1 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 transresveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. 2 An essential component of the aggregation process in platelets is an increase in intracellular free Ca 2+ ([Ca 2+ ] i ). Ca 2+ must enter the cell from the external media through speci®c and tightly regulated Ca 2+ channels in the plasma membrane. The objective of this study was to characterize what e ect trans-resveratrol had on the Ca 2+ channels in thrombin stimulated platelets. 3 In this study we showed that trans-resveratrol immediately inhibited Ca 2+ in¯ux in thrombinstimulated platelets with an IC 50 of 0.5 mM. trans-Resveratrol at 0.1, 1.0 and 10.0 mM produced 20+6, 37+6 and 57+4% inhibition respectively of the e ect of thrombin (0.01 u ml 71 ) to increase [Ca 2+ ] i . 4 trans-Resveratrol also inhibited spontaneous Ba 2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 mM trans-resveratrol producing 10+5, 30+5 and 50+7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca 2+ channel activity in the platelets in the absence of agonist stimulation. 5 trans-Resveratrol also inhibited thapsigargin-mediated Ca 2+ in¯ux into platelets. This suggests that the store-operated Ca 2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca 2+ stores to initiate in¯ux of Ca 2+ into the cell. 6 The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory e ects against thrombin similar to those seen with trans-resveratrol. 7 We conclude that trans-resveratrol is an inhibitor of store-operated Ca 2+ channels in human platelets. This accounts for the ability of trans-resveratrol to inhibit platelet aggregation induced by thrombin.

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 Ca 2ϩ 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 N3 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 Ca 2ϩ influx in human platelets, with the 2,2-diphenyl-1,3,2-oxazaborolidine-5-one derivative having the weakest activity at 100 M, 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 thrombininduced calcium release. We concluded that extensive modifications of the oxazaborolidine ring in 2-APB can be made, and Ca 2ϩ -blocking activity was maintained.2-Aminoethoxydiphenyl borate (2-APB; systematic name: 2,2-diphenyl-1,3,2-oxazaborolidine, compound 1) was originally described as an inhibitor of inositol 1,4,5-trisphosphate receptors (IP 3 R) in a variety of cells (Maruyama et al., 1997) and has been extensively used as a probe for examining calcium influx processes. 2-APB was inappropriately used in several studies to imply that IP 3 Rs were involved in storeoperated Ca 2ϩ entry (SOCE) . For a review of our current understanding of the nature of SOCE and how the SOCE channels are regulated, see Parekh and Putney (2005). It has been suggested that SOCE is mediated by members of the transient receptor potential canonical (TRPC), family although this has not been accepted universally (Parekh and Putney, 2005).Upon further investigation, it became evident that 2-APB, along with inhibition of IP 3 Rs, was also inhibiting SOCE directly in several cell types (Bakowski et al., 2001;Broad et al., 2001;Diver et al., 2001;Dobrydneva et al., , 2002Gregory et al., 2001;Iwasaki et al., 2001;Ma et al., 2001;Prakriya and Lewis, 2001;Bootman et al., 2002). The inhibition of SOCE seemed to be at an extracellular site by a mechanism not involving intracellular IP 3 Rs. In our study showing that 2-APB blocked SOCE in human platelets (Dobrydneva and Blackmore, 2001), we tested whether several other structurally related compounds were also inhibitors of SOCE. We Some of the findings in this manuscript were presented at two meetings of the Virginia Academy of Science (Dovel et al., 2002;Thadigiri et al., 2003) and ...