Roy L. Williams

trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets

The phytoestrogenic compound trans‐resveratrol (trans‐3,5,4′‐trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans‐resveratrol and pure trans‐resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans‐resveratrol had on the Ca2+ channels in thrombin stimulated platelets. In this study we showed that trans‐resveratrol immediately inhibited Ca2+ influx in thrombin‐stimulated platelets with an IC50 of 0.5 μM. trans‐Resveratrol at 0.1, 1.0 and 10.0 μM produced 20±6, 37±6 and 57±4% inhibition respectively of the effect of thrombin (0.01 u  ml−1) to increase [Ca2+]i. trans‐Resveratrol also inhibited spontaneous Ba2+ entry into Fura‐2 loaded platelets, with 0.1, 1.0 and 10.0 μM trans‐resveratrol producing 10±5, 30±5 and 50±7% inhibition respectively. This indicated that trans‐resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. trans‐Resveratrol also inhibited thapsigargin‐mediated Ca2+ influx into platelets. This suggests that the store‐operated Ca2+ channels are one of the possible targets of trans‐resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. The phytoestrogens genistein, daidzein, apigenin and genistein‐glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans‐resveratrol. We conclude that trans‐resveratrol is an inhibitor of store‐operated Ca2+ channels in human platelets. This accounts for the ability of trans‐resveratrol to inhibit platelet aggregation induced by thrombin.

Dietary phytoestrogens and their synthetic structural analogues as calcium channel blockers in human platelets.

Phytoestrogens have been shown to inhibit platelet activation by blocking platelet calcium channels. This study examined the effect of several synthetic derivatives of trans-resveratrol, genistein, and daidzein on plateletfree intracellular calcium ([Ca2+]i) elevation in thrombin-activated platelets and the possible mechanisms of this inhibitory effect. Studies were conducted on fresh human platelets from healthy volunteers. The fluorescent dye fura-2 was used to monitor [Ca2+]i in platelets. At 10 &mgr;M trans-resveratrol, triacetyl-trans-resveratrol, and trimethoxy-trans-resveratrol produced, respectively, 57 ± 4%, 40 ± 4%, and 21 ± 1% inhibition; genistein, acetylgenistein, and dihydrogenistein produced 51 ± 10%, 26 ± 7%, and 16 ± 2% inhibition, respectively; daidzein and diacetyldaidzein produced 56 ± 5% and 45 ± 10% inhibition of thrombin-induced [Ca2+]i elevation. The inhibitory effect was immediate and appeared to directly affect the calcium influx channels. Phytoestrogen action on [Ca2+]i did not cause alteration in nitric oxide signaling. Tyrosine phosphorylation was not involved in the inhibition of [Ca2+]i elevation by phytoestrogens, because the percent inhibition produced by the tyrosine kinase inhibitor genistein and its inactive analogue daidzein on thrombin-induced and thapsigargin-induced [Ca2+]i elevation was not significantly different for either compound at any concentration tested. Structure–activity relationship studies on this limited set of compounds reveal the requirements for the stilbene pharmacophore for the calcium-blocking activity.

Diethylstilbestrol and tetrahydrochrysenes are calcium channel blockers in human platelets: relationship to the stilbene pharmacophore

The effects of compounds with the stilbene pharmacophore [diethylstilbestrol (DES), DES derivatives, tetrahydrochrysene (THC), and THC derivatives] were examined for their ability to inhibit thrombin-induced Ca(2+) influx in human platelets. DES derivatives (DES dimethyl ether, DES dipropionate, dienestrol, and hexestrol) had lower inhibitory activity than DES. Esterification of DES with the bulky monobenzyl group eliminated inhibitory activity. Unsubstituted THC diol had the lowest inhibitory activity in the series of the THC derivatives bearing substituents in the 5,11 positions. These derivatives, either diethyl or dipropyl, cis or trans, were potent inhibitors of thrombin-induced [Ca(2+)](i) elevation (near 100% inhibition at 10 microM). Therefore, stilbene pharmacophore having bulk out of the plane of the double bond (from the twisting of the two aromatic rings or from addition of all substituents) seems to be requirement for the inhibitory activity. Free hydroxyl groups are also required for inhibitory activity, most likely for hydrogen bonding, since trans-diethyl tetrahydrochrysene dimethyl ether was inactive. Compounds bearing ethyl substituents (DES and THC derivatives) inhibited thrombin-induced release of calcium from the endoplasmic reticulum. These compounds also inhibited thapsigargin-induced Ca(2+) influx. This result implies that these compounds also block store-operated Ca(2+) influx directly, as well as internal Ca(2+) release. Compounds without ethyl substituents (trans-resveratrol, genistein, daidzein, and THC diol) only inhibited calcium influx into platelets.

