title: Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity.
abstract: BACKGROUND
  Activated platelets express a procoagulant surface when the asymmetric distribution of membrane phospholipids is scrambled, leading to phosphatidylserine (PS) exposure. PS expression, associated with apoptosis in nucleated cells, would be expected to be reversed by aminophospholipid translocase (APLT) activity.
  
  
  OBJECTIVE
  To determine whether the procoagulant surface of activated platelets persists after it forms; to examine whether PS expression on platelets is associated with loss of mitochondrial inner membrane potential (DeltaPsi(m)), a hallmark of apoptosis; and to investigate the role of APLT in persistence of PS expression.
  
  
  METHODS
  Platelets were stimulated with thrombin, collagen, a combination of both, or the Ca(2+)-ionophore A23187. Up to 4 h after activation, procoagulant surface expression was measured by annexin A5 binding by flow cytometry and by a prothrombinase assay. Flow cytometry was also used to measure PS expression concurrently with DeltaPsi(m) collapse, using CMXRos. APLT activity in annexin A5-negative and -positive platelets was measured flow cytometrically as the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet.
  
  
  CONCLUSIONS
  Procoagulant surface expression on activated platelets persisted in vitro for at least 4 h; if such persistence occurs in vivo, there are important implications for the propagation of thrombosis. With the physiological stimuli, only 10-20% of the activated platelets expressed PS on their surface, and of these, only a portion exhibited DeltaPsi(m) collapse, indicating that PS expression can be associated with platelet apoptosis, but can also occur independently. APLT activity was very low in the PS-expressing platelet subpopulation for up to 4 h after activation, indicating that the persistence of a procoagulant surface may be attributed, at least in part, to this reduced APLT activity.
authors.affiliation: Division of Haematology, Oncology and Physiology and Experimental Medicine Program, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Leung
authors.forename: A M
authors.initials: AM
authors.lastname: Gwozdz
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: K W A
authors.initials: KW
authors.lastname: Bang
authors.forename: M A
authors.initials: MA
authors.lastname: Packham
authors.forename: J
authors.initials: J
authors.lastname: Freedman
authors.forename: M L
authors.initials: ML
authors.lastname: Rand
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D003029:Coagulants / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005165:Factor V / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism; D013997:Time Factors
chemical_list: D003029:Coagulants; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; C022475:prothrombinase complex; D000001:Calcimycin; D005165:Factor V; D003094:Collagen; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa