Effects of different signaling pathway inhibitors on thrombin-induced decrease of the platelet cAMP. (A) Washed platelets (2 ×106/mL) were incubated with 2 μmol/L AktI, 0.1 μmol/L wortmannin, 10 μmol/L milrinone or IBMX, 10 μmol/L GF 109203, 10 μmol/L H89, or vehicle at conditions as described under “Materials and methods.” The samples were stimulated by thrombin (0.5 nmol/L) at 37°C for 3 minutes after incubation with 10 μmol/L forskolin for 3 minutes. The results are expressed as the percentage of baseline cAMP level in each condition without thrombin. (B–D) The changes of cAMP contents medicated by agonist thrombin (0.5 nmol/L at 37°C for 3 minutes), PMA (1 μmol/L at 37°C for 10 minutes), and TPO (100 ng/mL 37°C for 10 minutes) in the absence or in the presence of a variety of pharmacological agents were monitored, respectively. The results are normalized as the percentage of baseline cAMP level in each condition without agonist. cAMP levels were determined by Biotrak Enzyme Immunoassay kit. Data are from 4 independent experiments using platelets from different donors and are means plus or minus SEM. ∗, significant by ANOVA for wortmannin, AktI, milrinone, and IBMX compared with thrombin alone (P > .05).
