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Introduction
Platelets are key elements of human blood. They play a central role in the process of thrombus formation as well as an important role in atherogenesis and the progression of atherosclerotic lesions 38. When platelets come in contact with damaged or disrupted endothelium, they are activated, changing their shape and releasing their granules content 33. A great number of agents, including adenosine diphosphate, epinephrine, collagen, thrombin, arachidonic acid, antigen-antibody complexes, serotonin and vasopresin, can induce platelet aggregation 11,13. Collagen (C) is an extracellular matrix component, which directly binds to receptors that mediate platelet adhesion inducing activation and aggregation. Two receptors are involved in platelet response to collagen; integrin α2 β1 acts adhering platelets to collagen, allowing platelets to interact with the lower affinity receptor glycoprotein VI, which is mainly responsible for platelet activation 37. Adenosine diphos phate (APD), the first known low molecular weight platelet aggregating agent, is a weak platelet agonist. However, ADP plays an important role in platelet function given that, when released from the platelet dense granules, it amplifies the platelet responses induced by others agonists 18. Arachidonic acid (AA) is considered a key component in haemostasis and thrombosis, since the release of AA can trigger platelet aggregation and prostaglandin synthesis. AA in reaction with cyclooxigenase is converted to thromboxane A2, a potent platelet agonist 9. Regarding epinephrine (E), the mechanism by which it induces platelet aggregation is unknown. In some tissues, such as the respiratory epithelium, the activation of the A2AR increases transcellular sodium and chloride cotransport 42.
Several studies have approached platelet aggregation in response to in vitro stimulation of these agonists 9,22,37,42. The fact that platelet aggregation in vivo involves a combi nation of agonists, is well known. Consequently, to evaluate various agonists simultaneously according to their way of action and their potential synergistic interaction, becomes useful 23. Platelet aggregation is performed to identify abnormal platelet function, to quantify platelet response, and to monitor platelet inhibition through therapeutic drug 29. As an alternative source of natural antithrombotic drugs, the pharmaceutical industry is currently considering garlic to develop better and safer drugs with lower and less side effects. However, the mechanisms by which aqueous garlic extract elicit their antiplatelet effect are not fully known. Garlic (Allium sativum L.) has been used for its antioxidant properties that prevent heart disease including atherosclerosis. Also reduces both plasma cholesterol level and blood pressure 12 and has been recognized as an antiplatelet agent that may contribute to the prevention of cardiovascular disease 34. Raw garlic and some of its preparation like aqueous extracts, garlic oil, aged garlic extract and garlic powder inhibit human platelet aggregation in vitro17 and in vivo (4, 20, 40). Many of these physiological effects exerted by garlic are attributed to volatile sulfur-containing flavor compounds, particularly to thio sulfinates, which are responsible for its pungent aroma 24. Allicin (diallylthiosulfinate) is the most abundant compound, representing about 70% of the overall thiosulfinates formed upon crushing garlic cloves 26. It is produced by the interaction of the nonprotein amino acid alliin (+S-allyl-L-cysteine sulfoxide), with the enzyme alliinase (alliin lyase, EC 4.4.1.4) 24. Allicin and other thiosulfinates provide nearly all the antiplatelet activity of raw garlic homogenates in whole blood aggregometry 27. Variability in antiplatelet activity exerted by garlic cultivars belonging to different argentine ecophysiological groups using collagen as agonist has previously been described by our group 15. Perla INTA has the highest organosulfur levels, pungency and in vitro antiplatelet activity, being the most suitable garlic cultivar to obtain pharmacological products 15. In vitro platelet aggregation can be assayed with an electrical impedance aggregometer using different agonist and several donors. Results may exhibit large variances both among and within platelet donors. Conse quently, it is desirable to use an experimental design with several agonists and donor’s in order to make their results comparable.
Therefore, this study was conducted to determine the in vitro antiplatelet activity of aqueous extract of Perla INTA cultivar induced by different agonists and their mixtures.
