Secretion is ablated in the absence of SNAP23.

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Presentation transcript:

Secretion is ablated in the absence of SNAP23. Secretion is ablated in the absence of SNAP23. (A) Washed platelets at 200 × 109/L were stimulated under nonstirring conditions at 37°C in a Tecan Infinite M200Pro plate reader in the presence of 1/100 Chronolume luciferin luciferase. Median traces of platelets stimulated (arrow) with 0.4 units/mL thrombin (i) and 20 µg/mL collagen-related peptide (CRP) (ii), respectively. (iii) Maximum ATP released upon stimulation (WT [n = 6-8], KO [n = 5]). Data were compared by 2-way ANOVA with Sidak’s multiple comparisons test. (B) Washed platelets at 200 × 109/L were stimulated with thrombin (i) and CRP (ii) under nonstirring conditions at 37°C for 10 minutes before supernatant was harvested, run on a PF4 ELISA, and read at 450 nm. Data were compared by 2-way ANOVA with Sidak’s multiple comparisons test. WT (n = 3-4), KO (n = 3). (iii) Lysed basal platelets were used to determine total platelet PF4 content (n = 6). (C) Washed platelets at 20 × 109/L were stimulated with thrombin (i) and CRP (ii) under nonstirring conditions for 10 minutes at room temperature in the presence of FITC-conjugated anti–P-selectin. Curves were compared by 2-way ANOVA with Sidak’s multiple comparisons test. WT (n = 7-8), KO (n = 5). (iii) P-selectin binding at maximal thrombin (Thr; 0.4 units/mL) when costimulated with 10 µM ADP. WT (n = 3-7), KO (n = 4-5). Data were compared by 2-way ANOVA with Sidak’s multiple comparisons test. (D) Washed platelets at 200 × 109/L were stimulated with thrombin (i) and CRP (ii) under nonstirring conditions at 37°C for 10 minutes before supernatant was harvested, run through a β-hexosaminidase colorimetric assay, and read at 405 nm. Data were compared by 2-way ANOVA with Sidak’s multiple comparisons test. WT (n = 3-4), KO (n = 3). (iii) Lysed basal platelets were used to determine total platelet β-hexosaminidase content. WT (n = 7), KO (n = 6). *P < .05, **P < .01, ***P < .001, ****P < .0001, *****P = .002. Christopher M. Williams et al. Blood Adv 2018;2:3627-3636 © 2018 by The American Society of Hematology