Impaired lysosomal enzyme release upon thrombin simulation of HPS model platelets. Washed WT, pallid, pearl, or light ear platelets (8 × 10⁷) were stimulated with varying concentrations of thrombin (A and C) for 10 minutes in the absence or presence of 10 μM ADP as indicated. Supernatants were collected and analyzed for activity of the lysosomal enzymes β-hexosaminidase (A) or β-glucuronidase (C) using colorimetric or fluorogenic substrates, respectively. Panels A′,C′ (insets) show values at 0.025 U/mL of thrombin with or without ADP on an expanded scale. Untreated platelets (8 × 10⁷) were lysed in 100 μL of lysis buffer and analyzed directly for β-hexosaminidase (B) or β-glucuronidase (D) activity. Data represent mean corrected A₄₀₅ (A, A′, and B) or fluorescence (C, C′, and D) values (mean ± standard deviation)for undiluted supernatant, normalized to the highest value in a given experiment, from at least 3 separate experiments. *P < .05; **P < .01; ***P < .005. (E) Lysates from washed WT, pallid, pearl, or light ear platelets were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting for syntaxin-11, VAMP-8, or SNAP-23 (top row), or β-actin (bottom row) as a control. Bands from 3 separate experiments were quantified and plotted as the mean signal ± standard deviation for syntaxin-11, VAMP-8, or SNAP-23 relative to β-actin.