title: Ca2+ concentration during binding determines the manner in which annexin V binds to membranes.
abstract: Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with thrombin can induce the association of intracellular annexin V with membranes in two distinct ways. First, in such a way that it can be eluted from the membrane with EGTA and secondly in a manner such that it is tightly bound to the membrane and requires the non-ionic detergent Triton X-100 for its solubilization. We report that exposure of platelets to the calcium ionophore A23187 mimics the relocation induced by stimulation with thrombin. In separate experiments we demonstrate that a calcium ion concentration [Ca2+] of 0.8 microM is sufficient for maximum binding of the EGTA-resistant form to membranes. In contrast a higher [Ca2+] was required to induce maximal binding of the annexin V which could be extracted with EGTA. We demonstrate that following temperature-induced phase separation in Triton X-114, the membrane-associated annexin V partitions predominantly into the aqueous phase. We also show that the isoelectric point of annexin V does not change following membrane association. These observations suggest that a covalent modification, of annexin V itself, is not responsible for its association with the membrane. Millimolar [Ca2+] is required for maximal binding of purified annexin V to phospholipid vesicles. We show that binding to phospholipids can be reversed entirely by subsequent treatment with EGTA. This suggests that the EGTA-resistant form of annexin V is binding to a membrane component other than phosphatidylserine. Annexin V has previously been shown to bind to protein kinase C. Relocation of annexin V to membranes paralleled that of protein kinase C in thrombin-stimulated cells but not in cells treated with A23187, suggesting that these proteins are not functionally linked in platelet activation. Using bifunctional cross-linking reagents we have identified an 85 kDa complex containing annexin V. This may represent an association between annexin V and an annexin V-binding protein with a molecular mass of approximately 50 kDa.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
authors.forename: P J
authors.initials: PJ
authors.lastname: Trotter
authors.forename: M A
authors.initials: MA
authors.lastname: Orchard
authors.forename: J H
authors.initials: JH
authors.lastname: Walker
mesh_terms: D017304:Annexin A5 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008081:Liposomes; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D003432:Cross-Linking Reagents; D008081:Liposomes; D010743:Phospholipids; D004533:Egtazic Acid; D011493:Protein Kinase C; D013917:Thrombin; D002118:Calcium