Expression and phosphorylation of CD84 in platelets. (A) Top panel: FACS analysis of α-CD84 IgG-FITC stained resting (broken line) or Thrombin Receptor Activating Peptide (TRAP)–activated (solid line) platelets. Dotted line indicates staining with nonspecific IgG. Bottom panel: platelet activation confirmed by α-CD62P IgG-PE staining of resting (solid line) and activated (broken line) platelets. (B) Western blotting of immunoprecipitates from resting (lane 1) or agonist-induced platelet aggregates formed in the absence (lanes 2-4) or presence (lanes 5-7) of 2 μM eptifibatide. (C) Western blotting of immunoprecipitates from washed human platelets activated with either α-CD84-IgG (lanes 1, 3, and 4) or nonspecific IgG (lane 2) compared with platelet aggregation induced with 5 μM TRAP (lane 5). Antibody-induced signaling induced with 15 μg/mL α-mouse whole-IgG (lanes 1 and 2) or α-mouse Fab′2 (lane 3). For both B and C, lysed platelets were immunoprecipitated with α-CD84-IgG and immunoblotted with α-phosphotyrosine antibodies PY20 and 4G10 (top panel) or α-CD84 (bottom panel). Data are representative of experiments repeated at least 3 to 5 times.