Platelets from Unc13d^Jinx mice show severe defects in thrombin-stimulated granule secretion. (A) Platelets from wild-type (WT) 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 (final concentration 0.7mM) followed by addition of thrombin (final concentration is indicated). After a 1-minute incubation (RT), reactions were stopped by the addition of excess hirudin, and the extent of secretion from dense granules (DG), α-granules (Alpha), and lysosomes was analyzed and percent secretion was calculated as described.¹²  (B) Experiments were carried out as in panel A using 0.05 U/mL thrombin; reactions were stopped at the indicated time points and analyzed. (C-D) Five microliters of FITC-conjugated P-selectin antibody was added to equal amounts of platelets from wild-type and Unc13d^Jinx mice. Thrombin was added (gray fill) or not (black lines) and the samples were incubated for 1 minute (RT). The reactions were stopped with excess hirudin and analyzed by flow cytometry. (E-F) Experiments in panel C were repeated using a PE-conjugated CD107a/LAMP-1 antibody. Representative traces were shown in panels C and E. Data in panels D and F represent the mean ± SD from triplicates.