of lipoproteins in the development of atherosclerotic lesions is a subject of intense interest, as is the effect of antioxidative measures in its prevention. We recently studied the effect of aged garlic extract (AGE) versus placebo in a group of hypercholes-terolemic men (22). The results of the platelet-aggregation and adhesion studies reported here were collected from a subgroup of those individuals. In view of the delay in the effect of garlic or its extracts on lipids, we considered it important similarly to evaluate platelet function in a long-term intervention study. AGE, a garlic preparation that has lost the characteristic odor of freshly crushed garlic, produced a reduction of platelet adhesion to fibrinogen and also some inhibition of platelet aggregation induced by epinephrine and collagen. In addition, AGE showed a tendency to reduce the susceptibility of lipoproteins to oxidation. This study is the first demonstration of an inhibitory effect of a garlic preparation on platelet adhesion and to our knowledge is also the first long-term investigation of the effect of a garlic preparation on platelet aggregation in an ex vivo setting.
Hypercholesterolemic men, aged 30-65 years, were recruited for an 11-month double-blind crossover study in which individuals were randomized to receive either AGE or placebo for a period of 6 months and the other dietary supplement for 4 months. All participants were advised to follow the National Cholesterol Education Program Step I diet. After a 4-week baseline period, the 15 subjects in the study received either nine capsules of AGE (Wakunaga of America Co., Mission Viejo, CA, U.S.A.), each 800 mg containing primarily water-soluble organosulfur compounds (23) and a variety of nonsulfur compounds, or an equal number of placebos, each containing 600 mg cornstarch, 99.5 mg microcrystalline cellulose, 0.5 mg caramel, and 3.5 mg magnesium stearate, in three divided doses daily. These study subjects were selected at random from the larger group of 41 men described elsewhere (22) and also underwent lipid-profile measurement. Of the 20 individuals randomly selected, 15 were willing to abstain from taking nonsteroidal antiinflammatory agents and donate the extra amount of blood necessary for the studies. Platelet adhesion was tested twice during the first (at 3 and 6 months) and once during the second intervention period (3 months after switching over from the first supplement they received). At 2-3 months after the termination of the study, the study subjects were tested again. Because of a limitation of the amount of blood available, platelet aggregation and susceptibility of lipoproteins to oxidation was studied in only 10 of these individuals during both arms of the study. Compliance was checked by pill counting, as described previously (22).
This investigation was reviewed and approved by the Human Studies Review Board of the institution where it was conducted. All volunteers signed an informed consent form before enrollment in the study.
Blood was collected in 1/10 volume 3.8% sodium citrate as anticoagulant after discarding the first milliliter. Aggregation studies were performed as previously described (24) by using platelet-rich plasma (PRP), which was separated at 260 g for 15 min. Aggregation of 0.4 ml PRP was monitored turbidimetrically on an aggregometer (Chrono-Log Corp., Havertown, PA, U.S.A.) connected to a recorder. All aggregation measurements were performed at 37°C with PRPs having platelet counts of 3 × 108/ml. Arachidonic acid, collagen I, epinephrine, and adenosine diphosphate (ADP; all products of Sigma Chemical Co., St. Louis, MO, U.S.A.) were the platelet stimulants. Arachidonic acid was used solely as a check on the abstention from nonsteroidal antiinflammatory agents. Each aggregating agent was tested over a range of concentrations, and threshold doses were determined for epinephrine, collagen I, and ADP. Threshold dose denotes the minimal concentration of agonist capable of inducing complete aggregation of the platelets, including generation of the first and second wave of aggregation when epinephrine and ADP were the stimulants and maximal aggregation with arachidonic acid and collagen as the aggregation inducers. All aggregation studies were performed without delay but always within 2 h of collecting the blood.
Measurement of platelet adhesion
Platelet-adhesion studies were performed with citrated blood as described within 2 h of blood collection by using a laminar flow chamber (Hele-Shaw type), previously described in detail (25). Different from our previous investigations, however, we perfused whole blood circulated from a reservoir for 5 min at a shear rate of ∼1,250 s−1. At the end of the perfusion, the blood was replaced by phosphate-buffered saline (PBS), which was run through the flow chamber at half the shear rate of the blood. A total of 2.5 volumes of PBS was washed through the flow chamber. The upper deck of the flow chamber consisted of a glass slide coated with human fibrinogen, which was prepared as previously described (25). At the end of the washing period, the flow chamber was opened, and the area that had been exposed to flowing blood was repeatedly rinsed with 2% SDS in 0.068 M TRIS buffer, pH 6.8. Proteins were concentrated and resolved by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (26). Platelet actin was then identified by immunoblotting with monoclonal antibody (Sigma) by using a chemiluminescence method (ECL kit, Amersham, Arlington Heights, IL, U.S.A.) detection and densitometric quantification. The number of platelets was estimated from a calibration curve.
