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. PIP(2) binds many cytoskeletal proteins and may oppose microparticle formation through associations with these proteins. To determine whether PIP(2) effects microparticle formation, PIP(2) was incorporated into platelet membranes prior to activation-induced microparticle formation. Incorporation of PIP(2) into platelet membranes inhibited activation-induced microparticle formation by >or=90%. Inhibition was dose-dependent with an IC(50) of 12-18 microM. A permeabilized platelet system was next used to assess the effect of modulation of endogenous PIP(2) levels on microparticle formation. Infusion of type IIbeta 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 mu-calpain cleaved recombinant type IIbeta PIP kinase and inhibited its ability to phosphorylate PI(5)P. In permeabilized platelets, incubation of purified mu-calpain reduced PIP(2) levels, while exposure to calpeptin increased PIP(2) levels. Calpain has previously been implicated in platelet microparticle formation. These studies show that calpain may help limit PIP(2) formation following platelet activation and that PIP(2) content is an important determinant of platelet microparticle formation.
O'Connell
Daniel J
DJ
Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Rozenvayn
Nataliya
N
Flaumenhaft
Robert
R
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002154:Calpain / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000097:blood; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000097:blood* / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D015539:Platelet Activation; D011994:Recombinant Proteins / Q000378:metabolism
D008563:Membrane Lipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D002154:Calpain