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.

Materials and 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 α-granule proteins was assessed in platelets by confocal microscopy, TIMP-1, -2, and -4 were localized as distinct fluorescent patches apart from the established α-granule markers and largely independent of platelet metalloproteinases. TIMP-3 differed for it also had an α-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 α-granules, showed sparse labeling of von Willebrand factor and fibrinogen confined to vestigial α-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 α-granule pools.