Luciferin luciferase reagent

The platelet aggregation and release assay were performed as previously reported [41 (link)]. Washed mouse platelets were treated with isaridin E (6.25, 12.5, 25, 50, 100, or 200 μM) or vehicle or clopidogrel (20 μM) at 37 °C for 30 min, followed by stimulation with ADP (01905, Sigma-Aldrich, St. Louis, MO, USA, 5 μM), collagen (C7661, Sigma-Aldrich, St. Louis, MO, USA, 1 μg/mL) or thrombin (T6884, Sigma-Aldrich, St. Louis, MO, USA, 0.02 U/mL). The platelet aggregation was assessed using a lumi-aggregometer (700-2, Chrono-Log, Havertown, PA, USA) with a stirring speed of 1200 rpm at 37 °C. The release of ATP was simultaneously monitored using Luciferin/luciferase reagent (395, Chrono-Log, Havertown, PA, USA). Aggregation traces and ATP release traces were monitored continuously for at least 10 min.

Platelet aggregation was conducted in the presence of fibrinogen (0.5 mg/ml) as described previously (Luo et al., 2019 (link); Wei G. et al., 2020 (link)). After matrine treatment, platelet aggregation induced by CRP (0.1 μg/ml) or thrombin (0.04 U/ml) was analyzed in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, United States) at 37°C with stirring (1,000 rpm). Platelet aggregation was presented as a percentage of maximum platelet aggregation. ATP release was monitored in parallel with platelet aggregation after addition of luciferin/luciferase reagent (Chrono-log Corporation) according to the manufacturer’s instructions and quantified relative to the vehicle (0 mg/ml matrine) treatment.

The platelets were placed in modified HEPES-Tyrode buffer and then grown with either 0.01% DMSO or one of three concentrations of SA (50, 100, or 200 µg/mL) for 10 min at 37 °C. They were then activated via various agonists, including collagen (1 µg/mL), CRP (0.5 µg/mL), thrombin (0.05 U/mL), U46619 (3 µM), or ADP (2.5 µM). In some experiments, PRP was preincubated with either 0.01% DMSO or one of three concentrations of SA (50, 100, or 200 µg/mL) for 10 min at 37 °C. They were then stimulated with collagen (1 µg/mL) or CRP (0.5 µg/mL). In the case of ADP-facilitated aggregation, human FG (30 µg/mL) was added to the platelet suspension prior to ADP induction. Platelet aggregation was determined with a 4-channel platelet Lumi-aggregometer (Chrono-Log Corp., Havertown, PA, USA) at 37 °C with stirring at 1000 rpm. Platelet secretion was controlled by measuring that of ADP/ATP through the addition of luciferin/luciferase reagent (Chrono-log) to the platelet suspension [36 (link),37 (link)].

After salidroside treatment, human platelet aggregation in response to thrombin (0.03 U/ml) or CRP (0.1 μg/ml) was assessed in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, USA) at 37°C with stirring (1000 rpm). ATP release was monitored in parallel with platelet aggregation after addition of luciferin/luciferase reagent (Chrono-log Corporation) in accordance with the manufacturer's instructions. ATP release was presented as a percentage relative to the vehicle (0 μM salidroside) treatment. For mouse platelet aggregation, thrombin (0.02 U/ml) or CRP (0.05 μg/ml) was used.

Platelet aggregation was performed in a Lumi-Aggregometer Model 700 (Chrono-log Corporation, Havertown, PA, USA) at 37 °C with stirring (1000 rpm) before and after stimulation. The ATP secretion was monitored in parallel with platelet aggregation after adding luciferin/luciferase reagent (Chrono-log Corporation) to platelet suspensions. ATP secretion was quantified relative to control mouse platelets.

20(S)-protopanaxadiol (PPD, purity of ≥98% by HPLC) was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Hemocoagulase was purchased from Jinzhou Ahon Pharmaceutical Co, Ltd. (Liaoning, China). Prothrombin (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Luciferin/luciferase reagent and thrombin were purchased from Chrono-Log Corporation (Pennsylvania, USA). Vorapaxar, ticagrelor, and seratrodast were purchased from MedChemExpress (New Jersey, USA). Cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and human protease activated receptor 1 ELISA kits were purchased from Sino Best Biological Technology Co., Ltd. (Shanghai, China). FITC-conjugated anti-human CD62P (P-selectin) and FITC-conjugated anti-human PAC-1 were purchased from BioLegend (California, USA). Fluo-3 AM calcium indicators and lactate dehydrogenase (LDH) cytotoxicity assay kits were purchased from Beyotime Biotechnology (Shanghai, China).