Materials and methods
Chemicals
All chemicals used were of analytical grade. Collagen (1mg mL-1) was purchased from Chrono-log Corp, adenosine diphosphate, arachidonic acid and epinephrine were obtained from Helena Laboratory (Beaumont, TX, USA). Stock solutions of each agonist were prepared in deionized water and mixed gently until completely dissolved at the following concentra tions: 10 μM ADP, 0,5 mM AA, epinephrine 50 μM. These solutions were stored at -20 °C, except for the collagen solution, which was stored at 4 °C.
Preparation of aqueous garlic extracts
Fresh garlic bulbs of Perla INTA cultivar were chosen from the germplasm collection of INTA La Consulta, Mendoza, Argentina. The bulbs were peeled and blended for 1 min in deionized water (1:10 w/v). The aqueous garlic extract was collected, filtered and kept at room temperature for 15 min to allow enzymatic hydrolysis of the flavor precursors. After that, the extracts were kept frozen at -80 °C.
Aqueous garlic extracts characterization
The characterization of Perla INTA aqueous garlic extract was carried out according to their flavor precursor levels, thiosulfinates, allicin content, and pungency as previously described González et al. (2009).
Platelet aggregation
Platelet aggregation was measured in vitro using a whole blood electrical impedance aggregometer 10. Blood was collected from nine non-smoker healthy donors (5 women, 4 men), between 26-45 years old, who had no any previous disease and had not taken aspirin or any medication, and all had given informed consent. They also had abstained from eating Alliums or other known platelet-inhibitory food or beverages in the previous 2 weeks, for at least 7 days prior to venipuncture. A complete blood count, biochemical analysis and the clotting time was carried out following the Ivy method. Clotting times less than 9.5 minutes were considered normal.
Blood samples were collected by venipuncture (Hematology Service, Central Hospital, Mendoza, Argentina) in tubes containing sodium citrate anticoagulant (3.8%, 1 vol of anti coagulant per 9 vol of blood). Afterward,blood samples were diluted in equal volume of TRIS buffered saline (TBS, pH 7.4) and vortexed. The diluted blood was maintained at room temperature during the experiment and used within 2 h of donation.
Aliquots of 1mL of blood/TBS were incubated for 3 min at 37 °C, then a dose of aqueous garlic extract was added and platelet aggregation was induced by the addition of different agonists or their mixtures. Changes in the impedance was recorded over 6 min. The change in impedance is proportional to the amount of platelet aggregation. All treatments were eval uated with each donor’s blood four times. In vitro antiplatelet activity (AAI) was expressed as percentage of inhibition of platelet aggregation, compared to control samples prepared in the same way but without adding aqueous garlic extract. The control is considered as 100 % the aggregation or 0% the inhibition for each agonist.
Table 1 shows agonists concentrations prepared as described Bordia et al. (1996); Soloviev et al. (1999); Rahman and Billington, (2000).
Based on previous results a concentration of 7 mg of aqueous garlic extract per mL of blood was chosen for platelet aggre gations reactions 15. This concentration is equivalent to a daily consumption of 1 clove (1.5 g of garlic for a person weighing 70 kg) 43.
Statistical analysis
Values were expressed as means ± SEM. Data were analyzed by analysis of variance (ANOVA) to test the significant differences. Means were compared using Tukey’s test. The results were considered significant at P ≤ 0.05 unless specified otherwise. Statistical analysis was performed using the software STATISTICA for Windows 6.0. Means of each treatment were compared by least significant difference (LSD) test; P ≤ 0.05 were considered to be significant.
Results
None of the donors included in the assay showed a value of impedance higher than 5 Ω in platelet aggregation induced by ADP. Consequently, this agonist did not cause a significant aggregation in the respective samples. A similar behavior was observed with epinephrine.
Inhibition percentage in platelet aggregation showed significant differences (Figure 1, page 50) among different agonists as well as among their combinations.
Figure 1: In vitro inhibition of platelet aggregation exerted by Perla INTA aqueous extracts on platelet aggregation induced by different agonists and their mixtures.
