Blood samples were collected from eight nonsmoking healthy male volunteers at ages from 32 to 68 years. The study design and consent forms of all the procedures performed were approved by the ethics committee for human participants of the Niigata University School of Medicine (Niigata, Japan) in accordance with the Helsinki Declaration of 1964 as revised in 2013.

Peripheral blood (~ 9 mL) was collected into plastic vacuum plain blood collection tubes (Neotube; NIPRO, Osaka, Japan) containing 1 mL of the A-formulation of ACD (ACD-A; Terumo, Tokyo, Japan) and was immediately centrifuged at 530×g for 10 min. The upper plasma fraction was collected, transferred to fresh tubes, and served as a PRP fraction [10]. The numbers of platelets and other blood cells in whole-blood samples and PRP preparations were determined on an automated hematology analyzer (pocH 100iV, Sysmex, Kobe, Japan).

Evaluation of platelet surface antigen expression by immunofluorescence (IT) staining

Platelet concentrates were prepared from citrated whole-blood samples, pretreated with 5 μg/mL prostaglandin E₁ (PGE₁; Cayman Chemical Co., Ann Arbor, MI, USA) for 5 min, rinsed, and resuspended in PBS in sample tubes. Washed platelets were then treated with CaCl₂ at a final concentration of 0.1% and incubated in polystyrene culture dishes for up to 30 min at ambient temperature. At the end of the incubation, the reaction was stopped by adding 10% of neutralized formaldehyde. Platelets were washed twice, blocked with 0.1% Block-Ace (Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan) in 0.1% Tween 20-containing Tris-buffered saline (TBS) (T-TBS) for 1 h, and treated with a mouse monoclonal anti-human CD41, anti-CD62P, or anti-CD63 antibody (1:20 dilution; BioLegend, San Diego, CA, USA) overnight at 4 °C. At the end of treatment, the platelets were again washed twice with T-TBS and were then probed with a secondary antibody, i.e., a goat anti-mouse IgG H&L antibody (conjugated with Alexa Flour 555; 1:50 dilution; Abcam, Cambridge, MA, USA), for 30 min at ambient temperature. Finally, after a subsequent PBS wash, the platelets were mounted with an antifade mounting medium (Vectashield; Vector laboratories, Burlingame, CA, USA), and CD41, CD62P, and CD63 expression levels were examined under a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera (Nikon, Tokyo, Japan).