[Ca²⁺]_i measurements. Gel-filtered platelets at 5 × 10⁷/mL were loaded with 2 μM fura-2/am and resuspended in Tyrode buffer with 1 mM CaCl₂ and 1 mM MgCl₂. Platelets were stimulated with 0.01, 0.05, or 1 U/mL thrombin and fluorescence signals at excitation wavelengths of 340 and 380 were collected and the 340/380 ratio was analyzed using Felix software from Photon Technology. (A) Representative traces showing changes in F340/F380 ratio in platelets stimulated with 0.01, 0.05, and 1 U/mL thrombin. The solid and dotted lines represent WT and Akt-1-null platelets, respectively. Arrow indicates the point of thrombin addition. Curves were smoothed using the Loess technique with the sampling proportion 0.1 and with the polynomial degree 3 in SigmaPlot 8.0. (B) The average changes in fura-2 ratio (Δratio) in platelets following the addition of thrombin at indicated concentrations in the presence of 0.5 mM EGTA or 1 mM Ca²⁺. The Δratio was calculated as the difference between the peak ratio after agonist was added, and its level immediately before agonist addition. Data (means ± SD) were summarized from 3 independent experiments. ^*Significant difference between WT and Akt-1-null platelets (P < .05).