Munc13-4 is a limiting factor for platelet exocytosis. (A) Platelets were permeabilized by incubation with Streptolysin-O (SLO). Platelets from wild-type (WT) and Unc13d^Jinx mice were incubated with recombinant full-length (FL) Munc13-4 or ΔC2B as indicated. Samples in which Munc13-4 was omitted were included as controls. CaCl₂ (10μM final) was added as indicated and secretion from dense granules (DG), α-granules (Alpha), and lysosomes was monitored and plotted as in Figure 2. Data are represented as the mean ± SD, n = 3. (B) Platelets from wild-type (WT, black traces) or Unc13d^Jinx heterozygous mice (Unc13d^Jinx Het, gray traces) were prepared as previously described in the legend of Figure 4. Secretion of ATP from dense granules (Luminescence) was monitored after addition of the indicated agonists. (Inset) The expression levels of Munc13-4 in platelets from wild-type (WT), Unc13d^Jinx, and Unc13d^Jinx heterozygous mice as determined by Western blotting with actin serving as loading control. (C-D) Human platelets were permeabilized with SLO as describe above and incubated with the indicated concentrations of Munc13-4 (C), ΔC2B (D), or with proteins that had been heat-inactivated (2.5b). Secretion from dense granules (DG), α-granules (Alpha), and lysosomes was monitored and normalized to controls in which no exogenous Munc13-4 was added. A Student t test (SigmaPlot 9.0) was used to assess the statistical significance of the differences between groups (**P < .01).