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Evidence of a colocalisation of osteoprotegerin (OPG) with von Willebrand factor (VWF) in platelets and megakaryocytes alpha granules. Studies from normal and grey platelets

Osteoprotegerin (OPG), a secreted basic glycoprotein of 60 kD, is a critical modulator of bone resorption. OPG acts as a decoy receptor for the receptor activator of nuclear factor jB ligand (RANKL) in the osteoclasts, inhibiting osteoclast differentiation. OPG is secreted by osteoblastic, endothelial and haematopoietic cells. In endothelial cells, OPG is localised in the Weibel Palade bodies and is associated with von Willebrand factor (VWF) (Zannetino et al, 2005). Grey platelet syndrome (GPS) is a rare inherited platelet disorder, characterised by a mild thrombocytopenia and abnormal platelet morphology. The platelets lack alpha granules and their constituents; thus they have a grey appearance on Wright-Giemsa stained blood smears. Megakaryocytes (MKs) present defects in the alpha granule biogenesis with absence or low levels of the proteins synthesised or endocytosed by MKs, and stored in the alpha granules (Nurden & Nurden, 2007). Because of the endothelial localisation of OPG in the Weibel Palade bodies (Zannetino et al, 2005), and due to their similarity with platelet alpha granules, we hypothesised a possible platelet localization of OPG the in alpha granules. To confirm our hypothesis, after informed consent, as required by the French legislation, platelets were isolated from a known GPS patient, already extensively studied (Levy et al, 1981; Nurden & Nurden, 2007) who had a current platelet count at 30 · 10/l and whose platelet alpha granules constituents (fibrinogen, VWF) remained stable over time. Western Blot analysis was performed using a goat polyclonal anti-human OPG (R&D Systems, Minneapolis, MN, USA). The primary antibody was detected using a monoclonal mouse anti-goat IgG (Sigma, St Louis, MO, USA). Although a band of 55 kD was obtained in control platelets, no band was easily observed in GPS platelets (Fig 1A). These results suggested an alpha granule localisation of OPG leading us to perform a confocal microscopy (Olympus Fluoview FV1000) in normal platelets. Polyclonal goat anti-human OPG (R&D), sheep anti-human VWF: fluorescein isothiocyanate (FITC) (AbD Serotec, Kidlington, UK) and rabbit anti-human Fibrinogen:FITC (Dako, Glostrup, Denmark)were used. The primary OPG antibody was detected using an Alexa Fluor 555 donkey anti-goat IgG (Invitrogen, Eugene, OR, USA). Colocalisation of OPG with VWF and, to a lesser extent, with fibrinogen, was clearly observed (Fig 1B, C). Our data are in agreement with recent studies reporting evidence for the existence of different subtypes of platelet alpha granules whose release depends on different stimuli; VWF and fibrinogen were demonstrated to belong to different subtypes of alpha granules (Italiano et al, 2008). Our data showed that OPG preferentially colocalized with VWF rather than with fibrinogen. Due to a chronic worsening of thrombocytopenia in the GPS patient, a bone marrow biopsy had been performed during a minor surgical procedure under platelet transfusion, in order to evaluate the role of myelofibrosis in the progression of thrombocytopenia. Bone marrow histological examination (A)