Fig. 3. Diagrammatic representation of the biogenesis of WPBs. (1) VWF tubulation and AP1/clathrin coats are necessary for WPB formation at the TGN. P-selectin is incorporated into WPBs by a direct interaction with VWF. Some other integral membrane proteins may also be recruited at this point. (2) Further extension of the WPBs from the TGN occurs. Tubulation of VWF continues while the WPB remains attached to the TGN by a `stalk-like' connection. (3) The immature WPB is in the perinuclear region of the cell. Uncoating of clathrin occurs, although the tubules remain disorganised. (4) VWF tubules appear longer and more ordered. Clathrin-coated buds can be seen, often towards the tips of WPBs; this is presumably a sorting step to remove misdirected proteins. (5) Mature WPBs are identified at the ultrastructural level by their electron density and tight packing of VWF tubules, whereas at the light-microscopic level the recruitment of Rab27a, Rab3D, as well as the AP3-dependent recruitment of CD63 are the distinguishing features in this final maturation step. Although in both cases the WPBs are now in the periphery of the cell, whether these steps precisely coincide is not yet known.