2-Aminoethoxydiphenyl Borate as a Prototype Drug for a Group of Structurally Related Calcium Channel Blockers in Human Platelets

We have synthesized a series of 2-aminoethoxydiphenyl borate (2-APB, 2,2-diphenyl-1,3,2-oxazaborolidine) analogs and tested their ability to inhibit thrombin-induced Ca2+ influx in human platelets. The analogs were either synthesized by adding various substituents to the oxazaborolidine ring (methyl, dimethyl, tert-butyl, phenyl, methyl phenyl, and pyridyl) or increasing the size of the oxazaborolidine ring to seven- and nine-membered rings. NMR analysis of the boron-containing analogs suggests that each of them exist as a ring structure through the formation of an N→B coordinate bond (except for the hexyl analog). The possibility that these boron-containing compounds formed dimers was also considered. All compounds dose-dependently inhibited thrombin-induced Ca2+ influx in human platelets, with the 2,2-diphenyl-1,3,2-oxazaborolidine-5-one derivative having the weakest activity at 100 μM, whereas the (S)-4-benzyl and (R)-4-benzyl derivatives of 2-APB were approximately 10 times more potent than the parent 2-APB. Two nonboron analogs (3-methyl and 3-tert-butyl 2,2-diphenyl-1,3-oxazolidine) were synthesized; they had approximately the same activity as 2-APB, and this implies that the presence of boron was not necessary for inhibitory activity. All of the compounds tested were also able to inhibit thrombin-induced calcium release. We concluded that extensive modifications of the oxazaborolidine ring in 2-APB can be made, and Ca2+-blocking activity was maintained.

Diethylstilbestrol and other non-steroidal estrogens: Novel class of store-operated calcium channel modulators.

Background: Compounds with the stilbene pharmacophore and other nonsteroidal estrogens have previously been shown to inhibit thrombin-induced elevation of intracellular free calcium ([Ca2+]i) in human platelets. Thrombin elevates [Ca2+]i in platelets predominantly by activating a store-operated Ca2+ entry (SOCE) mechanism, probably involving STIM1 and Orai1 although other components may be involved. Methods: Human platelets were loaded with the Ca2+ sensitive indicator fura-2, various concentrations of stilbene compounds and other nonsteroidal estrogens were added to the platelets, and thrombin was added to elevate [Ca2+]i. The degree of inhibition by each compound was determined at the peak increase in [Ca2+]i induced by thrombin. Results: The additional compounds that were examined in the present study were analogs of diethylstilbestrol (DES), namely trans-resveratrol, hexestrol, tetrahydrochrysene (THC), indenestrol, isoflavones, flavones, and flavanones. DES, indenestrols, and substituted THC diols had the highest inhibitory activity. Dietary polyphenols were less active, and isoflavones were more active than flavones. Glycosides of flavones, flavanones, and isoflavones were inactive. Equol (a product of isoflavone metabolism) had low activity. Among the compounds with a stilbene moiety the presence of unsubstituted phenyl hydroxyls in the para- position were required for activity. Esterification of hydroxyls and bulky substituents at a hydroxyl group diminished or abolished activity. Presence of the ethyl side chains enhanced activity, and shortening or removal of these side chains was detrimental to activity. Presence of the conjugated double bound was necessary for activity. Reduction of the double bond (in fused rings such as equol, dihydrogenistein, indanestrol, or in open chain stilbene derivatives) or repositioning of this double bond outside the stilbene moiety was detrimental to activity, because phenyl rings are not co-planar and have to be at a certain angle to each other. Conclusion: DES likely represents the pharmacophore of this group of nonsteroidal estrogens as an inhibitor of SOCE in platelets.

Seed enhancement studies with Chardonnay wines

Reports regarding the positive health effects associated with the moderate consumption of red wine continue to be reinforced in the current scientific literature. These observations have prompted our laboratory to investigate the development of a white wine which would be enriched with the antioxidant, polyphenolks, associated with the protective influence of red wines. We report here the production of a seed‐enhanced Chardonnay wine which is relatively high in polyphenolks. The analysis of the seed‐enhanced wines for the levels of six important polyphenolks was carried out by high‐pressure liquid chromatography.