Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Thrombin-induced Secretion is Normal in Cellubrevin/VAMP-3^-/- Platelets. Platelets from Wild-Type (WT, closed circles) and VAMP-3^-/- (V3^-/-, open circles) mice were prepared as described in Methods and Materials. After adding 0.7 mM CaCl₂, aliquots (50 μl; 2.5x10⁸/mL) were incubated with the indicated concentration of thrombin at room temperature for 1 min (A) or 0.05 U/mL thrombin for the indicated time points (B). Reactions were stopped by the addition of hirudin. Release of ³H-5-HT from dense core granules (DC), PF4 from alpha granules (Alpha) and β-hexosaminidase from lysosomes (Lysosome) was measured and percent secretion was calculated. Each data point represents triplicate samples and the standard deviation is indicated.
• Supplemental Figure 2 - Thrombin-induced Secretion is Normal in Synaptobrevin/VAMP-2^+/- Platelets. Platelets from Wild-Type (WT, closed circles) and VAMP-2^+/- (V2^+/-, open circles) mice were prepared as described in Methods and Materials. After adding 0.7 mM CaCl₂, aliquots (50 μl; 2.5x10⁸/mL) were incubated with the indicated concentration of thrombin at room temperature for 1 min (A) or 0.05 U/mL thrombin for the indicated time points (B). Reactions were stopped by the addition of hirudin. Release of ³H-5-HT from dense core granules (DC), PF4 from alpha granules (Alpha) and β-hexosaminidase from lysosomes (Lysosome) was measured and percent secretion was calculated. Each data point represents triplicate samples and the standard deviation is indicated.
• Supplemental Figure 3 - Thrombin-induced Secretion is Normal in Synaptobrevin/VAMP-2^+/-/Cellubrevin/VAMP-3^-/- Platelets. Platelets from VAMP-2^+/- (V2^+/-, closed circles) and VAMP-2^+/-/VAMP-3^-/- (V2^+/-/V3^-/-, open circles) mice were prepared as described in Methods and Materials. After adding 0.7 mM CaCl₂, aliquots (50 μl; 2.5x10⁸/mL) were incubated with the indicated concentration of thrombin at room temperature for 1 min (A) or 0.05 U/mL thrombin for the indicated time points (B). Reactions were stopped by the addition of hirudin. Release of ³H-5-HT from dense core granules (DC), PF4 from alpha granules (Alpha) and β-hexosaminidase from lysosomes (Lysosome) was measured and percent secretion was calculated. Each data point represents triplicate samples and the standard deviation is indicated.
• Supplemental Figure 4 - P-Selectin Exposure is Unaffected by Tetanus Toxin Light Chain Treatment of Permeabilized Human Platelets. SLO-permeablized human platelets were prepared as described in Materials and Methods. Aliquots (100 μL; 5x10⁸/mL) were incubated with 25 μM Tetanus toxin light chain (+TeNT LC, grey bars) or boiled Tetanus toxin light chain as a control (-TeNT LC, black bars). The samples were then stimulated with indicated levels of free calcium for 5 min at RT and fixed for flow cytometry analysis. P-selectin exposure was measured and graphed as mean fluorescence intensity values (A). Each data point represents triplicate data and the standard deviation is indicated. A separated set of samples were probed by western blot for VAMP-3 and actin (B).
• Supplemental Figure 5 - Strain Differences are Not Responsible for the Defect in Secretion Seen in Endobrevin/VAMP-8^-/- Platelets. The secretion time course data from all VAMP-8^+/+ mouse strains (WT, VAMP-2^+/-, VAMP-3^-/-, VAMP-2^+/-/VAMP-3^-/-; Figures 5, and 1S-3S) were pooled and averaged (V8^+/+, closed circle). This was compared with the secretion time course of VAMP-8^-/- platelets (V8^-/-, open circle). Each data point represents the average ±SEM of 21-26 samples for VAMP-8^+/+ and 3-6 samples for VAMP-8^-/-. For each pair of time points, a student’s unpaired t-test (SigmaPlot 9.0) was performed to asses the statistical significance of the differences between the two groups. The P values of each pairwise analysis were indicated (P<0.01=**, P<0.0001=***).