The inhibition of specific flavonoid on the in vitro platelet aggregation induced by collagen, arachidonic acid and thromboxane A2 (TxA2) agonist, seems to be related mostly to their ability to compete for binding to the TxA2 receptor (TP). The aim of this study was to analyze the effect of soy isoflavone and equol in terms of inhibiting the platelet aggregation and sCD40L release stimulated by ristocetin, an in vitro-activator of glycoprotein Ib/IX/V, in platelets from postmenopausal women. When platelets were stimulated by 0.75 mg/ml ristocetin, equol (10 μM) exhibited a greater inhibitory activity on platelet aggregation (~68%) than genistein or daidzein. The effect of equol was dependent on the concentration of platelet aggregation agonist. In the presence of ristocetin (0.75 mg/ml, 1.125 mg/ml and 1.5 mg/ml), the inhibitory effect of 10 μM equol was 68% ± 5%, 54% ± 4% and 31% ± 5%, respectively. Equol (10 μM) was a potent inhibitor (~35%) of sCD40L release when stimulated with 1.5 mg/ml ristocetin. However, no significant differences were noted in platelets incubated in the presence of genistein or daidzein and stimulated by ristocetin. On the other hand, SQ29548, a high TP antagonist, also inhibited the sCD40L release stimulated by ristocetin. Finally, 10 μM of genistein, daidzein or equol did not significantly affect the thromboxane B2 production when platelets were incubated with 1.5 mg/ml ristocetin. The relevance of this study was to find that equol exhibits a potent activity by inhibiting ristocetin-induced sCD40L release, suggesting that soy isoflavone has important biological effects on the hemostatic system. However, clinical trials will be necessary to assess the effect of equol on platelet and their impact on inflammatory markers.
The bean of soy produces isoflavones, principally, genistein, daidzein and glycitein. Also, daidzein is metabo- lized by intestinal microbiota and transformed into equol. Isoflavones have been suggested as the principal chemical constituents responsible for the potential preventive effect of soy bean against risk factor of hormone- related diseases and cardiovascular disease (CVD) [
Platelets initially interact with the sub endothelium via adhesive receptors such as glycoprotein Ib/IX/V receptors, which mediate rolling and tethering of the platelets to von Willebrand factor on the sites of vascular in- jury and induce glycoprotein IIb/IIIa activation, resulting in platelet aggregation [
In the present study, we analyzed the effect of genistein, daidzein and equol in the action of ristocetin release of sCD40L from platelets of postmenopausal women. In addition, the effect of isoflavone and equol on ristocetin- stimulated platelets aggregation was examined to provide the antiplatelet effect.
The study protocol was reviewed and approved by the Institutional Review Board for Research on Human Subjects of the Institute of Nutrition and Food Technology, University of Chile.
Twenty-five women, aged 40 - 60 years, were recruited from Santiago Metropolitan Area, and gave informed consent according to the Declaration of Helsinki. To be eligible for this study, women had to be in menopause at least 6 months, have FSH levels over 20 IU/L, without any type of hormonal treatment during previous 6 months, and not currently using soybean-derived products, or herbal supplement diets. Exclusion criteria included: cigarettes smoking within the last 5 years, diabetes, heavy alcohol consumption (more 30 g/day), hypertension and coexistent major illnesses.
Genistein, daizein and ristocetin were purchased from Sigma-Aldrich Co. (St. Louis, MA, USA). Equol was obtained from Cayman Chemical, Co. (Ann Arbor, MI, USA). The other materials and chemicals were obtained from commercial sources.
Human platelets were prepared as previously described [
A turbidimetric method was applied to measure platelet aggregation, using a Lumi-aggregometer (Chrono-log Corp, Havertown, PA, USA). Platelet suspensions (0.25 ml) were preincubated with various concentrations of genistein, daidzein, equol or an isovolumetric solvent control (0.5% DMSO) for 15 min before the addition of ristocetin. The reaction was allowed to proceed for 6 min, and the extent of aggregation was expressed in light- transmission units. The percentage of transmittance of the isolated platelets was recorded as 0%, and platelet- poor plasma (blank) was recorded as 100%.
The measurement of sCD40L was performed as described by Enomoto et al. [
Values shown in the text, tables, and figures are means ± SD. One way ANOVA test was applied for compari- sons of means, and then Student’s t test was performed. p < 0.05 was considered significant.
In a preliminary assay, we first tested the inhibitory capacity of genistein, daidzein and equol (10 µM) on platelets aggregation induced by 0.75 mg/ml ristocetin. As observed in
When was analyzed the effect of 10 µM isoflavone on sCD40L release reaction stimulated by 1.5 mg/mL of ristocetin (
Ristocetin (1.5 mg/mL) induces a marked increase in TXB2 compared to control (0.80 ± 0.21 vs. 2.79 ± 0.61 ng/ml n = 12). However, 10 µM genistein, daidzein or equol did not affect significantly TxB2 stimulated by
Condition | sCD40L, ng/mL Mean ± SD, N= 6 |
---|---|
Basal | 0.38 ± 0.13 |
Ristocetin | 4.86 ± 0.63 |
Ristocetin + Genistein | 4.71 ± 0.57 |
Ristocetin + Daidzein | 4.81 ± 0.51 |
Ristocetin + Equol | 3.20 ± 0.32* |
Ristocetin + SQ29548 | 0.95 ± 0.21* |
*p < 0.001 compared with ristocetin (control) condition.
ristocetin; 2.85 ± 0.42 ng/ml, 2.93 ± 0.43 ng/ml and 2.76 ± 0.31 ng/ml, respectively.
Besides the classic inflammatory markers, CD40L has been introduced as a new inflammatory marker. Studies on the cellular distribution of CD40L indicate that more than 95% of the circulating CD40L exist in platelets. Platelets express CD40L on their surface upon stimulation, and CD40L is then cleaved and circulates as sCD40L. When expressed on the surface of platelets and exposed to CD40-bearing vascular cells, platelet-associated CD40L is capable of initiating various inflammatory responses [
The main interest of the present study was to investigate the effect of soy isoflavones and equol in terms of inhibiting the aggregation and sCD40L release stimulated by ristocetin on platelets from postmenopausal women. The results of these studies confirm that GPIb/IX/V activity induces sCD40L release from human platelets [
Under normal physiological conditions, platelets play a primary role in hemostasis through their aggregation. However, alteration of platelets activity regulation is an important factor in CVD and thrombosis. Evidence of platelets involvement in these pathologies includes increased TxA2 production and increased TP density [
Several studies, including ours, have demonstrated that inhibition of specific flavonoid of in vitro platelet ag- gregation induced by collagen, arachidonic acid and thromboxane A2 agonist, seems to be related, to a great extent, to their ability to compete for binding to the TP [
Soy-derived isoflavone have received wide attention because of its potential beneficial effects on chronic diseases, such as CVD [
The present study shows new experimental evidence to the biological plausibility of the epidemiological finding that soy-isoflavones decreases CVD morbidity and mortality. The present finding, although preliminary, show that equol exhibits a potent activity by inhibiting ristocetin-induced sCD40L release, suggesting that soy isofla- vone have biologically significant effects on the hemostatic system. However, clinical trials will be necessary to assess the effect of equol on platelet and their impact on inflammatory markers.
This work was supported by grant No 1100299 from National Fund for Scientific and Technological Development of Chile (FONDECYT), grant No ENL007/14 from Universidad de Chile.