⁶²Gap27 inhibits GPVI signaling in human platelets.⁶²Gap27 (50 and 100 μg/mL) or scrambled peptide (S; 100 μg/mL) pretreated human washed platelets (4 × 10⁸ cells/mL) were stimulated for 90 seconds with CRP-XL (1 μg/mL) under nonaggregation conditions in the presence of indomethacin (20 µM), cangrelor (1 µM), MRS2179 (100 µM), and EGTA (1 mM). Samples were lysed in the Laemmli sample buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, and tested for (A) total tyrosine phosphorylation, (B) Syk phosphorylation (Tyr^525/526), (C) LAT phosphorylation (Tyr²⁰⁰), (D) PLCγ phosphorylation (Tyr¹²¹⁷), and (E) Fura-2-acetoxymethyl ester (Fura-2AM)–loaded washed platelets (4 × 10⁸ cells/mL) were treated with ⁶²Gap27 or scrambled peptide (S; 100 μg/mL) for 5 minutes before stimulation with CRP-XL (0.25 μg/mL). Spectrofluorimetry was used to measure the release of calcium from intracellular stores. (F) PKC substrate phosphorylation. Representative blots for the phosphorylation levels are shown. The bar graph represents mean normalized phosphorylation values relative to actin or 14-3-3-ζ levels. Representative traces of calcium mobilization over a period of 5 minutes and quantified data (peak calcium levels) are shown. Results are mean ± SEM (n ≥ 3). *P < .05; **P < .01 (calculated by 1-way ANOVA).