Susceptibility of lipoproteins to oxidative modification
Blood was drawn into EDTA-containing tubes. Samples of plasma were treated with dextran sulfate (MW, 50,000; Genzyme, Cambridge, MA, U.S.A.), 0.02 mM, and MgCl2, 50 mM, both final concentrations, to precipitate LDL and very low density lipoprotein (VLDL). After removal of the supernatant, the precipitate was washed with 6% bovine serum albumin and 0.01 mM dextran sulfate and was then dissolved in 4% NaCl. A volume of redissolved precipitate containing 100 μg non-high-density lipoprotein (HDL) cholesterol was combined with 4% NaCl solution to give a total volume of 0.5 ml. This solution was dialyzed in the dark and prepared for oxidation as described by Suzukawa et al. (27). To initiate the latter, freshly prepared CuSO4, 0.05 mM final concentration, was added, and samples were incubated for 3 h at 37°C in a shaking waterbath. Thiobarbituric acid-reactive substances were measured as previously described (28).
All results are reported as means ± SD and were evaluated by analysis of variance for significance by using the SPSS software program (ANOVA); p values >0.05 were considered nonsignificant.
Platelet-aggregation studies showed a highly significant increase in the threshold dose of epinephrine after AGE administration (Fig. 1 and Table 1). There was also a modest but significant increase in collagen concentration required to induce complete aggregation in subjects receiving AGE compared with placebos (Fig. 2 and Table 1). On the other hand, ADP-induced aggregation was not changed by AGE administration. The 95% confidence interval for the median effective dose (ED50) of collagen was 0.63-0.77 μg/ml for placebo recipients and 0.86-1.14 μg/ml for subjects taking AGE. For epinephrine as platelet stimulant, the 95% confidence intervals for the placebo and AGE group were 1.1-1.3 and 4.9-6.9 μM, respectively. The postintervention threshold doses of the different platelet agonists were in the same range as those during placebo supplementation.
Platelet adhesion to fibrinogen measured at moderately high shear rate (1,250 s−1), with whole blood perfusing the laminar flow chamber, was significantly reduced in subjects taking AGE (Table 2). The average reduction was somewhat >30%. There was no significant difference between the adhesion measurements at 3 and 6 months when subjects were receiving AGE or placebo. The fact that there was no significant difference between the 3-month (which include samples of subjects during the first and second intervention arm) and 6-month adhesion measurements in the placebo group is evidence in favor of no longterm carryover effects of the garlic treatment. Whereas the adhesion measurements at 3 months were done in all individuals tested, the 6-month measurements, because of the shorter duration of the second intervention period, were done in only seven and eight subjects for placebo and AGE groups, respectively. The 95% confidence interval for fibrinogen adhesion in the placebo group was 0.57-0.90 × 106 adherent platelets/cm2 and for the AGE group of subjects, 0.88-1.32 × 106 adherent platelets/cm2. Postintervention measurements of platelet adhesion were in a similar range as those obtained while study subjects received placebo supplements.
The tendency of lipoproteins to undergo oxidation stimulated by copper salts was evaluated by determining the malonyldialdehyde production. While taking AGE, study subjects showed a decreased accumulation of malonyldialdehyde compared with the accumulation when they were receiving placebos or after intervention (Table 3). Although the difference between these two intervention treatments did not reach statistical significance, there appears to be a trend toward lower susceptibility in subjects taking AGE.
The reduction in platelet aggregation induced by epinephrine and collagen and in platelet adhesion to fibrinogen-coated surfaces is of interest as there are few if any ex vivo studies measuring these platelet functions in subjects taking long-term dietary supplementation with garlic preparations. Short-term investigations of a variety of garlic preparations ranging from commercially available garlic powders (14) to diethyl ether extracts of fresh garlic (17) gave variable results. Although some of them had flawed study designs (i.e., uncontrolled or nonrandomized), most of the studies tested the response to platelet agonists at fixed concentrations, thus making it difficult to recognize small inhibitory effects of the test preparations. The major difference between these and our studies is the long-term administration of stable organosulfurs (e.g., S-allylcysteine and S-allylmercaptocysteine (23) contained in AGE that are quite different from the thioallyls of fresh garlic extracts and of various dried-garlic preparations). A further point of difference is the far higher dosage of the garlic preparation given in our study.
