F 4500 spectrometer

This study was approved by the Institutional Review Board of Shin Kong Wu Ho-Su Memorial Hospital (Approval No. 20160705R) and conformed to the principles outlined in the Declaration of Helsinki. All volunteers provided informed consent. Human platelet suspensions were prepared as previously described [9 (link),10 (link)]. In brief, blood was collected from healthy volunteers, who had taken no medicine during the preceding two weeks, and mixed with an acid–citrate–dextrose solution (9:1, v/v). After centrifugation, the supernatant (platelet-rich plasma) was supplemented with prostaglandin E1 (0.5 μM) and heparin (6.4 IU/mL). Washed platelets were suspended in Tyrode’s solution containing bovine serum albumin (BSA; 3.5 mg/mL). The final concentration of Ca²⁺ in Tyrode’s solution was 1 mM.
Platelet aggregation was measured through turbidimetry [9 (link),10 (link)] using a Lumi-Aggregometer (Payton, Scarborough, ON, Canada), and adenosine triphosphate (ATP) release was detected through the measurement of luminescence using the F-4500 spectrometer (Hitachi, Osaka, Japan). Platelet suspensions (3.6 × 10⁸ cells/mL) were preincubated with various concentrations of VU1, VU2, or an isovolumetric solvent control (0.1% DMSO, final concentration) for 3 min before the addition of agonists. The reaction was allowed to proceed for 6 min.

Solvents were dried and deoxygenated by refluxing over the appropriate reagents before use. Elemental analyses were obtained from a PE 2400 series II CHNS/O analyzer (PerkinElmer, Boston, MA, USA) or an Elementar Vario EL cube analyzer (Elementar Americas Inc., Ronkonkoma, NJ, USA). ¹H-NMR and TOF-MS (ESI-MS) were recorded on a Bruker 400 MHz NMR and a Bruker MicrOTOF II (Bruker, Billerica, MA, USA), respectively. Powder X-ray diffraction patterns were obtained from a Bruker D2 PHASER diffractometer (Bruker, Billerica, MA, USA) with CuK_α (λ_α = 1.54 Å) radiation. IR spectra (KBr disk) were recorded on a Jasco FT/IR-460 plus spectrometer (JASCO, 28600 Mary’s Court City, MD, USA). The UV-Vis absorption spectra and emission spectra were obtained in the solid state at room temperature by using an SP-1901 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan) and a Hitachi F-4500 spectrometer (Hitachi, Tokyo, Japan), respectively. Thermal gravimetric analysis (TGA) was carried out on a SII EXSTAR6000 TG/DTA 6200 (Seiko Instruments Inc., Tokyo, Japan) under N₂ atmosphere at a heating rate of 10 °C min⁻¹ in the temperature range of 30 to 1000 °C.