Functional bioassays

Tested and reference compounds were dissolved in DMSO at a concentration of 10^–2 M. Serial dilutions were prepared in 96-well microplate in assay buffer and eight to ten concentrations were tested.

A cellular aequorin-based functional assay was performed with recombinant Chinese hamster ovary cells expressing mitochondrially targeted aequorin, human G-protein-coupled receptors, and the promiscuous G protein α16 for 5-HT_2A. The full description of this assay is in Supplementary materials.

Isolated rat aorta was used to determine the antagonistic activity of studied compounds for 5HT_2A-receptors. The male Wistar rats weighting 200–350 g were anesthetized with thiopental sodium (75 mg/kg i.p.) and the aorta was dissected and placed in a Krebs–Henseleit solution (NaCl 118 mM, KCl 4.7 mM, CaCl₂ 2.25 mM, MgSO₄ 1.64 mM, KH₂PO₄ 1.18 mM, NaHCO₃ 24.88 mM, glucose 10 mM, C₃H₃O₃Na 2.2 mM, EDTA 0.05 mM), denuded of the endothelium, cleaned of surrounding fat tissues and cut into 4-mm-long rings. The aortic rings were incubated in 30 ml chambers filled with a Krebs–Henseleit solution at 37 °C and pH 7.4 with constant oxygenation (O₂/CO₂, 19:1). Two stainless steel stirrups were inserted through the lumen of each aortic segment: one stirrup was attached to the bottom of the chamber and the other to an isometric FDT10-A force–displacement transducer (BIOPAC Systems, Inc., COMMAT Ltd., Turkey). The aortic rings were stretched and maintained at optimal tension of 2 g and allowed to equilibrate for 2 h.

After the equilibration period, the aortic rings were contracted to maximal tension with KCl (60 mM). The depolarizing solution KCl (60 mM) was obtained by equimolar substitution of NaCl for KCl. Then the cumulative concentration–response curves to 5-HT were determined in the absence and presence of antagonist. Tissues were incubated with antagonists for 30 min.

Concentration–response curves were analyzed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). Contractile responses to 5-HT (in the presence or absence of tested compounds) are expressed as a percentage of the maximal KCl effect reached in the concentration–response curves obtained before incubation with the tested compounds (Emax = 100%). Data are the mean ± SEM of three separate experiments. The affinity was estimated with the equation pK_B = log(concentration ratio–1)-log(molar antagonist concentration), where the concentration ratio is the ratio of equi-effective agonist concentrations in the absence and in the presence of the antagonist.

In vitro aggregation assays were performed using freshly drawn whole rat blood with a Multiplate platelet function analyzer (Roche Diagnostic), the five-channel aggregometer based on measurements of electric impedance. Blood was collected from carotid arteries with a hirudin blood tube (Roche Diagnostic). 300 μl of hirudin anticoagulated blood was mixed with 300 μl prewarmed isotonic saline solution containing studied compound or vehicle (deionized water or DMSO 0.1%) and preincubated for 3 min at 37 °C with continuous stirring. Aggregation was induced by adding collagen (Hyphen-Biomed, France) (final concentration 1.6 µg/ml), 5-HT (30 µM) and sub-threshold concentration of collagen (0.8 µg/ml), 5-HT (6 µM) and sub-threshold concentration of ADP (ADP test, Roche Diagnostic), (1.6 µM). The aggregation process was recorded for 6 min. Ketanserin (ketanserin ( +) tartrate salt, Sigma-Aldrich, Germany), sarpogrelate (sarpogrelate hydrochloride, Sigma-Aldrich, Germany), and aspirin (Tocris, UK) were used as reference compounds. The Multiplate software analyzed the area under the curve (AUC) of the clotting process of each measurement and calculated the mean values. Each concentration of studied compounds was tested at least three times. The exact sample size (n value) is presented on Figs. 2–4. Concentration-inhibition curves were constructed and analyzed by non-linear curve fitting using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).

Data were presented as mean ± standard error the mean (SEM). Statistical comparisons were made by the one-way analysis of variance (one-way ANOVA) and the significance of the differences between the control group and treated groups was determined by the Dunnet post hoc test. p < 0.05 was considered significant.