Substructure of human von Willebrand factor.

Section: Generation Of Recombinant Viruses and Transfection Of Sf9mentioning

confidence: 55%

Defective dimerization of von Willebrand factor subunits due to a Cys-> Arg mutation in type IID von Willebrand disease.

Schneppenheim¹,

Brassard²,

Krey³

et al. 1996

“…(1), and tubules usually extend their entire length (22,23). The helical packing of the dimeric DЈD3 domains, with 4.2 repeating units per 11-nm turn, suggests that every 1 m of tubule length might correspond to a disulfide-linked multimer of Ϸ780 VWF subunits (2 subunits per repeating unit), having a mass of Ϸ195 million Da and an extended length of Ϸ47 m (Ϸ60 nm per subunit) (27). The largest plasma VWF multimers observed by gel electrophoresis or EM are perhaps one-tenth this size (27), but filaments of VWF secreted acutely by endothelial cells in culture or in vivo are often several hundred micrometers long (13,28,29).…”

Section: Discussionmentioning

confidence: 99%

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

Huang

Wang²,

Roth³

et al. 2008

149

285

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.

“…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. 4F) as in rotary shadowing images (37).…”

Section: Multimeric Von Willebrand Factor Tubules Are Helical and Form Amentioning

confidence: 99%

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

Berriman

Hewlett³

et al. 2009

140

In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.electron cryomicroscopy ͉ paracrystal ͉ von Willebrand factor ͉ tomography E ndothelial cells line the inner surfaces of blood vessels and play important roles in hemostasis, thrombosis, and inflammation. Some of these roles are achieved by secretion of the large, multimeric blood glycoprotein von Willebrand factor (VWF). VWF has multiple ligands and on acute release functions as an adhesive protein to bind platelets to sites of vascular injury. VWF circulating in the bloodstream also functions as a carrier for coagulation Factor VIII, increasing its lifetime. Defects in VWF and its storage are responsible for bleeding disorders including von Willebrand's disease (1).VWF is synthesized as a 350-kDa precursor (proVWF) that forms disulfide-linked dimers in the ER through its C-terminal cysteine knot domain. Proteolytic cleavage of proVWF in the Golgi gives rise to the N-terminal propolypeptide (a 100-kDa protein called proregion) and to mature VWF dimers that form large homo-oligomers through disulfide-links near each of its mature N-termini, a process catalyzed by proregion (2, 3). VWF and proregion remain non-covalently associated and are stored together in specialized secretory organelles called WeibelPalade bodies (WPBs), first identified by EM of fixed tissue sections as rod-shaped organelles containing fine tubules (4). Secretagogues stimulate WPB exocytosis, releasing VWF and other low molecular weight molecules such as cytokines and chemokines into the bloodstream (5), although mature VWF and its proregion account for greater than 95% of the protein in the granule (6). On release, VWF multimers are able to unfurl to strings up to 100 m long and associate with multiple ligands on platelet and endothelial cell surfaces at the site of vascular injury to help form a platelet plug. Mechanical shear exposes ligand binding sites on VWF as well as sites for cleavage by the protease ADAMTS13, which regulates the length of VWF multimers in the bloodstream (7).Like most other secretory granules, WPBs are thought to form at the trans-Golgi network (TGN) in a pH-dependent process. P-selectin is also recru...