Potential and limitations of PKA/ PKG inhibitors for platelet studies

Cyclic nucleotides (cAMP and cGMP) and corresponding protein kinases, protein kinase A (PKA) and protein kinase G (PKG), are the main intracellular mediators of endothelium-derived platelet inhibitors. Pharmacological PKA/PKG inhibitors are often used to discriminate between these two kinase activities and to analyze their underlying mechanisms. Previously we showed that all widely used PKG inhibitors (KT5823, DT3, RP isomers) either did not inhibit PKG or inhibited and even activated platelets independently from PKG. In this study, we examined several PKA inhibitors as well as inhibitors of adenylate and guanylate cyclases to reveal their effects on platelets and establish whether they are mediated by PKA/PKG. The commonly used PKA inhibitor H89 inhibited both PKA and PKG but PKA-independently inhibited thrombin-induced platelet activation. In our experiments, KT5720 did not inhibit PKA and had no effect on platelet activation. PKI inhibited PKA activity in platelets but also strongly PKA-independently activated platelets. Inhibition of adenylate and guanylate cyclases may be an alternative approach to analyze PKA/PKG function. Based on our previous and presented data, we conclude that all results where the mentioned PKA inhibitors were used for the analysis of PKA activity in intact platelets should be considered with caution.