title: Involvement of Ca2+ Activated Cl- Channel Ano6 in Platelet Activation and Apoptosis.
abstract: OBJECTIVE
  The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP), which trigger increase of cytosolic Ca2+-activity ([Ca2+]i), production of reactive oxygen species (ROS), degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i), degranulation (P-selectin exposure), integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume.
  
  
  METHODS
  Platelets from mice lacking Ano6 (ano6-/-) were compared to platelets from corresponding wild-type mice (ano6+/+). [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbβ3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter.
  
  
  RESULTS
  Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbβ3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml) and CRP (2 or 5 µg/ml) increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbβ3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets.
  
  
  CONCLUSIONS
  Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis of blood platelets.
authors.forename: Guoxing
authors.initials: G
authors.lastname: Liu
authors.forename: Guilai
authors.initials: G
authors.lastname: Liu
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Oliver
authors.initials: O
authors.lastname: Borst
authors.forename: Meinrad
authors.initials: M
authors.lastname: Gawaz
authors.forename: Andrea
authors.initials: A
authors.lastname: Vortkamp
authors.forename: Rainer
authors.initials: R
authors.lastname: Schreiber
authors.forename: Karl
authors.initials: K
authors.lastname: Kunzelmann
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D000075368:Anoctamins; D017209:Apoptosis / Q000187:drug effects; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002097:C-Reactive Protein / Q000494:pharmacology; D002118:Calcium / Q000032:analysis; D051379:Mice; D018345:Mice, Knockout; D019007:P-Selectin / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000172:deficiency / Q000235:genetics; D015539:Platelet Activation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry
chemical_list: C576687:ANO6 protein, mouse; D000814:Aniline Compounds; D000075368:Anoctamins; D019007:P-Selectin; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D017382:Reactive Oxygen Species; D014966:Xanthenes; C059715:Fluo-3; D002097:C-Reactive Protein; D013917:Thrombin; D002118:Calcium