Ginkgolide b

A mix of terpene lactones from G. biloba: bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J (100 μg/mL each component in acetonitrile) was obtained from Sigma-Aldrich. Serial concentrations (1.5–9 μg/ml) were prepared in DMEM 2% FBS for experiments. Acetonitrile (9% and below) does not cause viral inactivation (control). Final concentrations of acetonitrile in tested cells were ≤ 0.9% (non-toxic concentration, control).

Analysis of platelet aggregation in human platelet-rich plasma (hPRP) was carried out on a Chronolog-490 two channel turbidimetric platelet aggregometer (Havertown, PA, USA), coupled to the accompanying AGGRO/LINK software package. 20G safety needles and evacuated sodium citrate S-monovettes for blood sampling were purchased from Sarstedt Ltd. (Wexford, Ireland). Platelet aggregation related consumables were purchased from Labmedics LLP (Abingdon on Thames, UK). Standard PAF, thrombin, BSA, and ginkgolide B were purchased from Sigma-Aldrich (Wicklow, Ireland), while collagen and ADP from Chronolog (Havertown, PA, USA) and aspirin (Bayer, Berlin, Germany) was purchased from a pharmacy (Bayer, Berlin, Germany). 1-hexadecyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine standard (PCS) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Centrifugations were carried out on an Eppendorf 5702R centrifuge (Eppendorf Ltd., Stevenage, UK). Spectrophotometric analysis was carried out on a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan) using a quartz 1 cm cuvette. All glass and plastic consumables, reagents, and solvents were of analytical grade and were purchased from Fisher Scientific Ltd. (Dublin, Ireland).

Drugs used in the present study were 5 µm dl-2-amino-7-phosphonovalerate (dl-APV), 5 µm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 1 µm 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid (SR95531), 1 µm strychnine, 20–40 μg/ml gramicidin D, 100 µm or 1M glycine, 5 µg/ml brefeldin A (BFA), 1 µm ginkgolide B, 0.5 µm mecamylamine hydrochloride, 10 µm BAPTA-AM, 30 µm KN-62, 10 µm Rp-adenosine 3, and 1 µm ginkgolide monophosphorothioate triethylammonium salt (all from Sigma-Aldrich Japan); 50 nm GF 109203X (GFX; Enzo Life Sciences); and 0.3 µm tetrodotoxin (TTX; Latoxan). Drug-containing solutions were locally applied via a Y-tube perfusion system (Murase et al., 1989 (link)) in electrophysiological recordings and via bath application in imaging experiments.