Mice Lacking Pleckstrin or Pleckstrin-2 Each Have Unique Platelet Secretion Defects.

Approximately 1% of total cellular protein within platelets and leukocytes is pleckstrin, a protein best known for containing the two prototypic Pleckstrin Homology (PH) domains. Following platelet activation, PKC rapidly phosphorylates pleckstrin, inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Platelets also contain a widely expressed paralog of pleckstrin, called pleckstrin-2. Although the activity of pleckstrin is regulated through protein phosphorylation, pleckstrin-2 is not a phosphoprotein, but is instead activated by binding a specific PI3K generated phospholipid, phosphatidylinositol 3,4 bisphosphate (PI3,4P2). To define the role of individual pleckstrin isoforms in vivo, we generated mice containing loss of function mutations within either the pleckstrin or pleckstrin-2 genes. Mice lacking either isoform exhibited no spontaneous hemorrhage. Studies of pleckstrin knockout platelets demonstrated that they displayed normal aggregation and secretion in response to collagen and thrombin. In contrast, pleckstrin null platelets had no secretion of dense or alpha granules following exposure to the PKC stimulant, PMA. This demonstrates that pleckstrin is the critical effector for PKC-mediated platelet secretion. Our findings also suggest that an alternative pathway in pleckstrin knockout platelets is able to compensate for the secretion defect when the cells are stimulated with an agonist such as thrombin. We reasoned that the compensatory pathway might utilize another PIP2-dependent second messenger. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet secretion, we tested whether a PI3K-dependent pathway compensated for the loss of pleckstrin. We found that pleckstrin knockout platelets completely failed to secrete in response to stimulation of the thrombin receptor in the presence of a PI3K inhibitors (LY294002 or wortmannin.) This demonstrates that second messengers generated by PI3K are able to compensate for the secretion defect induced by the loss of pleckstrin. In contrast to the findings observed with pleckstrin knockout platelets, we have found that platelets lacking pleckstrin-2 aggregated and secreted normally in response to thrombin, collagen, and PMA. Additionally, unlike the effect seen on pleckstrin knockout platelets, inhibitors of PI3K had no effect on the aggregation or secretion of pleckstrin-2 knockout platelets. However, again in contrast to pleckstrin knockout platelets, pleckstrin-2 null platelets failed to secrete in response to thrombin when they were exposed to inhibitors of either PLC or PKC. This demonstrates that pleckstrin-2 knockout platelets compensate for their secretion defect by a pathway dependent on PLC and PKC. It is notable that PI3K or PKC inhibitors only minimally affected the thrombin-induced secretion of wild-type platelets unless both inhibitors were used together. Together, our results show that both pleckstrin and pleckstrin-2 are essential components of thrombin-mediated platelet secretion. These data also demonstrate that thrombin-induced platelet secretion can be mediated by one of two parallel pathways, the first involving PKC and pleckstrin, and the second involving PI3K and pleckstrin-2.