Platelet-derived MPs contain functional Ago2•miR-223 effector complexes. (A-C) Human platelets (PLT) were activated with thrombin (0.1 U/mL) for 60 minutes, and the MP fraction was isolated by ultracentrifugation. Unstimulated platelets served as the control.³  (A) The presence of Ago2 was assessed by immunoblot (IB) analysis using an anti-Ago2 antibody. (B) Protein extracts derived from the MP fraction were subjected to immunoprecipitation using anti-Ago2 antibody, followed by quantitative miR-223 detection by qPCR. Results were normalized by the 2^{^-ΔΔCt} method, using RNU6 as a reference,²⁷  and expressed as a percentage of the input (mean ± SEM; n = 4 experiments). *P < .05 vs normal isotypic IgG, which was used as an IP control (Student t test). (C) The supernatant (S100) (50 µg proteins) fraction of protein extracts derived from PLT or the MP fraction of thrombin-activated platelets were subjected to RNA target cleavage assays, using a 5′ end, ³²P-labeled miR-223 RNA sensor. RNA was isolated by phenol/chloroform extraction and ethanol precipitation, separated by denaturing 8% polyacrylamide gel electrophoresis PAGE/7 M urea and visualized by autoradiography (n = 2 experiments). *The 39-nucleotide RNA product expected from Ago2-mediated endonucleolytic cleavage of the sensor. -, uncleaved ³²P-labeled miR-223 RNA sensor.