⁶²Gap27 inhibits platelet activation and function specifically through Cx62. Washed human platelets (4 × 10⁸ cells/mL) were treated with ⁶²Gap27 or scrambled peptide (S; 100 μg/mL) for 5 minutes before their stimulation with (A) CRP-XL (EC₅₀, 0.2-0.4 µg/mL) or (B) thrombin (EC₅₀, 0.05-0.08 U/mL). Aggregation was measured using optical light transmission aggregometry for 180 seconds. Representative aggregation traces and quantified data shown (samples treated with scrambled peptide represent 100% aggregation). (C) Effects of ⁶²Gap27 on CRP-XL (0.25 µg/mL) and thrombin (0.05 U/mL) mediated fibrinogen binding compared with the scrambled peptide (S; 100 μg/mL) was evaluated in platelets (in platelet-rich plasma [PRP]) using flow cytometry. (D) PRP from Cx57^+/+ and Cx57^−/− mice was treated with ⁶²Gap27, ^37,43Gap27, ⁴⁰Gap27 (100 µg/mL), or scrambled peptide (S; 100 μg/mL) for 5 minutes. Fibrinogen binding levels were evaluated after stimulation with CRP-XL (1 µg/mL). (E) PRP from Cx57^+/+ and Cx57^−/− mice was stimulated with CRP-XL (1 µg/mL) and fibrinogen binding was measured. (F) P-selectin exposure was measured in ⁶²Gap27 or scrambled peptide (S; 100 μg/mL) treated human platelets (in PRP) after stimulation with CRP-XL (0.25 μg/mL) or thrombin (0.05 U/mL). (G) Changes in ATP release were monitored for 5 minutes in washed platelets (4 × 10⁸ cells/mL) incubated with ⁶²Gap27 or scrambled peptide (S; 100 μg/mL) and stimulated with CRP-XL (0.5 µg/mL) or thrombin (0.05 U/mL). (H) The levels of TxB₂ were measured by immunoassay in human washed human platelets (4 × 10⁸ cells/mL) treated with scrambled peptide (S; 100 μg/mL) or ⁶²Gap27 after stimulation with CRP-XL (0.5 µg/mL) or thrombin (0.05 U/mL). Data represent mean ± SEM (n ≥ 3). ns, not significant. *P < .05; **P < .01; ***P < .001 (calculated by 1-way ANOVA). †P < .05 (calculated by Student t test).