ABSTRACT
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.
Subject(s)
Blood Platelets/drug effects , Calcium Channel Blockers/pharmacology , Chrysenes/pharmacology , Diethylstilbestrol/pharmacology , Adult , Barium/metabolism , Blood Platelets/metabolism , Calcium/metabolism , Calcium Channels/drug effects , Calcium Channels/metabolism , Calcium Signaling/drug effects , Chrysenes/chemistry , Diethylstilbestrol/chemistry , Female , Fura-2/metabolism , Humans , Hydrogen Bonding , Ion Transport/drug effects , Ionophores/chemistry , Ionophores/pharmacology , Isoflavones/chemistry , Isoflavones/pharmacology , Male , Middle Aged , Molecular Structure , Phytoestrogens , Plant Preparations/chemistry , Plant Preparations/pharmacology , Platelet Activation/drug effects , Strontium/metabolism , Structure-Activity Relationship , Substrate Specificity , Thapsigargin/pharmacology , Thrombin/pharmacologyABSTRACT
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 platelet free 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 microM-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.