title: Tissue inhibitors of matrix metalloproteinases in platelets and megakaryocytes: a novel organization for these secreted proteins.
abstract: OBJECTIVE
  Expression of tissue inhibitors of matrix metalloproteinases (TIMPs) is one way that activated platelets intervene in tissue remodeling and angiogenesis. Our study was designed to investigate their synthesis in megakaryocytes (MKs) and their storage in platelets.
  
  
  METHODS
  TIMP expression in MKs derived from blood CD34(+) progenitor cells of normal donors and a megakaryocytic cell line (CHRF-288-11) grown in serum-free conditions and platelets from normal donors or two patients with gray platelet syndrome was studied by immunofluorescence labeling, reverse transcription-polymerase chain reaction, and western blotting.
  
  
  RESULTS
  Biosynthesis of TIMPs 1-4 in MKs was indicated by presence of their messenger RNAs as shown by polymerase chain reaction and of their proteins. Immunofluorescence labeling suggested a primarily granular localization of TIMPs in MKs and platelets. But when colocalization with von Willebrand factor, fibrinogen, P-selectin, and other alpha-granule proteins was assessed in platelets by confocal microscopy, TIMP-1, -2, and -4 were localized as distinct fluorescent patches apart from the established alpha-granule markers and largely independent of platelet metalloproteinases. TIMP-3 differed for it also had an alpha-granule location. Western blotting confirmed the presence of TIMPs 1-4 in platelets and thrombin activation resulted in their extensive release to the medium. Platelets from two patients with gray platelet syndrome, congenitally deficient in alpha-granules, showed sparse labeling of von Willebrand factor and fibrinogen confined to vestigial alpha-granules; however, localization of the TIMPs was unchanged.
  
  
  CONCLUSIONS
  TIMPs are synthesized and organized in MKs and platelets independently of other secreted proteins present in alpha-granule pools.
authors.affiliation: INSERM U889 and Université Victor Segalen Bordeaux 2, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Villeneuve
authors.forename: Anna
authors.initials: A
authors.lastname: Block
authors.forename: Marie-Caroline
authors.initials: MC
authors.lastname: Le Bousse-Kerdilès
authors.forename: Sébastien
authors.initials: S
authors.lastname: Lepreux
authors.forename: Paquita
authors.initials: P
authors.lastname: Nurden
authors.forename: Jean
authors.initials: J
authors.lastname: Ripoche
authors.forename: Alan T
authors.initials: AT
authors.lastname: Nurden
mesh_terms: D000368:Aged; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D016895:Culture Media, Serum-Free; D005260:Female; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism*; D015539:Platelet Activation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019714:Tissue Inhibitor of Metalloproteinases / Q000096:biosynthesis / Q000097:blood*
chemical_list: D016895:Culture Media, Serum-Free; D019714:Tissue Inhibitor of Metalloproteinases