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School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, England, UK
3 To whom correspondence should be addressed. E-mail: k.rahman{at}livjm.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • aged garlic extract, platelets • A23187 • calcium ions • fura-2 AM
One of the factors that contributes to cardiovascular disease is an increased tendency of platelets to aggregate. Platelets adhere to the exposed collagen, laminin, and von Willebrand factor in the injured vessel, a process that is known as platelet activation. This result can also be achieved through agonists, such as ADP, collagen, and thrombin. It is now recognized that garlic and its various preparations have the ability to inhibit platelet aggregation both in vivo and in vitro (1–3). One such garlic preparation is an aged garlic extract (AGE),4 which inhibits platelet aggregation in vivo (4–7). AGE also inhibits platelet aggregation when platelets are stimulated by agonists such as ADP, collagen, and epinephrine (4–7). However, the mechanisms by which AGE inhibits platelet aggregation have not yet been established.
Calcium (Ca2+) mobilization is a critical step in various aspects of platelet activation such as aggregation, shape change, and secretion (8–13). Stimulation of human platelets with various agonists elevates Ca2+ in 2 ways, i.e., the release of Ca2+ from intracellular stores and the activation of Ca2+ entry through plasma-membrane channels. Agonists interact with receptors coupled to phospholipase C (PLC) via G-proteins contained within the plasma membrane, leading to the formation of diacylglycerol (DAG) and inositol-triphosphate (IP3), which then stimulate the release of Ca2+ from intracellular stores. The emptying of the Ca2+ stores regulates Ca2+-conducting channels within the plasma membrane and initiates the influx of external Ca2+ into the platelet cytosol. This sustained level of Ca2+ activates phospholipase A2 (PLA2), leading to the formation of thromboxane A2 and the subsequent release of secretory granules to complete the aggregatory process (8–13).
Ca2+ plays a pivotal role in platelet aggregation. Hence, a possible mechanism by which AGE may inhibit platelet aggregation is by interfering with Ca2+ mobilization, either by blocking the influx of this ion into the platelet or by its chelation within the platelet cytosol. AGE is a complex mixture and was reported previously to have metal-chelating properties (14). In this preliminary in vitro study, we report the effects of AGE on platelet aggregation and Ca2+ mobilization, using the fluorescence calcium indicator dye, fura-2 acetoxymethyl ester (fura2 AM), in ADP- and A23187-activated platelets.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ca2+ measurement. Fura-2 AM at a final concentration of 2 µmol/L was added to PRP, and the mixture was incubated for 45 min at 37°C. After washing, fura 2-loaded platelets were resuspended in HEPES-Tyrode's buffer, pH 7.4 at a concentration of 2.5 x 105 cells/mL.
Due to the dark color of AGE, a diethyl ether extract was prepared by diluting 1 part AGE with 2 parts diethyl ether; this was left to stand at room temperature for 5 min, after which the diethyl ether extract was removed and dried under oxygen-free nitrogen gas. The residue was resuspended in PBS, pH 7.2, to its original volume.
Washed platelets were incubated with either AGE or its diethyl ether extract at a final concentration of 25% (v:v) for 10 min at 37°C. These experiments were performed in the presence of 1 mmol/L calcium chloride, 1 mmol/L calcium chloride plus 1 mmol/L EGTA, or EGTA alone. Finally, the platelets were stimulated by the addition of ADP at a final concentration of 8 µmol/L or by the Ca2+ ionophore A23187 at a final concentration of 5 µmol/L. Fura-2 fluorescence was measured using a VARIAN Cary Eclipse Fluorescence Spectrophotometer with an excitation wavelength alternating every 0.5 s from 340 to 380 nm; the emission wavelength was set at 510 nm. The [Ca2+]i values were determined from the ratio of fura-2 fluorescence intensity at 340 and 380 nm, using the ratio-scan function and the software version 1.1(132) for the Cary Eclipse machine.
Platelet aggregation. Platelets loaded with fura-2 that had been treated with AGE were also tested for their ability to aggregate in the presence or absence of external calcium. Aggregation was measured as the total percentage of aggregation (%) using a PAP-4D Platelet Aggregation Profiler (Bio/Data) and compared with the control for aggregation induced by ADP (8 µmol/L).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the presence of ADP, 
70% platelet aggregation occurred (Fig. 1). In the experiments designed to observe calcium mobilization, ADP-induced platelet aggregation increasd intracellular calcium ions, such that a peak was observed at 0.5 min (Fig. 2). This finding confirms earlier studies indicating a link between an increase in intracellular calcium ions and platelet aggregation (9–13). When AGE was included in these experiments, significant inhibition of platelet aggregation occurred and the increase in intracellular calcium ions was also suppressed. In fact, platelets that had been preincubated with AGE had significantly lower calcium ion concentrations, thereby suggesting that AGE is acting as a possible calcium-ion chelator (Fig. 2).
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0.5 min (Fig. 3). AGE had effects on platelet aggregation similar to those observed in the experiments in which ADP was used as an agonist. Platelet aggregation was significantly reduced, as was the increase in intracellular calcium-ion concentration. A23187 is an ionophore, which results in the influx of calcium ions into platelets (15). Thus, it is likely that AGE interferes either with this step or with steps involved within platelets in the generation of second messengers.
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Fura-2–loaded platelets, when stimulated with ADP, had lower percentages of aggregation than platelets that had not been exposed to the fura-2 (Fig. 1). The addition of external calcium ions did not cause an increase in the aggregatory response to ADP. The addition of external calcium ions decreased aggregation, compared with fura-2–loaded platelets in the absence of calcium ions. In the presence of AGE and external calcium, aggregation was reduced in a similar manner to fura-2–loaded platelets in the presence of both external calcium ions and EGTA (Fig. 1).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We found that AGE appeared to suppress calcium mobilization and platelet aggregation induced by both ADP and the calcium ionophore A23187. There was significant variation among individuals in their responsiveness to the individual agonists. Although an inhibitory effect on calcium mobilization was apparent for the results shown here, the magnitude varied among the individual blood donors. However, the data suggest that AGE may inhibit platelet aggregation by suppressing calcium mobilization. Another probable explanation is a "quenching" effect caused by the dark coloring of AGE on the fluorescence signal emitted by fura-2. To rule out this possible artifact, a diethyl ether extract of AGE was prepared and tested. Dillon et al. (14) reported previously that AGE has metal-chelating properties. In the present study, a diethyl ether extract of AGE did not inhibit platelet aggregation and did not suppress agonist-induced calcium mobilization. The results together imply that the inhibitory effects of AGE on platelet aggregation may be due to its metal-chelating properties. It is also possible that AGE or one of its constituents alters intracellular messenger activity.
The variation in results could be due to many different factors. There are numerous problems associated with the use of human volunteers, such as gender (17,18), age, availability of viable platelets, diet, any undisclosed medication, and preexisting medical conditions that may affect platelet function.
Although this is a preliminary study, the results presented suggest that AGE exerts its inhibitory effects on platelet aggregation by suppressing calcium mobilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosure: No relationships to disclose.
4 Abbreviations used: AGE, aged garlic extract; DAG, diacylglycerol; fura2 AM, fura-2 acetoxymethyl ester; IP3, inositol-triphosphate; PLA2, phospholipase A2; PLC, phospholipase C; PRP, platelet-rich plasma.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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