scholarly journals RGS4 controls secretion of von Willebrand factor to the subendothelial matrix

2020 ◽  
Vol 133 (14) ◽  
pp. jcs247312
Author(s):  
Francesca Patella ◽  
Daniel F. Cutler
Keyword(s):  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Endothelial Damage ◽  
Rna Seq ◽  
G Protein Signalling ◽  
Functional Consequences ◽  
Subendothelial Matrix ◽  
Storage Granules ◽  
Von Willebrand ◽  
Willebrand Factor

ABSTRACTThe haemostatic protein von Willebrand factor (VWF) exists in plasma and subendothelial pools. The plasma pools are secreted from endothelial storage granules, Weibel–Palade bodies (WPBs), by basal secretion with a contribution from agonist-stimulated secretion, and the subendothelial pool is secreted into the subendothelial matrix by a constitutive pathway not involving WPBs. We set out to determine whether the constitutive release of subendothelial VWF is actually regulated and, if so, what functional consequences this might have. Constitutive VWF secretion can be increased by a range of factors, including changes in VWF expression, levels of TNF and other environmental cues. An RNA-seq analysis revealed that expression of regulator of G protein signalling 4 (RGS4) was reduced in endothelial cells (HUVECs) grown under these conditions. siRNA RGS4 treatment of HUVECs increased constitutive basolateral secretion of VWF, probably by affecting the anterograde secretory pathway. In a simple model of endothelial damage, we show that RGS4-silenced cells increased platelet recruitment onto the subendothelial matrix under flow. These results show that changes in RGS4 expression alter levels of subendothelial VWF, affecting platelet recruitment. This introduces a novel control over VWF function.

Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles

Blood ◽  
2009 ◽  
Vol 113 (13) ◽  
pp. 3102-3109 ◽  
Author(s):  
Maartje van den Biggelaar ◽  
Alexander B. Meijer ◽  
Jan Voorberg ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Hek293 Cells ◽  
Binding Studies ◽  
Cargo Proteins ◽  
Storage Granules ◽  
Factor Type ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that coexpression of factor VIII (FVIII) with VWF results in costorage of both proteins. However, whether cotrafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of cotargeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly with VWF.

Distinct Functions of pH-Sensing Histidine Residues in the Multimer Assembly and Storage of Von Willebrand Factor in Weibel-Palade Bodies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3311-3311
Author(s):  
Jing Huang ◽  
Lisa A Westfield ◽  
J. Evan Sadler
Keyword(s):  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Conflicts Of Interest ◽  
Hek293 Cells ◽  
Ph Sensing ◽  
Wild Type ◽  
Length Width ◽  
Storage Granules ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3311 The hemostatic function of von Willebrand factor (VWF) depends on the formation of disulfide-linked multimers. We have previously shown that several evolutionarily conserved His residues within the VWF propeptide are critical for pH-dependent VWF multimerization. For example, the charge-neutralization mutations H395A and H460A in the VWF D2 domain prevented multimer assembly, while the charge-stabilization mutation H460K preserved it, suggesting that protonation at these sites is essential. In this study, we investigated the role of these His residues in VWF targeting to storage granules and agonist-induced secretion. Full-length VWF was expressed in transiently-transfected HEK293 cells. If Weibel-Palade body-like storage granules (WPBs) were formed, their morphology was analyzed by immunofluorescence microscopy. A WPB was defined as “long” if length/width was > 2, or “round” if length/width was ≤ 2. To analyze the assembly of VWF multimers, we stimulated VWF secretion by incubating cells with 100 ng/ml of phorbol myristate acetate for 1 hour and collected cell lysates and supernatants for multimer gel electrophoresis. Expression of wild type VWF and VWF H460K induced the formation of approximately 60% long WPBs. Treatment of cells with monensin increases the pH of the Golgi, and as expected monensin reduced the storage of wild type VWF in long WPBs. However, monensin did not impair the storage of VWF H460K or reduce the fraction of long WPBs, indicating that a positive charge at residue 460 can sustain tubular storage of VWF despite neutralization of the late secretory pathway. Both WT VWF and VWF H460K were secreted with a normal distribution of multimers. VWF H395A and VWF H460A formed predominantly round pseudo-WPBs, with only 10–20% long granules, and these mutations also disrupted VWF multimerization. In contrast, VWF H395R and VWF H395A/H460R did not assemble multimers but nevertheless formed long WPBs. Insertion of an extra Gly between the vicinal cysteines in the D1 domain (159CGLC162 to 159CGGLC162) was reported to prevent multimerization but allow storage in long WPBs in AtT-20 cells (Mayadas & Wagner, PNAS 1992; 89:3531). We found that this mutation also inhibited VWF multimerization and resulted in the formation of long WPBs in HEK293 cells. These results indicate that VWF multimerization and packing in elongated WPBs both depend on the acidic pH of the late secretory pathway. However, these processes are independent, with distinct structural requirements, and they can be dissociated by mutations of specific His residues. VWF construct Multimers Granule Shape Granule Shape (with monensin) Wild type Yes Long Round H395A No Round H395R No Long H395A/H460R No Long H460A No Round H460K Yes Long Long 159CGGLC162 No Long Disclosures: No relevant conflicts of interest to declare.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

scholarly journals Von Willebrand factor (vWF): marker of endothelial damage and thrombotic risk in COVID-19?

2020 ◽  
Vol 20 (5) ◽  
pp. e178-e182 ◽  
Author(s):  
Eleni E Ladikou ◽  
Helena Sivaloganathan ◽  
Kate M Milne ◽  
William E Arter ◽  
Roshan Ramasamy ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Endothelial Damage ◽  
Thrombotic Risk ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Insights Into Immunothrombosis: The Interplay Among Neutrophil Extracellular Trap, von Willebrand Factor, and ADAMTS13

2020 ◽  
Vol 11 ◽  
Author(s):  
Junxian Yang ◽  
Zhiwei Wu ◽  
Quan Long ◽  
Jiaqi Huang ◽  
Tiantian Hong ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Therapeutic Strategy ◽  
Endothelial Activation ◽  
Endothelial Damage ◽  
Neutrophil Extracellular Trap ◽  
Activated Neutrophils ◽  
A Disintegrin And Metalloproteinase ◽  
Von Willebrand ◽  
Willebrand Factor

Both neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) are essential for thrombosis and inflammation. During these processes, a complex series of events, including endothelial activation, NET formation, VWF secretion, and blood cell adhesion, aggregation and activation, occurs in an ordered manner in the vasculature. The adhesive activity of VWF multimers is regulated by a specific metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Increasing evidence indicates that the interaction between NETs and VWF contributes to arterial and venous thrombosis as well as inflammation. Furthermore, contents released from activated neutrophils or NETs induce the reduction of ADAMTS13 activity, which may occur in both thrombotic microangiopathies (TMAs) and acute ischemic stroke (AIS). Recently, NET is considered as a driver of endothelial damage and immunothrombosis in COVID-19. In addition, the levels of VWF and ADAMTS13 can predict the mortality of COVID-19. In this review, we summarize the biological characteristics and interactions of NETs, VWF, and ADAMTS13, and discuss their roles in TMAs, AIS, and COVID-19. Targeting the NET-VWF axis may be a novel therapeutic strategy for inflammation-associated TMAs, AIS, and COVID-19.

scholarly journals The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells

1992 ◽  
Vol 286 (2) ◽  
pp. 631-635 ◽  
Author(s):  
M A Carew ◽  
E M Paleolog ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Selective Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.

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