The inhibitory effect on platelet aggregation induced by collagen ranged between 22.5-100%. After adding collagen before aggregation onset, aggregation slopes showed a delay of about 60 s or more, then followed by a steady increase in aggregation. This delay was greater than control samples when garlic extract was added (data no shown).
Antiplatelet aggregation, when AA was used, ranged between 75.7-100%. The aggre gation slopes showed a monophasic aggregation response to AA.
On the other hand, antiplatelet effects exerted by Perla INTA aqueous extract were studied by using different agonist mixtures. When a collagen-AA mixture was used as platelet agonist, antiplatelet activity ranged between 3.84-26%. In addition, in vitro Perla INTA aqueous extract suppressed platelet aggregation induced by collagen-epinephrine and ADP-epinephrine induced human platelet in a wide range, 10.23-48.48% and 20-80%, respectively. Significant synergistic interaction was observed with collagen in combination with epinephrine, AA and ADP, and ADP in combination with epinephrine when added simultaneously at the selected concentration. Thus, higher impedance values were observed when aggregation was induced by two agonists combination (Figure 2, page 50).
Figure 2: Aggregation platelet in vitro: synergistic effect exerted by different combinations of agonists.
In general, the antiaggregatory effect showed by Perla INTA aqueous extract on platelet aggregation induced by paired of agonists were minor than antiaggregatory effects observed when platelet aggregation was induced by each agonist alone (Figure 1, page 50).
Taking into account average antiplatelet activity of all agonists and mixtures used, this biological activity varied between 18.51% ± 2.43 and 84.40% ± 2.15. The highest antiaggregatory effect was observed with AA and the lowest effect with collagen-AA mixture. ANOVA analysis revealed an interaction effect between donor and agonist (or mixtures) at P ≤ 0.05 (Table 2).
Table 2: ANOVA table of in vitro antiplatelet activity variation due to donor, agonist, and their interaction.
ANOVA analysis of donor, agonist and their interaction resulted in 15.2%, 54.74% and 26.2 % of variation to the AAI in this study, respectively, indicating that agonist was the main factor determining AAI, and that donor and donor and agonist interaction, played a minor role. There were not significant differences in platelet aggregation inhibition response between women and men.
The chemical profile of the main organosulfur compound present in Perla INTA aqueous garlic extract is shown in Table 3.
Table 3: S-alk(en)yl-L-cysteine sulfoxides (ACSOs), thiosulfinates, allicin, and pungency of Perla INTA garlic cultivar.
Alliin was the most abundant ACSO, representing more than 70% of the total content of ACSOs, whereas isoallin content was nearly to 23% and methiin content represented less than 10%. Also, this cultivar showed high levels of thiosul finates, allicin, and pungency.
Discussion
Under pathological conditions, platelets may become more proadhesive/procoagulant, which leads platelets to adhere to an injured area, and to subsequently release a lot of active constituents inducing plug formation 16. For that reason, antiplatelet therapy is used as a strategy to prevent vascular thrombosis and vascular disorders. Several researchers have shown that garlic and their sub products have in vivo1,2,8,44 and in vitro7,28,35 antiplatelet activity. These studies have involved the use of collagen, ADP, AA or epinephrine as agonists, but not their combinations. In vivo, more than one agonist and different combi nations are involved in platelet activation. Therefore, the study of the antiplatelet effect of aqueous garlic extracts over platelet activity induced by different agonists and their combi nations was considered of great contribution to the understanding of the factors involved in this activity. Two types of aggregation can be distinguished in vitro: primary aggregation that is reversible and occurs without the release reaction and secondary aggregation that is irreversible and is associated with the release reaction 36. The reversibility of platelet aggregation induced by each dose of agonists included in this study was assessed from the shape of aggregation tracings. A monophasic curve indicating irreversible aggregation was observed with all the agonist and the combinations evaluated. We found that when platelet aggregation was induced by ADP or epinephrine, the aggregation induced was lower than 40% and 30% respectively. Similar results have been reported 6,14,25. Mammalian platelets vary widely in their responses to epinephrine, in part due to they can act via excitatory α receptors and inhibitory β receptors 41. Some people have reduced platelet responsiveness to epinephrine due to the lack of these receptors 39. This fact might explain the results obtained in our work. On the other hand, when these agonists were used in combination, a synergistic effect was observed. This was evidenced by higher responses than the expected for ADP or epinephrine alone. As it has been previously reported by other authors 19,36.
