Thrombin stimulates PDE3A by activating Akt. (A) PDE3A, PI3K/Akt inhibitors reverse thrombin-induced activation of PDE3A. Washed platelets (2 ×10⁸/mL) were first incubated with AktI (2 μmol/L), wortmannin (0.1 μmol/L), milrinone (10 μmol/L), H89 (10 μmol/L), or vehicle as described under “Materials and methods.” Thrombin (0.5 nmol/L) or vehicle was then added at 37°C for 3 minutes. PDE3A activity in the platelet lysate was determined in the presence of 20 μmol/L EHNA to remove the effect of PDE2A. Results are from 4 experiments using different donor platelets. (B) The correlation of thrombin-stimulated Akt and PDE3A activation. Activities of Akt and PDE3A in platelets were measured either after 5 minutes of thrombin stimulation (i) (thrombin concentration-dependent) or with thrombin at 0.5 nmol/L (iii) (time-dependent). The closed squares represent Akt activity and the open squares represent PDE3A activity. The data of (i) and (iii) was from 3 individual experiments. Correlation of Akt kinase and PDE3A activities in dose-response of thrombin (ii) was plotted with Microsoft Excel software (Microsoft, Redmond, WA). Values are means plus or minus SEM.