To our knowledge, there have been no studies in which platelet adhesion in humans was investigated. The adhesion studies reported in animals (13) are difficult to compare with our own, as they involved not only a different species but also used short-term infusions of garlic preparations or of ajoene, a highly unstable compound. Also the adhesive surface differed, as in our study, fibrinogen-coated surfaces were tested. Decrease of adhesion was reported at "low" shear rate (212 s−1) but not at high shear rate (1,690 s−1; 13)
The reproducibility of adhesion measurements by laminar flow chamber is extremely good. This can be seen from a comparison of measurements during placebo administration with those performed after intervention. In previous studies using platelet rich plasma as perfusate (25), we have also been able to show the high degree of reproducibility of this method when tested over a period of months. The differences in platelet adhesion noted between placebo- and AGE-administration periods are probably not due to dietary changes, as the participants did not alter their diets during the intervention trial. As previously shown (22), the weight of the study participants remained stable during the study period, and cholesterol and other lipid levels varied <10% from their baseline values. Increasing the content of cholesterol in the platelet membrane has been reported to have stimulatory effects on platelet aggregation, especially induced by epinephrine (29). Whether the reduction in total and LDL cholesterol observed in response to AGE administration was sufficient to alter platelet cholesterol and thereby responsiveness to epinephrine is unknown at this time, but we must keep this in mind as a possible explanation of the aggregation results obtained.
Most if not all of the biologic effects of garlic have been ascribed to the organosulfur compounds in it (18). Invariably these compounds contain an allyl group at one end of the molecule, a variable number of sulfurs in the center, and an allyl, methyl, cysteine, or other group at the other end of the molecule. Organosulfurs are readily cleaved in vivo, separating into SH-reactive compounds. This may in fact be one of the principal mechanisms of action of these garlic compounds. It certainly could explain the reduction in platelet adhesion to fibrinogen. The major platelet integrin, gpIIb/IIIa, has a high content of SH groups and has been shown by Apitz-Castro et al. (30) to be very sensitive to the thioallyl compound ajoene. This heterodimer protein is the receptor for fibrinogen on the platelet surface and plays a prominent role in platelet aggregation and adhesion (31). The obvious discrepancy between the effect of AGE on fibrinogen-mediated adhesion and ADP-induced platelet aggregation, both dependent on the major platelet integrin, deserves some comment. Whereas adhesion is mediated by the interaction of a relatively small fraction of the total number of receptors, aggregation leads to the activation of the entire complement of gpIIb/IIIa heterodimers. Assuming some correspondence between the functional activity and the observed inhibition, we can see that a majority of the heterodimers remain functionally intact and thus can interact with fibrinogen. We submit that a small reduction of functionally competent receptors affects adhesion as platelets become more susceptible to early dislodgment, whereas a normal aggregation process can be sustained in the presence of a majority of unaffected gpIIb/IIIa molecules. Epinephrine-induced platelet aggregation was found to be highly sensitive to AGE, far more than the aggregation induced by collagen. The epinephrine receptor on platelets, which belongs to the α-adrenergic group of receptors, as well as receptor for collagen, has SH groups important for their biologic functions.
Oxidative changes in lipoproteins are thought to play a role in atherosclerotic processes (32). It was therefore of interest that administration of a garlic preparation reduced the susceptibility of lipoproteins to oxidation or at least produced a downward trend. Even though our studies did not attain statistical significance, the number of subjects studied was small, and a larger study population could have produced a significant reduction. AGE has been shown to reduce in vitro oxidation of tocopherols and of ascorbic acid (33); to protect cultured hepatocytes from injury by carbon tetrachloride, which is mediated by free radicals (34); and to inhibit formation of thiobarbituric acid-reactive substances and emission of low-level chemiluminescence in butyl hydroperoxide-initiated liver microsomes (23). Our results are consistent with these observations.
We conclude from these results that AGE administration can produce an inhibition of some of the platelet functions important for initiating thromboembolic events in the arterial circulation. Together with its reduction of total and LDL cholesterol and blood pressure, AGE provides a combination of therapeutic effects directed against the major pathogenic factors of atherosclerosis and associated thrombotic events.
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