Adenosine diphosphate (adp)

The fluorescence polarization assay was performed as previously described (Chen et al., 2021 (link)). The reaction was prepared with 0–25 μM recombinant protein, 10 nM FAM-labeled [AG]₁₀ RNA, 1 mM AMP-PNP (Roche), 1 mM MgCl₂, 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol, and 1% DMSO (as a solvent of RocA) with or without 50 μM RocA/aglafoline. After incubation at room temperature for 30 min, the mixture was transferred to a black 384-well microplate (Corning), and the fluorescence polarization was measured by an Infinite F-200 PRO (Tecan). Under ADP + Pi conditions, 1 mM ADP (Fujifilm Wako Chemicals) and 1 mM Na₂HPO₄ were used as substitutes for AMP-PNP. The data were fitted to the Hill equation to calculate K_d values and visualized by Igor Pro v8.01 (WaveMetrics). The affinity fold change was calculated as the fold reduction in the K_d of RocA compared to the K_d of DMSO.

P-PRP and other platelet suspensions were serially diluted with equal volume of acellular plasma or the corresponding buffer solutions. Series of diluted platelet suspensions were measured using a pocketable spectrophotometer (PiCOSCOPE, Ushio Inc., Tokyo, Japan) [6 (link)]. The spectrophotometer can be operated by remote control through a specific application installed on smart devices, including the iPad Air (Apple, Cupertino, CA, USA). Platelet suspensions were transferred into 0.2 mL highly transparent PCR tubes (Nippon Genetics Co., Ltd., Tokyo, Japan) and treated with 5 μM ADP (Wako Pure Chemicals, Osaka, Japan).
To prepare dysfunctional platelet models, we pretreated P-PRP with 0.1 mg/mL aspirin (acetylsalicylic acid; Wako Pure Chemicals, Osaka, Japan) or 10 μM H₂O₂ (Wako) for 30 min at 22–24 °C. The absorbance was measured at an interval of one minute at 615 nm (range of wavelength: 570–660 nm). At the end of measurement, each blank was measured as the absorbance of 100% aggregation.

Aggregation responses to HBSS, poly P (0.5 mg/mL), and ADP (40 μM) (FUJIFILM Wako Pure Chemical) were measured (n = 5) using an EnSpire (PerkinElmer Japan, Kanagawa, Japan).