The Role of Akt3 in Platelet Activation

Abstract

Akt is a family of serine/threonine kinases that are activated downstream of Phosphoinositide 3 Kinases (PI3K). There are three known Akt isoforms: Akt1, Akt2, and Akt3. Both Akt1 and Akt2 have been shown to be expressed in platelets and play important roles in PI3K-dependent platelet activation signaling. Previous studies have concluded that Akt3 is not expressed in platelets; however, we show that Akt3 is in fact a major Akt isoform present in platelets, and demonstrate that Akt3 plays an important role in platelet activation. In order to determine if Akt3 mRNA is present in platelets, RNA was isolated from platelets and RT-PCR experiments were performed using Akt3 specific primers. Akt3 mRNA was detected in both human and mouse platelets, and the possibility that Akt3 mRNA came from leukocyte contamination was excluded because Akt3 was not detected with RT-PCR using RNA purified from the same number of leukocytes as that in platelet preparation. To assess if Akt3 protein is present in platelets, western blot analysis was performed using an Akt3 specific antibody. Results revealed that Akt3 is highly expressed in human and mouse platelets. In order to assess the relative amount of Akt3 present in platelets, total Akt levels and phosphorylated Akt levels were measured in Akt3 knockout mouse platelets in comparison with wild type mouse platelets. Akt3 knockout mouse platelets showed a significant decrease in total Akt, and also a significant reduction in phosphorylated Akt, as indicated by Western blot analysis of phospho-Thr308-Akt and phospho-Ser473-Akt antibodies. Thus, our data indicates Akt3 is a major Akt isoform expressed in platelets. To determine the role of Akt3 in platelet activation, platelets from wild type mice and Akt3 knockout mice were stimulated with low concentrations of platelet agonists. Akt3 knockout mouse platelets exhibited impaired aggregation and ATP secretion in response to low dose thrombin and collagen compared with wild type controls, indicating that Akt3 plays an important role in promoting platelet activation. In order to assess the signaling molecules downstream from Akt3, Akt3 knockout and wild type mouse platelets were stimulated with low dose thrombin and phosphorylation of GSK-3β, an enzyme recently reported to negatively regulate platelet function downstream from Akt1 or Akt2 in platelets, was assessed. Western blot for phospho-Ser9 of GSK-3β in Akt3 knockout mouse platelets stimulated with thrombin showed less phosphorylation compared to Akt1 or Akt2 knockout mouse platelets, indicating that Akt3 is the major Akt isoform responsible for GSK-3β phosphorylation. Thus, our data shows that Akt3 is a major Akt isoform in platelets and plays an important role in platelet activation. Since phosphorylation of GSK-3β at Ser9 has been reported to reverse the inhibitory effect of GSK-3β on platelet function, our data also suggest that the stimulatory role of Akt3 in platelet activation may be mediated by negative regulation of GSK-3β function.