Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

Abstract

Lengthwise sections show longitudinal striations, and cross sections reveal closely spaced Ϸ20-nm diameter tubules separated by a less-dense matrix (1). Weibel-Palade bodies are composed almost entirely of von Willebrand factor (VWF) (2, 3), which is a multimeric plasma glycoprotein that can exceed 20 million Da in mass and 4 m in length. Megakaryocytes synthesize large VWF multimers and package them into platelet ␣-granules that are roughly spherical rather than cigar-shaped. Nevertheless, the VWF multimers in ␣-granules are organized into clusters of tubules with dimensions similar to those of VWF tubules in Weibel-Palade bodies (4). VWF is the largest known protein in the blood, and the very largest VWF multimers bind connective tissue and mediate platelet adhesion at sites of vascular injury. Weibel-Palade bodies play a critical role in hemostasis by delivering VWF multimers into the circulation. Defects in VWF multimer structure cause several forms of von Willebrand disease, the most common inherited bleeding disorder worldwide (5).VWF is synthesized as an Ϸ350-kDa precursor with a signal peptide and five kinds of structural domains arranged in the order D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK ( Fig. 1) (5). In the endoplasmic reticulum (ER), proVWF dimerizes through disulfide bonds between C-terminal CK domains (6-8).The resultant ''tail-to-tail'' proVWF dimers are transported to the Golgi, where the propeptide (domains D1D2) is cleaved by furin and additional ''head-to-head'' disulfide bonds form between D3 domains, yielding multimers that condense into tubules and form Weibel-Palade bodies (6, 9).In fact, the expression of VWF determines the existence and also the cigar-like shape of Weibel-Palade bodies. Without VWF, endothelial cells lack Weibel-Palade bodies (10, 11), and the expression of VWF in other cell types that have a regulated secretory pathway results in the generation of organelles that are indistinguishable from Weibel-Palade bodies (12, 13). This self-organizing behavior depends on a conserved set of Nterminal domains that have the dual function of promoting multimer assembly and directing tubular storage.Multimer assembly (14-16) and tubular packing (12, 17) both depend on the N-terminal D1D2DЈD3 domains and require the acidic pH of the late secretory pathway (13). Furthermore, the tubular morphology of Weibel-Palade bodies is essential for . VWF multimers are held together by intersubunit disulfide bonds between CK domains, which form in the ER, and by intersubunit disulfide bonds between D3 domains, which form in the Golgi. The structures of polypeptides encoded by the plasmids used in these studies are indicated.

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

“…The structural organization of VWF in WPBs suggests a specific model by which VWF can drive formation of a granule. VWF is organized as a helix in WPBs, similar in structure to helices formed by assembly of the D1D2/D'D3 domains of VWF in vitro (25). Covalent addition of a new VWF dimer to the end of a growing helix, catalyzed by proregion, provides a strategy for packaging the long strings of covalently-linked VWF without entanglement.…”

“…4E) under low pH and divalent calcium conditions similar to those present during VWF assembly in the TGN (25). The repeating subunit of the in vitro structure has been interpreted as consisting of a covalent D'D3 domain dimer and two proregions (D1D2).…”

“…Highly extended C-terminal polypeptides, disulfide-linked at a cysteine knot (CK) domain, connect the repeating subunits of the core helix (25) but are likely poorly ordered. Multimerization is required for VWF function following release by exocytosis although not for tubule (25) or granule formation (36). Disassembly of the helix during granule exocytosis would then produce mature, multimeric VWF with globular domains separated by more extended polypeptide giving the appearance of beads on a string (schematic, Fig.…”

“…We address structural aspects of two problems posed by VWF trafficking: how can a long multimeric protein be organized for dense storage in WPBs, and how can the packaging of this protein determine the identity and morphology of this unique secretory granule? We combine images and tomograms to show that VWF is packaged as a helix in WPBs similar to those that can be assembled in vitro from only proregion (domains D1D2) and the N-terminal D'D3 domains of mature VWF (25). We build a 3D model describing the higherorder assembly of VWF tubules and the surrounding membrane in a WPB.…”

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

“…6 In the trans-Golgi and post-Golgi compartments, the VWF propeptide catalyzes the formation of VWF multimers via disulfide bonds near the VWF N terminus (C1099-C1099 and C1142-C1142) and is cleaved from proVWF by furin. [7][8][9] The resulting mature VWF multimers condense into long, helical structures that characterize the shape of WeibelPalade bodies and are released into the circulation as long, linear polymers with multiple concatemerized subunits. Each VWF multimer is composed of 2 to .60 subunits 10 (with each monomer containing a single FVIII-binding site).…”