Phosphatidylinositol 4,5-bisphosphate regulates activation-induced platelet microparticle formation

While the role of the cytoskeleton in microparticle formation is well-described, the role of membrane phospholipids in regulating this process is poorly defined. PIP2 binds many cytoskeletal proteins and may oppose microparticle formation through associations with these proteins. To determine whether PIP2 effects microparticle formation, PIP2 was incorporated into platelet membranes prior to activation-induced microparticle formation. Incorporation of PIP2 into platelet membranes inhibited activation-induced microparticle formation by >= 90%. Inhibition was dose-dependent with an IC50 of 12-18 mu M. A permeabilized platelet system was next used to assess the effect of modulation of endogenous PIP2 levels on microparticle formation. Infusion of type II PIP kinase into permeabilized platelets inhibited microparticle formation by 75 +/- 8%. In contrast, incubation of permeabilized platelets with PI-specific phospholipase C augmented microparticle formation by greater than 3-fold. Evaluation of PIP kinases following platelet activation demonstrated that they were lost from platelets in a calpain-dependent manner during microparticle formation. Purified gamma-calpain cleaved recombinant type II beta PIP kinase and inhibited its ability to phosphorylate PI(5)P. In permeabilized platelets, incubation of purified mu-calpain reduced PIP2 levels, while exposure to calpeptin increased PIP2 levels. Calpain has previously been implicated in platelet microparticle formation. These studies show that calpain may help limit PIP2 formation following platelet activation and that PIP2 content is an important determinant of platelet microparticle formation.