TNAP-positive matrix vesicles are enriched in Annexin II and tetraspanins

Matrix vesicles (MVs) provide a specialised niche for the nucleation of biological apatite. While the mineralisation enabling enzyme tissue non-specific alkaline phosphatase (TNAP) is commonly applied as a marker, its organisation and relative abundance on MVs remains incompletely defined. The relationship between MVs and unbound exosomes/microvesicles recovered from culture media (mEVs) also remains a point of contention. We show that MVs expressed markers of ESCRT (endosomal sorting complexes required for transport) biogenesis common to exosomes/microvesicles, including Alix, TSG101 and CD63. TEM and AFM analysis revealed heterogeneity in the MV population, with pro-mineralising electron-dense and non-mineralising MVs observed. Few MVs (< 7%) expressed TNAP independently of CD63. Applying a bespoke immuno-isolation protocol, we showed that TNAP was only expressed in 36% (MV) and 10% (mEV) of the total vesicle population. Super-resolution microscopy revealed the majority of TNAP⁺ MVs (mean of 82.3%) and mEVs (mean of 72.7%) localised with the calcium channelling protein annexin II. Annexin V (alternative calcium channel) was not upregulated in the TNAP⁺ fraction. Both MVs and mEVs were capable of mineral nucleation, though only MVs isolated at earlier time points (days 7 and 9) appeared capable of generating hydroxyapatite-like material. In conclusion, we identify for the first time that mineralisation-competent TNAP⁺ MVs are enriched in annexin II and express common markers of endosomal biogenesis. We show that a TNAP⁺/annexin II⁺ population is also present in mEV fractions but only quantifiable using high-resolution techniques. These results provide new insights into the composition of pro-mineralising vesicles and give further credence to the hypothesis that MVs may represent anchored exosomes.