scholarly journals Modulation of endothelial organelle size as an antithrombotic strategy

2020 ◽  
Author(s):  
Francesco Ferraro ◽  
Joana R. Costa ◽  
Robin Ketteler ◽  
Janos Kriston-Vizi ◽  
Daniel F. Cutler
Keyword(s):  
Von Willebrand Factor ◽  
Pharmacological Treatment ◽  
Antithrombotic Therapy ◽  
Secretory Granules ◽  
Dramatic Reduction ◽  
Endothelial Surface ◽  
Wide Range ◽  
Novel Target ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractIt is long-established that Von Willebrand Factor (VWF) is central to haemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), uniquely rod-like exocytic organelles generated in a wide range of lengths (0.5 to 5.0 µm). It has been shown that WPB size responds to physiological cues and pharmacological treatment and that, under flow, VWF secretion from shortened WPBs produces a dramatic reduction of platelet and plasma VWF adhesion to an endothelial surface. WPB-shortening therefore represents a novel target for antithrombotic therapy acting via modulation of VWF adhesive activity. To this aim, we screened a library of licenced drugs and identified several that prompt WPB size reduction. These compounds therefore constitute a novel set of potentially antithrombotic compounds.SummaryThe size of the endothelial secretory granules that store Von Willebrand Factor correlates with its activity, central to haemostasis and thrombosis. Here, human-licenced drugs that reduce the size of these secretory granules are identified, providing a set of novel potential anti-thrombotic compounds.

scholarly journals Weibel-Palade body size modulates the adhesive activity of its von Willebrand Factor cargo in cultured endothelial cells

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Francesco Ferraro ◽  
Mafalda Lopes da Silva ◽  
William Grimes ◽  
Hwee Kuan Lee ◽  
Robin Ketteler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Granules ◽  
Vascular Wall ◽  
Endothelial Cell Surface ◽  
Endothelial Surface ◽  
Wide Range ◽  
Storage Organelle ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall.

scholarly journals Actomyosin II contractility expels von Willebrand factor from Weibel–Palade bodies during exocytosis

2011 ◽  
Vol 194 (4) ◽  
pp. 613-629 ◽  
Author(s):  
Thomas D. Nightingale ◽  
Ian J. White ◽  
Emily L. Doyle ◽  
Mark Turmaine ◽  
Kimberly J. Harrison-Lavoie ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Secretory Granules ◽  
Myosin Ii ◽  
Human Endothelial Cells ◽  
Spatiotemporal Resolution ◽  
Regulated Secretion ◽  
Experimental Systems ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Extracellular Environment

The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel–Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament–based prevention and promotion of specific exocytic steps during regulated secretion.

scholarly journals von Willebrand factor biosynthesis, secretion, and clearance: connecting the far ends

Blood ◽  
2015 ◽  
Vol 125 (13) ◽  
pp. 2019-2028 ◽  
Author(s):  
Peter J. Lenting ◽  
Olivier D. Christophe ◽  
Cécile V. Denis
Keyword(s):  
Life Cycle ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Current Knowledge ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Proinflammatory Response ◽  
Wide Range ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract To understand the placement of a certain protein in a physiological system and the pathogenesis of related disorders, it is not only of interest to determine its function but also important to describe the sequential steps in its life cycle, from synthesis to secretion and ultimately its clearance. von Willebrand factor (VWF) is a particularly intriguing case in this regard because of its important auxiliary roles (both intra- and extracellular) that implicate a wide range of other proteins: its presence is required for the formation and regulated release of endothelial storage organelles, the Weibel-Palade bodies (WPBs), whereas VWF is also a key determinant in the clearance of coagulation factor VIII. Thus, understanding the molecular and cellular basis of the VWF life cycle will help us gain insight into the pathogenesis of von Willebrand disease, design alternative treatment options to prolong the factor VIII half-life, and delineate the role of VWF and coresidents of the WPBs in the prothrombotic and proinflammatory response of endothelial cells. In this review, an update on our current knowledge on VWF biosynthesis, secretion, and clearance is provided and we will discuss how they can be affected by the presence of protein defects.

scholarly journals Arf GTPase-Activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor

2021 ◽  
Author(s):  
Asano Watanabe ◽  
Hikari Hataida ◽  
Naoya Inoue ◽  
Kosuke Kamon ◽  
Keigo Baba ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Intracellular Transport ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Adp Ribosylation ◽  
Gtpase Activating Proteins ◽  
Summary Statement ◽  
Golgi Network ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractArf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors, which are important for intracellular transport. ArfGAPs have been shown to be critical for cargo sorting in the Golgi-to-ER and post-Golgi traffic. However, their roles in the sorting of secretory proteins remains unclear. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPBs are formed at the trans-Golgi Network, and this process is thought to be coupled with the sorting of vWF. WPB biogenesis was reported to be regulated by ADP-ribosylation factors and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. These results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. However, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA)-stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected processes regulated by ArfGAPs for vWF transport.Summary StatementThe ArfGAP proteins SMAP1 and AGFG2 were identified as regulating WPB size and vWF exocytosis.

scholarly journals von Willebrand factor proteolytic processing and multimerization precede the formation of Weibel-Palade bodies

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3536-3544 ◽  
Author(s):  
UM Vischer ◽  
DD Wagner
Keyword(s):  
Von Willebrand Factor ◽  
Healing Process ◽  
Proteolytic Processing ◽  
Wound Healing Process ◽  
Endothelial Surface ◽  
High Calcium ◽  
High Calcium Concentration ◽  
Golgi Network ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We investigated the intracellular site of pro-von Willebrand factor (pro-vWF) cleavage and multimerization, as well as the fate of the propolypeptide (von Willebrand antigen II) after cleavage. Analysis of subcellular fractions of endothelial cells metabolically labeled with sulfate showed that both cleavage and covalent multimerization occur after sulfation and precede the formation of Weibel-Palade bodies. Because sulfation is a processing step localized to the trans-Golgi network (TGN), our results indicate that multimerization and prosequence cleavage also occur in this organelle. After cleavage, the propolypeptide remains noncovalently associated with the mature vWF subunit. This association is promoted by a high calcium concentration and an acidic pH (conditions thought to prevail in the TGN) and explains the 1:1 stoichiometry of the propolypeptide and mature vWF found in Weibel-Palade bodies. The propolypeptide remains an integral part of the large multimeric vWF aggregates in the Weibel-Palade body until secretion. When secretion occurs under slightly acidic conditions, such as may be found in poorly perfused wounds, the propolypeptide remains associated with the endothelial surface-bound vWF, and may thus participate in the wound healing process.

Morphological and Functional Studies of Ultra Long Von Willebrand Factor (ULVWF) Anchored on Endothelial Surface.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1784-1784
Author(s):  
Jing Huang ◽  
Rodger P. McEver ◽  
John Heuser ◽  
J. Evan Sadler
Keyword(s):  
Electron Microscopy ◽  
Shear Stress ◽  
Von Willebrand Factor ◽  
Protein Interactions ◽  
Fluid Shear Stress ◽  
Flow Direction ◽  
Flow Chamber ◽  
Endothelial Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand factor multimers that are tethered on endothelial cells are ultra large, hyperactive and susceptible to ADAMTS13 cleavage. However, the morphology of acutely secreted ULVWF that are deposited on endothelial surface and the mechanism by which they persist under fluid shear stress remain elusive. In this study, we use immunofluorescence and electron microscopy to characterized HUVEC surface associated VWF. By conducting perfusion assays in a parallel plate flow chamber in the presence of an agonist and a fluorescent anti-VWF antibody, we directly visualized VWF secretion and dynamics in real time. Upon histamine or forskolin stimulation, VWF multimers secreted by confluent HUVEC formed extended strings parallel to flow direction. While strings formed independent of platelet adhesion, they bound platelets without requiring platelet activation. Quick-freeze, deep-etch electron microscopy of immuno-gold labeled VWF showed that multiple single strings can merge and form parallel or twisted bundles, suggesting lateral association between individual VWF multimers. These “strings” or “bundles of strings” were tethered to the plasma membrane by a limited number of anchoring points, remained stable for over 30 minutes of continuous laminar flow at the shear stress of 2.5dyn/cm2, and about 50% of these strings were disrupted (bent or washed away) by a reversal of flow direction. Static adhesion assays indicated that both “RGDS” binding integrin and P-selectin were involved in binding of CHO-P cells to ULVWF containing HUVEC supernatant. However, “RGDS” peptide, but not soluble P-selectin or a polyclonal P-selectin antibody by itself, could interfere with the formation of fluorescent VWF strings under flow. These observations suggest that “RGDS” binding integrins are implicated in the presentation of ultra-long VWF strings on endothelial lumen, and that multiple protein-protein interactions could contribute to this process.

