Tissue inhibitors of matrix metalloproteinases in platelets and megakaryocytes: a novel organization for these secreted proteins.
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.
Villeneuve
Julien
J
INSERM U889 and Université Victor Segalen Bordeaux 2, France.
Block
Anna
A
Le Bousse-Kerdilès
Marie-Caroline
MC
Lepreux
Sébastien
S
Nurden
Paquita
P
Ripoche
Jean
J
Nurden
Alan T
AT
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*
D016895:Culture Media, Serum-Free; D019714:Tissue Inhibitor of Metalloproteinases