Differential Kinetics of Cell Surface Loss of von Willebrand Factor and Its Propolypeptide after Secretion from Weibel-Palade Bodies in Living Human Endothelial Cells

Hannah, Matthew J.; Skehel, Paul; Erent, Muriel; Knipe, Laura; Ogden, David; Carter, Tom

doi:10.1074/jbc.m412547200

Cited by 56 publications

(97 citation statements)

References 29 publications

(23 reference statements)

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“…ssEGFP mobilities were the same in both cell types (P0.19). Coexpression of ProVWF-mRFP did not change D (P0.31; (Erent et al, 2007) it will not interact strongly with ProVWF (Babich et al, 2009;Hannah et al, 2005). Consistent with this, Proregion-EGFP was significantly more mobile than ProVWF-EGFP (P<10 -4 ).…”

Section: Wpb Core Proteins Are Mobile In the Er Of Huvecssupporting

confidence: 71%

“…Primary HUVECs were purchased, cultured, nucleofected and processed for immunocytochemistry as previously described (Babich et al, 2008;Erent et al, 2007;Hannah et al, 2005). For live cell imaging nucleofected cells were plated at confluent density in culture medium onto 35-mm diameter glass-bottomed culture dishes (MatTeK Corp.) or 25-mm diameter glass coverslips (no.…”

Section: Cell Culture and Solutionsmentioning

confidence: 99%

“…Secondary antibodies coupled to fluorophores were from Jackson ImmunoResearch (USA). ProVWF-EGFP, Proregion-EGFP, Proregion-mRFP, tPA-EGFP, P-selectin-EGFP, EGFP-CD63 and eotaxin-3-EGFP were made or obtained as previously described (Babich et al, 2009;Babich et al, 2008;Hannah et al, 2005;Manneville et al, 2003). EGFPRab27a and mRFP-Rab27a were constructed using full-length human Rab27a cDNA obtained from the cDNA Resource Center (www.cdna.org).…”

Section: Reagents and Immunocytochemistrymentioning

confidence: 99%

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Protein mobilities and P-selectin storage in Weibel–Palade bodies

Kiskin¹,

Hellen²,

Babich

et al. 2010

Journal of Cell Science

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Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel–Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion–VWF paracrystalline core by prolonged incubation with NH4Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion–VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

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Section: Wpb Core Proteins Are Mobile In the Er Of Huvecssupporting

confidence: 71%

Section: Cell Culture and Solutionsmentioning

confidence: 99%

Section: Reagents and Immunocytochemistrymentioning

confidence: 99%

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Protein mobilities and P-selectin storage in Weibel–Palade bodies

Kiskin¹,

Hellen²,

Babich

et al. 2010

Journal of Cell Science

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“…Human Rab27a cDNA (gift from Guthrie cDNA resource centre www.cdna.org) was cloned into pEGFP-C3 (Clontech) using HindIII/ApaI. For fixed cell imaging, grids supporting cells Nucleofected with EGFP-hRab27a were fixed, mounted and imaged as previously described (51). For live-cell fluorescence microscopy, cells expressing EGFP-CD63 were imaged at 7 frames per s during histamine (100 M) stimulation at 37°C as previously described (5) and data analyzed in ImageJ Software (http://rsb.info.nih.gov/ij).…”

Section: Methodsmentioning

confidence: 99%

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

Berriman

Hewlett³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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140

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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...

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“…However, there were no significant differences between the I/R group and GPC group at different time points after reperfusion. Because P-selectin is stored both in the WeibelPalade bodies of endothelial cells and within platelet agranules, it can be very rapidly expressed on the cell surface following cell activation (Singh et al, 1999;Hannah et al, 2005;Harakawa et al, 2007;Oostingh et al, 2007). We hypothesize that the blockade of expression of P-selectin from Weibel-Palade bodies of endothelial cells and a-granules of platelets, rather than depression of Pselectin mRNA within the liver, may be the possible mechanism underlying the efficacy of GSH on the expression of P-selectin.…”

Section: Discussionmentioning

confidence: 99%

Protective Effect of Glutathione Against Liver Warm Ischemia‐Reperfusion Injury in Rats is Associated with Regulation of P‐Selectin and Neutrophil Infiltration

Wang

Pang

Liu

et al. 2008

The Anatomical Record

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We examined the effects of glutathione (GSH) preconditioning through the portal vein on rat warm liver ischemia reperfusion injury (I/R injury) and investigated the mechanisms involved. In rats with warm liver I/R injury, administration of GSH by means of the portal vein before ischemia increased the 7-day survival rates of rats after liver I/R from 38% to 75%. This effect was correlated with significantly improved liver function, depressed MDA content in the liver and fewer histologic features of hepatocyte injury. Intrahepatic expression of P-selectin and infiltration of neutrophils were increased significantly after liver I/R. GSH pretreatment decreased intrahepatic MPO content and the expression of P-selectin. However, it did not significantly affect the mRNA levels for P-selectin after liver I/R. Thus, preconditioning with GSH protects the liver against I/R injury by a mechanism dependent on free radical species scavenging, down-regulation of adhesion molecule expression and inhibition of neutrophil accumulation. These findings document the potential clinical utility of GSH to improve the overall success of diverse procedures, such as liver surgery and liver transplantation. Anat Rec, 291:1016Rec, 291: -1022Rec, 291: , 2008 2008 Wiley-Liss, Inc.Key words: ischemia reperfusion injury; liver; glutathione; P-selectinHepatic warm ischemia/reperfusion (I/R) injury, which can occur in diverse settings, including liver transplantation, trauma, hemorrhagic shock, or liver surgery, is a serious clinical complication that may compromise liver function because of extensive hepatocellular loss (Liu et al., 1996;Eghtesad et al., 2003;Miras et al., 2007;Fowell and Benyon, 2008). The mechanisms responsible for liver I/R injury are not well understood. Previous Abbreviations used: I/R 5 ischemia reperfusion; GSH 5 glutathione; ALT 5 alanine aminotransferase; AST 5 aspartate aminotransferase; MPO 5 myeloperoxidase; MDA 5 malondialdehyde; ROS 5 reactive oxygen species; PMN 5 polymorphonuclear leukocytes.

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Differential Kinetics of Cell Surface Loss of von Willebrand Factor and Its Propolypeptide after Secretion from Weibel-Palade Bodies in Living Human Endothelial Cells

Cited by 56 publications

References 29 publications

Protein mobilities and P-selectin storage in Weibel–Palade bodies

Protein mobilities and P-selectin storage in Weibel–Palade bodies

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

Protective Effect of Glutathione Against Liver Warm Ischemia‐Reperfusion Injury in Rats is Associated with Regulation of P‐Selectin and Neutrophil Infiltration

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