In vitro Modeling of matrix vesicle nucleation -: Synergistic stimulation of mineral formation by annexin a5 and phosphatidylserine

Annexins A5, A2, and A6 ( Anx-A5, -A2, and -A6) are quantitatively major proteins of the matrix vesicle nucleational core that is responsible for mineral formation. Anx-A5 significantly activated the induction and propagation of mineral formation when incorporated into synthetic nucleation complexes made of amorphous calcium phosphate ( ACP) and Anx-A5 or of phosphatidylserine ( PS) plus ACP ( PS-CPLX) and Anx-A5. Incorporation of Anx-A5 markedly shortened the induction time, greatly increasing the rate and overall amount of mineral formed when incubated in synthetic cartilage lymph. Constructed by the addition of Ca2(+) to PS, emulsions prepared in an intracellular phosphate buffer matched in ionic composition to the intracellular fluid of growth plate chondrocytes, these bio-mimetic PS- CPLX nucleators had little nucleational activity. However, incorporation of Anx-A5 transformed them into potent nucleators, with significantly greater activity than those made from ACP without PS. The ability of Anx-A5 to enhance the nucleation and growth of mineral appears to stem from its ability to form two-dimensional crystalline arrays on PS- containing monolayers. However, some stimulatory effect also may result from its ability to exclude Mg2(+) and HCO3- from nucleation sites. Comparing the various annexins for their ability to activate PS- CPLX nucleation yields the following: avian cartilage Anx-A5 > human placental Anx-A5 > avian liver Anx-A5 >= avian cartilage Anx-A6 >> cartilage Anx-A2. The stimulatory effect of human placental Anx-A5 and avian cartilage Anx-A6 depended on the presence of PS, since in its absence they either had no effect or actually inhibited the nucleation activity of ACP. Anx-A2 did not significantly enhance mineralization.