Regarding collagen, we found that a concentration of 1 μg mL-1 induces of 100% of platelet aggregation in vitro. In addition, we observed that the concentration of Perla INTA aqueous garlic extract used inhibited the platelet aggregation induced by this agonist. Aqueous garlic extracts contain mainly allicin 26. Allicin easily go through cellular membranes and can react with different sulfhydryl (SH)-containing proteins blocking their free sulfhydryl residues 30. We propose that the antiplatelet effect of Perla INTA aqueous garlic extract is due, at least partially, to the inhibition of collagen binding to specific receptors on platelet surface. Consequently, platelet adhesion and platelet activation could be inhibited. This results are in agreement with Manaster et al (2009), who demonstrated that allicin inhibit agonist-induced washed platelet activation via inhibition of platelet signaling. According to Baghalian et al. (2005) the minimum allicin content to ensure pharmaceutical and economic viability of garlic should be 4.5 mg g-1 fw. The higher level of allicin present in this cultivar could be taken as an opportunity to elaborate pharmacological products based on its health-benefit quality.
A monophasic curve in response to a concentration of 0.5 μM of AA was obtained. Both aqueous and organic garlic extracts have been found to inhibit several steps of the AA-cascade in platelets 3. Moreover, garlic extracts inhibit incorporation of labeled AA into platelet phospolipids and inhibit AA-metabolizing enzymes in platelets 15. Ali (1995) showed that raw garlic inhibited cyclooxygenase activity non-competitively and irreversibly. Our results confirm these observations. The highest antiaggregatory effects exerted by Perla INTA aqueous extract were observed when AA was used as agonist.
Platelet aggregation in vivo probably involves a combination of agonists, being collagen more important at the beginning, thrombin becomes important later on, and with other agonists in different mixtures throughout 19. Platelets exhibit diverse responses, including shape change, aggregation, and secretion in response to a variety of agonists. These agonists differ in their intrinsic ability to produce the effects on physiological responses by platelet 14,21. The degree of inhibition depends on the potential activity of each platelet agonist used in the present work. Perla INTA aqueous extract strongly inhibited platelet aggregation induced by a strong agonist like collagen and also by AA. However, the antiaggregatory effect was lower when the aggregation was induced by collagen together with AA. These results demonstrate the synergistic interaction exerted for the combination of agonists used. These results are in agreement with those reported by Razi et al (2005) and Huang and Detwiler (1981), who showed the potentiated response to platelet aggregation in combination of collagen and AA, and epinephrine. The synergism between the different platelet agonists increases platelet aggregations. Full platelet aggregation can also be induced by the simulta neous addition of subthreshold levels of platelet stimuli, which fail to induce platelet aggre gation on their own merit 21.
Other important aspect that was evaluated was the influence of donor, agonist and their interaction. Although, the analysis revealed an interaction effect between donor and agonist, the type of agonist was the main factor determining of in vitro antiplatelet activity. These results were consistent with previous reports that showed that the response of platelets to agonists has a large inter-individual variability within the population 31,32.
Conclusion
This study reveals the potential antiaggregatory effect of Perla INTA aqueous garlic extract, when this activity is induced by different agonists and their mixtures. Several mech anisms are involved in this inhibitory effect, and they include the modification of platelet membrane, a direct inhibitory effect on the AA metabolism and lipoxygenase enzyme and inhibition of calcium mobilization. The modification of platelet membrane inhibits the signaling pathway and consequently the platelet activation and platelet adhesion. When enzymes involved in several steps of AA-cascade are inhibited, endoperoxides interme diates are not converted to prostanglandins and thromboxane B2 which are potent platelet aggregators. Furthermore, a synergistic effect was evidenced. This aspect is important since the therapeutic effect of antiplatelet herbal drugs can be affected. However, the biochemical basis of these synergistic responses needs further exploration.