Antidote-Controlled Antithrombotic Therapy Targeting Factor IXa and Von Willebrand Factor

2009 ◽  
Vol 1175 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Richard C. Becker ◽  
Sabah Oney ◽  
Kristian C.D. Becker ◽  
Bruce Sullenger
Keyword(s):  
Von Willebrand Factor ◽  
Antithrombotic Therapy ◽  
Factor Ixa ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Selective and Signal-dependent Recruitment of Membrane Proteins to Secretory Granules Formed by Heterologously Expressed von Willebrand Factor

2002 ◽  
Vol 13 (5) ◽  
pp. 1582-1593 ◽  
Author(s):  
Anastasia D. Blagoveshchenskaya ◽  
Matthew J. Hannah ◽  
Simon Allen ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Von Willebrand Factor ◽  
De Novo ◽  
Secretory Granules ◽  
Synaptotagmin I ◽  
Organelle Membrane ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) is a large, multimeric protein secreted by endothelial cells and involved in hemostasis. When expressed in AtT-20 cells, vWF leads to the de novo formation of cigar-shaped organelles similar in appearance to the Weibel-Palade bodies of endothelial cells in which vWF is normally stored before regulated secretion. The membranes of this vWF-induced organelle, termed the pseudogranule, are uncharacterized. We have examined the ability of these pseudogranules, which we show are secretagogue responsive, to recruit membrane proteins. Coexpression experiments show that the Weibel-Palade body proteins P-selectin and CD63, as well as the secretory organelle membrane proteins vesicle-associated membrane protein-2 and synaptotagmin I are diverted away from the endogenous adrenocorticotropic hormone-containing secretory granules to the vWF-containing pseudogranules. However, transferrin receptor, lysosomal-associated membrane protein 1, and sialyl transferase are not recruited. The recruitment of P-selectin is dependent on a tyrosine-based motif within its cytoplasmic domain. Our data show that vWF pseudogranules specifically recruit a subset of membrane proteins, and that in a process explicitly driven by the pseudogranule content (i.e., vWF), the active recruitment of at least one component of the pseudogranule membrane (i.e., P-selectin) is dependent on residues of P-selectin that are cytosolic and therefore unable to directly interact with vWF.

scholarly journals How I treat type 2 variant forms of von Willebrand disease

Blood ◽  
2015 ◽  
Vol 125 (6) ◽  
pp. 907-914 ◽  
Author(s):  
Alberto Tosetto ◽  
Giancarlo Castaman
Keyword(s):  
Von Willebrand Factor ◽  
Bleeding Risk ◽  
Von Willebrand Disease ◽  
Major Surgery ◽  
Bleeding Tendency ◽  
Wide Range ◽  
Deficiency Type ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractType 2 von Willebrand disease (VWD) includes a wide range of qualitative abnormalities of von Willebrand factor structure and function resulting in a variable bleeding tendency. According to the current classification, 4 different subtypes can be identified, each with distinctive phenotypic and therapeutic characteristics. Current available laboratory methods allow a straightforward approach to VWD subtyping, and although the precise molecular characterization remains complex, it is not required for appropriate treatment of the vast majority of cases. Desmopressin can be useful only in a few type 2 cases compared with patients with actual quantitative deficiency (type 1), most often in variants with a nearly normal multimeric pattern (type 2M). However, since no laboratory test accurately predicts response to desmopressin, a trial test should always be performed in all type 2 VWD patients, with the exception of type 2B ones. Replacement therapy with plasma-derived von Willebrand factor-factor VIII concentrates represents the safe mainstay of treatment of all patients, particularly those not responding to desmopressin or requiring a sustained hemostatic correction because of major surgery or bleeding. A significant patient bleeding history correlates with increased bleeding risk and should be considered in tailoring the optimal antihemorrhagic prophylaxis in the individual patient.

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