Role of Prednisolone in Platelet Activation by Inhibiting TxA Generation through the Regulation of cPLA Phosphorylation.

Glucocorticoids have been commonly used in the treatment of inflammation and immune-mediated diseases in human beings and small animals such as cats and dogs. However, excessive use can lead to Cushing's syndrome along with several thrombotic and cardiovascular diseases. Although it is well-known that glucocorticoids exert a significant effect on coagulation, the effect of cortisol on platelet function is much less clear. Thus, we aimed to study the effects of prednisolone, one of the commonly used glucocorticoids, on the regulation of platelet function using murine platelets. We first evaluated the concentration-dependent effect of prednisolone on 2-MeSADP-induced platelet function and found that the 2-MeSADP-induced secondary wave of aggregation and dense granule secretion were completely inhibited from 500 nM prednisolone. Since 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by TxA generation, this result suggested a role of prednisolone in platelet TxA generation. Consistently, prednisolone did not affect the 2-MeSADP-induced aggregation in aspirinated platelets, where the secondary wave of aggregation and secretion were blocked by eliminating the contribution of TxA generation by aspirin. In addition, thrombin-induced platelet aggregation and secretion were inhibited in the presence of prednisolone by inhibiting the positive-feedback effect of TxA generation on platelet function. Furthermore, prednisolone completely inhibited 2-MeSADP-induced TxA generation, confirming the role of prednisolone in TxA generation. Finally, Western blot analysis revealed that prednisolone significantly inhibited 2-MeSADP-induced cytosolic phospholipase A (cPLA) and ERK phosphorylation in non-aspirinated platelets, while only cPLA phosphorylation, but not ERK phosphorylation, was significantly inhibited by prednisolone in aspirinated platelets. In conclusion, prednisolone affects platelet function by the inhibition of TxA generation through the regulation of cPLA phosphorylation, thereby shedding light on its clinical characterization and treatment efficacy in dogs with hypercortisolism in the future.