⁶²Gap27 modulates PKA activity independently of cAMP. (A) Resting and (B) CRP-XL stimulated (90 seconds) washed human platelets (4 × 10⁸ cells/mL) treated with scrambled peptide (S; 100 µg/mL) or ⁶²Gap27 (50 and 100 μg/mL) for 5 minutes were tested for VASP S157 phosphorylation (a marker of PKA activity). VASP S157 phosphorylation was also evaluated in washed platelets treated with ⁶²Gap27 (100 μg/mL) for 5 minutes in the presence of (C) H89 (10 µM), (D) PKI (10 µM), (F) Rp-8-CPT-cAMPS (200 µM), and (G) SQ 22536 (100 µM). Platelets treated with PGI₂ (1 μg/mL) for the stimulation of PKA-mediated phosphorylation were used as positive controls. Sample lysis was carried out using the Laemmli sample buffer before separation by SDS-PAGE. Samples were then transferred to PVDF membranes. 14-3-3-ζ was used as a loading control. (E) Levels of cAMP were measured in resting and CRP-XL (1 μg/mL) stimulated washed human platelets (4 × 10⁸ cells/mL) that had been preincubated with the scrambled peptide (S; 100 µg/mL) or ⁶²Gap27 (50 or 100 µg/mL) for 5 minutes. Platelets treated with PGI₂ (1 μg/mL) were used as a positive control. (H-I) Resting washed platelets (4 × 10⁸ cells/mL) from Cx57^+/+ and Cx57^−/− mice were treated with scrambled peptide (S) or ⁶²Gap27 (100 μg/mL) for 5 minutes in the presence of H89 (10 µM) or Rp-8-CPT-cAMPS (200 µM) and investigated for VASP-S157 phosphorylation. Platelets treated with PGI₂ (1 μg/mL) were used as a positive control. Results are mean ± SEM (n ≥ 4). **P < .01; ***P < .001 (calculated by 1-way ANOVA).