GPVI signaling is affected by the absence of SNAP23. (A) Washed platelets at 200 × 10⁹/L were stimulated with 0.075 units/mL Thr (i) or 2 µg/mL CRP (ii) at 1000 rpm in a Chronolog model 700 aggregometer. (iii) The maximum aggregation within 5 minutes was recorded. Data from at least 3 independent experiments were compared by unpaired Student t test. (B) Washed platelets at 20 × 10⁹/L were stimulated with (i) thrombin and (ii) CRP under nonstirring conditions for 10 minutes at room temperatures in the presence of phycoerythrin-conjugated antibody raised against activated α_IIbβ₃/CD41 (Jon/A). Curves were compared by 2-way ANOVA with Sidak’s multiple comparisons test. WT (n = 8), KO (n = 5). (iii) Jon/A binding at maximal thrombin (0.4 units/mL) and maximal CRP (20 µg/mL CRP) were assessed when costimulated with 10 µM ADP. WT (n = 3-6), KO (n = 6-10). Data were compared by 2-way ANOVA with Sidak’s multiple comparisons test. (C) Fura–PE3-labeled platelets at 100 × 10⁹/L were stimulated under nonstirring conditions and analyzed on a Tecan Infinite M200Pro plate reader. At the end of the run, samples were lysed with 0.2% TX100 to give a total value to which values were standardized. n = 4. Data were compared by 2-way ANOVA with Sidak’s multiple comparisons test. (D) Platelets at 400 × 10⁹/L were stimulated for 1 minute with 20 µg/mL CRP under nonstirring conditions at 37°C. Membranes were probed as indicated and imaged on a LI-COR Odyssey CLx. (i) Representative blots of WT (n = 3) and KO (n = 5). (ii) Densitometric analysis of pSyk/Syk ratio as a fold increase over basal. *P < .05, **P < .01, ***P < .001, ****P < .0001. AUC, area under the curve; ns, not significant.