Platelet Preparation and Flow‐Cytometry Analysis

Whole blood from patients and healthy donors was collected into tubes containing 3.8% sodium citrate (9:1) and was centrifuged at 150g for 15 minutes to obtain platelet‐rich plasma (PRP).⁷ Washed platelets were obtained from PRP. To avoid leukocyte contamination, only the top 75% of the PRP was collected. The purity of platelets was checked by platelet‐specific markers (CD45‐ve and CD61+) by flow cytometry, as described previously.²⁴ To perform the ex vivo tests, platelets from HC were diluted with Tyrode’s buffer to obtain the same number of platelets as that of the patient. Platelet aggregation, intracellular calcium flux (362561; BioLegend, San Diego, CA), anti‐procaspase‐activating compound‐1 (PAC‐1) (362803; BioLegend), P‐selectin (304903; BioLegend), and CD40L (310809; BioLegend) expression analyses were performed.⁷ Intracellular ROS were measured (109244‐58‐8; Cayman Chemical Co., Ann Arbor, MI)²⁵ (Supporting Methods).