title: Involvement of phosphatidylinositol 4,5-bisphosphate in phosphatidylserine exposure in platelets: use of a permeant phosphoinositide-binding peptide.
abstract: During platelet activation, phosphatidylserine (PS) exposure on the extracellular face of the plasma membrane is associated with increased procoagulant activity. PS externalization is generally attributed to an increase in intracellular Ca(2+). Various phospholipid transporters, such as specific scramblases or proteins from the family of multidrug resistance proteins, and cofactors such as phosphatidylinositol 4,5-bisphosphate (PIP2) have been proposed to participate in this process. In this study, we used a membrane-permeant polycationic peptide (RhB-QRLFQVKGRR), derived from the PIP2-binding site of gelsolin (GS 160-169) and linked to rhodamine B, to investigate the role of PIP2 in PS externalization in whole platelets. The peptide penetrated rapidly into the platelets, specifically bound to PIP2, and induced PS exposure to a similar extent as thrombin or collagen, but independently of changes in intracellular Ca(2+) or phosphoinositide 3-kinase activity. A pretreatment of platelets with quercetin, an inhibitor of phosphoinositide metabolism, drastically decreased PS exposure induced by agonists or peptide. In large unilamellar vesicles (LUVs), the presence of PIP2 was strictly required for the induction of scrambling of NBD-labeled phospholipids (PC and PS) by the peptide. In inside-out vesicles from erythrocytes (IOVs), the peptide also induced redistribution of PC and PS. Our data suggest that, in intact platelets, PIP2 acts as a target of polycationic effectors, including Ca(2+), to promote PS exposure. The use of a membrane-permeant and fluorescent peptide which binds to PIP2 is a promising tool to investigate the role of PIP2 in various cellular processes.
authors.affiliation: University of Pennsylvania, Institute for Medicine and Engineering, 1010 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, Pennsylvania 19104, USA. buckirob@mail.med.upenn.edu
authors.forename: R
authors.initials: R
authors.lastname: Bucki
authors.forename: P A
authors.initials: PA
authors.lastname: Janmey
authors.forename: R
authors.initials: R
authors.lastname: Vegners
authors.forename: F
authors.initials: F
authors.lastname: Giraud
authors.forename: J C
authors.initials: JC
authors.lastname: Sulpice
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D000328:Adult; D000595:Amino Acid Sequence; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D018260:Gelsolin / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000097:blood / Q000502:physiology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010718:Phosphatidylserines / Q000097:blood* / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism
chemical_list: C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D005456:Fluorescent Dyes; D018260:Gelsolin; D008081:Liposomes; C034296:N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D012235:Rhodamines; D019869:Phosphatidylinositol 3-Kinases; D009327:4-Chloro-7-nitrobenzofurazan; C029773:rhodamine B; D002118:Calcium