title: Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca2+-ATPase inhibitors.
abstract: The development of procoagulant activity and microparticle formation during platelet activation is known to depend on an increase in cytosolic Ca2+ levels. We have studied the mechanisms leading to these events using FITC-labeled recombinant annexin V, a protein which binds with a high affinity to aminophospholipids, in flow cytometry. In particular, we show that the Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid are as potent inducers of aminophospholipid exposure and microparticle formation as the ionophore A23187. In contrast, 2,5-di-tert-butyl-1, 4-benzohydroquinone induced negligible microparticle formation, although platelets abundantly bound annexin V-FITC. That platelet activation had occurred was confirmed by binding studies with VH10, a monoclonal antibody specific for the alpha-granule membrane glycoprotein GMP-140, and by prothrombinase activity measurements. These results demonstrate that microvesiculation is not an automatic response to aminophospholipid exposure. The Ca(2+)-ATPase inhibitors induced different intracellular Ca2+ levels as measured using fluo-3 as a calcium dye. These were 10 +/- 4 microM (n = 11) for thapsigargin (3 microM), 19.6 +/- 2.2 microM (n = 8) for cyclopiazonic acid (100 microM), and 0.619 +/- 0.137 microM (n = 8) for 2,5-di-tert-butyl-1,4-benzohydroquinone (100 microM). Calpain activity, as assessed in platelets by analyzing the degradation of cytoskeletal proteins, was only observed with agents that stimulated microparticle formation. Phospholipid transbilayer movement was studied by measuring annexin V binding during platelet activation. Results showed that aminophospholipid exposure induced by ionophore A23187 (t1/2 = 133 +/- 14 s) was more rapid than that induced by TG (t1/2 = 280 +/- 30 s), although the rate-limiting step in the assay was the binding of annexin V to activated platelets (t1/2 = 70-80 s). Interestingly, the presence of annexin V itself during the activation inhibited microparticle formation, although degradation of platelet proteins by calpain continued to occur. Our results clearly show (i) that aminophospholipid exposure and platelet microvesiculation are independent but closely regulated events and (ii) that while both processes are associated with an increase in intracellular Ca2+, microvesiculation additionally requires Ca(2+)-induced calpain activation and a fusion process inhibited by annexin V.
authors.affiliation: URA CNRS 1464, Hôpital Cardiologique, Pessac, France.
authors.forename: J
authors.initials: J
authors.lastname: Dachary-Prigent
authors.forename: J M
authors.initials: JM
authors.lastname: Pasquet
authors.forename: J M
authors.initials: JM
authors.lastname: Freyssinet
authors.forename: A T
authors.initials: AT
authors.lastname: Nurden
mesh_terms: D017304:Annexin A5 / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002154:Calpain / Q000378:metabolism; D003029:Coagulants / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008863:Microspheres; D015335:Molecular Probes; D019007:P-Selectin / Q000276:immunology; D010743:Phospholipids / Q000378:metabolism*; D015539:Platelet Activation; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D017304:Annexin A5; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D003029:Coagulants; D004791:Enzyme Inhibitors; D015335:Molecular Probes; D019007:P-Selectin; D010743:Phospholipids; D013925:Thromboplastin; D002154:Calpain; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium