Biosynthesis and packaging of VWF in WPBs. The biosynthesis of VWF distinguishes a series of sequential steps that ultimately lead to its incorporation into endothelial storage organelles, the WPBs. (Step 1) During synthesis of the VWF propolypeptide chain in the endoplasmic reticulum, intraprotein cysteine bonding occurs to facilitate folding of the individual domains. Subsequent tail-to-tail interprotein disulfide bridge formation involving the C-terminal CK domains allows the formation of prodimers. Furthermore, the first building blocks for N-linked glycosylation are coupled to the growing polypeptide chain. (Step 2) Upon arrival in the Golgi apparatus, the presence of a slightly acidic pH and relatively high Ca²⁺ concentration promote the organization of the prodimers into a dimeric bouquet structure, in which the dimers are aligned into a side-by-side manner. Moreover, this environment favors multimerization via disulfide bridging that couples adjacent N-terminal D3 domains, a process that is catalyzed by the propeptide. While the multimerization process takes place, the expanding multimer organizes into a right-handed helical structure, allowing 100-fold compaction of the protein. In this helical structure, the propeptide (D1-D2 domains) and the D′-D3 domains form the wall of the hollow tube. The remainder of the VWF protein (A1-CK domains) protrudes outward from the helical architecture, occupying the space between the tubules that characterize the electron-microscopic images of WPBs. VWF tubules assemble into so-called ministacks that represent the first WPB-like structure. During the passage of VWF through the Golgi, maturation of the N-linked glycans proceeds while O-linked carbohydrate structures are also added to the protein. (Step 3) An important gap in our knowledge of WPB formation is the location of the proteins that coreside with VWF in this organelle. For example, FVIII is known to interact with the D′D3 region, suggesting that FVIII may locate to the inner core of the helix. In contrast, osteoprotegerin (which binds to the A1 domain) and galectins-1 and -3 (which bind to VWF glycans) are more likely to be present in the intertubular space. (Step 4) In the Trans-Golgi network, copackaging of VWF-containing ministacks promotes maturation and formation of larger WPBs. In addition, furin mediates the proteolytic separation of the propeptide from the mature VWF subunits. Of note, under the slightly acidic conditions present within the Trans-Golgi network, the propeptide remains associated with mature VWF. Multimer analysis of endothelial VWF has revealed the presence of very large VWF multimers that exceed the size of multimers found in plasma.