Normal Presence of Tissue Inhibitors of Matrix Metalloproteinases in Platelets of Patients with the Gray Platelet Syndrome Suggests a Novel Organization for These Secreted Proteins.

Tissue inhibitors of matrix metalloproteinases (TIMPs) are a family of molecules that control extracellular matrix degradation through their ability to inactivate matrix metalloproteinases (MMPs). Non-MMP-dependent TIMP functions have also been recognized. Expression of TIMPs is an important way by which activated platelets may intervene in tissue remodeling and angiogenesis. We have studied the localization and release of TIMPs 1-4 from blood platelets, demonstrated their synthesis by megakaryocytes (MKs), and shown their normal presence in platelets of 2 patients with the Gray Platelet syndrome (GPS), an inherited disorder characterized by an absence of alpha-granules and marrow myelofibrosis. Bicolor confocal microscopy using paraformaldehyde-fixed and permeabilized resting platelets was first performed. A membrane glycoprotein (GPIbalpha) and the cytosolic protein beta-tubulin were used as controls. For each TIMP, colocalization with VWF or P-selectin, two alpha-granule proteins, was assessed using paired murine monoclonal or rabbit polyclonal antibodies detected using Alexa 458 or 488 (green) and Alexa 568 (red) conjugated species-specific secondary antibodies. The TIMPs were localized as fluorescent patches apparently distinct from the alpha-granules. These were often distributed at the platelet periphery and in proximity or associated with the membrane. TIMP-3 was also found to have an additional alpha-granule location. For the 2 patients with GPS, an expected sparse labeling of VWF was found in vestigial alpha-granules, but the peripheral expression and localization of the TIMPs remained unchanged and differed from that of residual P-selectin. Western blotting confirmed the presence of the TIMPs in resting platelets and thrombin activation resulted in a loss of TIMPs from platelet lysates with their simultaneous appearance in the platelet supernatants. Biosynthesis of the TIMPs by MKs was indicated by

• the presence of TIMP proteins in MKs derived in vitro from blood CD34+ progenitor cells of normal donors,

• in two megakaryocytic cell lines grown in serum-free conditions, and

• by the presence of mRNAs for each TIMP (RT-PCR).

Thus MKs are a likely source of at least some of the TIMPs found in platelets. The TIMP localization is an illustration of the heterogeneity in the topological organization of the platelet secretome. Further colocalization studies showed that the TIMPs were organized individually and that they were not present as mixed hetero-oligomers. They were also located separately from MMPs (MMP-2, -9, ADAM10 and ADAM17 were tested) that co-localized at least in part in alpha-granules. An exception was the additional alpha-granule localization of TIMP-3 that may agree with its unique property in the TIMP family to associate with extracellulat matrix components. Finally, as TIMPs are key actors in tissue fibrosis, it is interesting to note that they were present in platelets from GPS patients, suggesting that they are not liberated during MK maturation in the bone marrow, an observation that would tend to exclude such a mechanism in the pathophysiology of bone marrow fibrosis, a condition associated with GPS.