Platelet activation is closely associated with an increase in intracellular Ca(2+) concentration. Various compounds including Ca(2+) ionophores are able to trigger platelet aggregation by increasing intracellular Ca(2+) concentration in platelets. In the present study, we monitored the effect of the phytoestrogen ferutinin, which acts as a Ca(2+) ionophore in human blood platelets; its ionophore-like properties include upregulation of [Ca(2+)](in), activation of fibrinogen receptors and increased fibrinogen binding. Using spectrofluorometry and triple-color flow cytometry, we demonstrate that ferutinin increases [Ca(2+)](in) in both isolated platelets and platelets in whole blood from humans. This effect was almost completely blocked by the Ca(2+) chelator EGTA and was not sensitive to either Gd(3+) or econazole, which inhibit VOC and SOC channels, respectively. Nor was the effect sensitive to thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) ATPases. Ferutinin stimulated the expression of the active form of the GPIIb-IIIa complex and whole blood platelet aggregation only weakly and had no statistically significant effect on the binding of fibrinogen. These results demonstrate apparently inconsistent effects of ferutinin, which raises intraplatelet Ca(2+) concentration but fails to have an effect on spontaneous blood platelet aggregation. This pattern of responses may be caused by the combination of ferutinin's Ca(2+) ionophoric and estrogenic properties.

Calcium ionophoretic properties of ferutinin were re-evaluated in solvent-containing bilayer lipid membranes. The slopes of conductance-concentration curves suggest that in the presence of a solvent in the membrane the majority of complexes appear to consist of a single terpenoid molecule bound to one Ca ion. By contrast, the stoichiometry of ferutinin-Ca(2+) complexes in acetone determined using the conductometric method was 2 : 1. While the cation-cation selectivity of ferutinin did not change, the cation-anion selectivity slightly decreased in solvent containing membranes. FT-IR and NMR data together with DFT calculations at the B3LYP/6-31G(d) level of theory indicate that in the absence of Ca ions ferutinin molecules are hydrogen-bonded at the phenol hydroxyl groups. The variations of absorption assigned to -OH and -C-O stretching mode suggest that ferutinin interacts strongly with Ca ions via the hydroxyl group of ferutinol and carboxyl oxygen of the complex ether bond. The coordination through the carbonyl group of ferutinin was demonstrated by theoretical calculations. Taken together, ferutinin molecules form H-bonded dimers, while complexation of Ca(2+) by ferutinin ruptures this hydrogen bond due to spatial re-orientation of the ferutinin molecules from parallel to antiparallel alignment.

It is well known that the terpenoid ferutinin (4-oxy-6-(4-oxybenzoyloxy) dauc-8,9-en), isolated from the plant Ferula tenuisecta, considerably increases the permeability of artificial and cellular membranes to Ca-ions and produces apoptotic cell death in different cell lines in a mitochondria-dependent manner. The present study was designed for further evaluation of the mechanism(s) of mitochondrial effects of ferutinin using isolated rat liver mitochondria. Our findings provide evidence for ferutinin at concentrations of 5-27 µM to decrease state 3 respiration and the acceptor control ratio in the case of glutamate/malate as substrates. Ferutinin alone (10-60 µM) also dose-dependently dissipated membrane potential. In the presence of Ca-ions, ferutinin (10-60 µM) induced considerable depolarization of the inner mitochondrial membrane, which was partially inhibited by EGTA, and permeability transition pore formation, which was diminished partly by cyclosporin A, and did not influence markedly the effect of Ca on mitochondrial respiration. Ruthenium Red, a specific inhibitor of mitochondrial calcium uniporter, completely inhibited Ca-induced mitochondria swelling and membrane depolarization, but did not affect markedly the stimulation of these Ca-dependent processes by ferutinin. We concluded that the mitochondrial effects of ferutinin might be primarily induced by stimulation of mitochondrial membrane Ca-permeability, but other mechanisms, such as driving of univalent cations, might be involved.