title: BH3-mimetic ABT-737 induces strong mitochondrial membrane depolarization in platelets but only weakly stimulates apoptotic morphological changes, platelet shrinkage and microparticle formation.
abstract: BACKGROUND
  Depolarization of mitochondrial inner transmembrane potential (ΔΨm) is a key biochemical manifestation of the intrinsic apoptosis pathway in anucleate platelets. Little is known, however, about the relationship between ΔΨm depolarization and downstream morphological manifestations of platelet apoptosis, cell shrinkage and microparticle (MP) formation.
  
  
  OBJECTIVE
  To elucidate this relationship in human platelets.
  
  
  METHODS
  Using flow cytometry, we analyzed ΔΨm depolarization, platelet shrinkage and MP formation in platelets treated with BH3-mimetic ABT-737 and calcium ionophore A23187, well-known inducers of intrinsic platelet apoptosis.
  
  
  RESULTS
  We found that at optimal treatment conditions (90min, 37°C) both ABT-737 and A23187 induce ΔΨm depolarization in the majority (88-94%) of platelets and strongly increase intracellular free calcium. In contrast, effects of A23187 and ABT-737 on platelet shrinkage and MP formation are quite different. A23187 strongly stimulates cell shrinkage and MP formation, whereas ABT-737 only weakly induces these events (10-20% of the effect seen with A23187, P<0.0001).
  
  
  CONCLUSIONS
  These data indicate that a high level of ΔΨm depolarization and intracellular free calcium does not obligatorily ensure strong platelet shrinkage and MP formation. Since ABT-737 efficiently induces clearance of platelets from the circulation, our results suggest that platelet clearance may occur in the absence of the morphological manifestations of apoptosis.
authors.affiliation: Division of Transfusion Medicine, Department of Laboratory Medicine, The Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, ON, Canada.
authors.forename: Armen V
authors.initials: AV
authors.lastname: Gyulkhandanyan
authors.affiliation: Division of Transfusion Medicine, Department of Laboratory Medicine, The Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, ON, Canada.
authors.forename: Asuman
authors.initials: A
authors.lastname: Mutlu
authors.affiliation: Division of Transfusion Medicine, Department of Laboratory Medicine, The Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, ON, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: Allen
authors.affiliation: Division of Transfusion Medicine, Department of Laboratory Medicine, The Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, ON, Canada; Toronto Platelet Immunobiology Group, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Department of Medicine, University of Toronto, Toronto, ON, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Freedman
authors.affiliation: Division of Transfusion Medicine, Department of Laboratory Medicine, The Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, ON, Canada; Toronto Platelet Immunobiology Group, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Department of Medicine, University of Toronto, Toronto, ON, Canada; Department of Physics, Ryerson University, Toronto, ON, Canada. Electronic address: leytinv@smh.ca.
authors.forename: Valery
authors.initials: V
authors.lastname: Leytin
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D040761:Biomimetic Materials / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood; D055252:Cell-Derived Microparticles / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D051336:Mitochondrial Membranes / Q000187:drug effects* / Q000378:metabolism; D009596:Nitrophenols / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects*; D013449:Sulfonamides / Q000494:pharmacology*
chemical_list: C501332:ABT-737; D001713:Biphenyl Compounds; D009596:Nitrophenols; D010879:Piperazines; D013449:Sulfonamides; D000001:Calcimycin; D002118:Calcium
keywords: 3,3’-dihexyloxacarbocyanine iodide; Apoptosis in platelets; BH3-mimetic ABT-737; Calcium ionophore A23187; DMSO; DiOC(6)(3); FSC; GPRP; Gly-Pro-Arg-Pro peptide; MP; Microparticle formation; Mitochondrial inner membrane depolarization; PRP; PS; Platelet shrinkage; SSC; dimethylsulfoxide; forward scatter; microparticle; mitochondrial inner transmembrane potential; phosphatidylserine; platelet-rich plasma; side scatter; ΔΨm