Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

Rosenberg, Jonathan B.; Foster, Paul A.; Kaufman, Randal J.; Vokac, Elizabeth A.; Moussalli, Micheline J.; Kroner, Philip A.; Montgomery, RR

doi:10.1172/jci1250

Cited by 107 publications

(118 citation statements)

References 55 publications

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“…Finally, our findings on sequestering of P-selectin by multimeric vWF are also reminiscent of the data acquired by recent vWF and factor VIII coexpression studies in transfected AtT-20 cells and in bovine aortic endothelial cells. 30 In both cell types, colocalization of factor VIII and vWF was observed in storage granules, whereas no storage of factor VIII could be detected in the absence of vWF synthesis. In those studies, it was elegantly shown that intracellular vWF-mediated trafficking of factor VIII was due to actual binding of factor VIII to vWF in the trans-Golgi network.…”

Section: Discussionmentioning

confidence: 92%

Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin

Hop

Guilliatt

Daly

et al. 2000

ATVB

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Abstract-We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA-transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA-transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules. Key Words: von Willebrand factor Ⅲ P-selectin Ⅲ Weibel-Palade bodies Ⅲ protein sorting M ultimeric von Willebrand factor (vWF) is an obligatory glycoprotein for the formation of a hemostatic plug. It connects the platelet to the subendothelium that is exposed on injury of the vessel wall. vWF is synthesized by megakaryocytes and vascular endothelial cells. Endothelial cells display 2 secretory routes for newly synthesized vWF, namely, a constitutive and a regulated pathway. 1 Constitutively secreted vWF is rapidly transported in secretory vesicles toward the plasma membrane. This material consists of vWF dimers and low-molecular-weight multimers. Another portion of the newly synthesized vWF is encountered in specific storage organelles, denoted Weibel-Palade bodies (WPBs). These organelles fuse with the plasma membrane on activation of the protein kinase C signal transduction pathway, followed by release of their contents (regulated secretion). 2,3 vWF routed by this pathway consists of high-molecular-weight multimers that are Ϸ2 orders of magnitude more effective in mediating adhesion of platelets to the injured vessel wall than are vWF dimers or low-molecular-weight multimers. 4 The WPBs are considered to be endothelial cell-specific, rod-shaped, electron-dense storage organelles. 5 Apart from vWF multimers and the vWF propolypeptide, 6 -8 the WPB contains a number of other proteins, namely CD63, a transmembrane glycoprotein that is also present in lysosomal membranes of many cell types 9,10 ; interleukin-8 11 ; tissue plasminogen activator 12 ; and the transmembrane receptor P-selectin (also called GMP-140 or PADGEM). 13,14...

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Section: Discussionmentioning

confidence: 92%

Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin

Hop

Guilliatt

Daly

et al. 2000

ATVB

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“…3,21 Transfected AtT-20 cells were grown on 35-mm culture plates (Fisher Scientific, Itasca, IL) in Dulbecco modified Eagle medium/G418 media for 72 hours. Negative controls (nontransfected and mock-transfected cells) were processed in parallel with each immunofluorescence labeling experiment.…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

“…2 VWF undergoes a series of intracellular modifications, including dimerization, glycosylation, sulfation, N-terminal multimerization, VWFpp cleavage, and FVIII binding. 3,4 VWF is synthesized exclusively in endothelial cells and megakaryocytes. In these cells, VWF is packaged in releasable storage vesicles termed Weibel-Palade bodies in endothelium and ␣-granules in megakaryocytes and platelets.…”

Section: Introductionmentioning

confidence: 99%

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

Rosenberg

Haberichter

Jozwiak

et al. 2002

Blood

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While studying patient plasma containing an unusual pattern of von Willebrand factor (VWF) multimers, we discovered a previously unreported phenomenon: heavy predominance of dimeric VWF. Genomic analysis revealed a new congenital mutation (Tyr87Ser) that altered the final stages of VWF biosynthesis. This mutation in the propeptide (VWFpp) resulted in synthesis of dimeric VWF with an almost complete loss of N-terminal multimerization. The multimer pattern in patient plasma appears to result from separate alleles' synthesizing wild-type or mutant (dimeric) VWF, with homodimers composing the predominant protomeric species. We have expressed VWF protein containing the Tyr87Ser mutation and analyzed the intracellular processing and resulting VWF biological functions. The expressed dimeric VWF displayed a loss of several specific functions: collagen binding, factor VIII binding, and ristocetin-induced platelet binding. However, granular storage of dimeric VWF was normal, demonstrating that the lack of multimerization does not preclude granular storage. Although the tertiary structure of the VWFpp remains unknown, the mutant amino acid is located in a region that is highly conserved across several species and may play a major role in the multimerization of VWF. Our data suggest that one function of the highly cysteine-rich VWFpp is to align the adjacent subunits of VWF into the correct configuration, serving as an intramolecular chaperone. The integrity of the VWFpp is essential to maintain the proper spacing and alignment of the multiple cysteines in the VWFpp and N-terminus of the mature

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“…[15][16][17][18] Expression of FVIII in endothelial cells results in storage of FVIII in WPBs. 6,19,20 Co-storage of the VWF/FVIII complex in secretory granules and subsequent release of the VWF/FVIII complex upon agonist-induced stimulation has the potential of secreting large amounts of FVIII at sites of vascular injury as well as directly increasing FVIII half-life by protecting FVIII from premature clearance and proteolytic degradation.…”

Section: Introductionmentioning

confidence: 99%

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Biggelaar¹,

Bouwens²,

Kootstra³

et al. 2009

Haematologica

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BackgroundGene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion. Design and MethodsHuman blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human Bdomain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy. ResultsBlood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1×10 6 cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6-to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15-22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies. ConclusionsWe conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.

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Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

Cited by 107 publications

References 55 publications

Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin

Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

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