The morphology and response to activation are similar in platelets from wild-type and Unc13d^Jinx mice. (A) Platelets from wild-type and Unc13d^Jinx mice were prepared as described in “Platelet preparation” and diluted to approximately 2.5 × 10⁸/mL. CaCl₂ was added to each sample (0.7mM final). Panels i and ii represent platelets under resting conditions. Thrombin (final concentration 0.1 U/mL) was added to samples shown in panels iii through viii. After 2 minutes at RT, samples were fixed and analyzed by transmission electron microscopy. Scale bars represent 1 μm in panels iii and iv, and 200 nm for all others. (B) Washed platelets from wild-type (black trace) or Unc13d^Jinx mice (gray trace) were loaded with Fura-2/AM to measure intracellular [Ca²⁺]_i upon activation with thrombin (0.1 U/mL). Arrows indicate the addition of extracellular Ca²⁺ (0.7mM final) and thrombin. (C) Equal amounts of washed platelets from wild-type or Unc13d^Jinx mice were mixed with 5 μL of PE-conjugated-Jon/A antibody and 5 μL of FITC-conjugated CD61 antibody in the presence of 1.25mM CaCl₂ and 0.02 U/mL apyrase. Samples were incubated without thrombin (resting) or with thrombin (0.1 U/mL) for 5 minutes at RT. Reactions were stopped with excess hirudin and analyzed by flow cytometry. Data were plotted as 2-color dot plots (Jon/A and CD61). The percentage of platelets in each quadrant was indicated. (D) Equal amounts of washed platelets from wild-type or Unc13d^Jinx mice were either kept in the resting state or stimulated with the indicated agonists for 2 minutes at RT. The reactions were stopped by adding SDS sample buffer and the samples were analyzed by Western blot for phosphotyrosine (pY). Ponceau S red staining of actin serves as loading control (bottom panel).