MOLECULAR CLONING OF THE HUMAN GENE FOR VON WILLEBRAND FACTOR AND IDENTIFICATION OF THE TRANSCRIPTION INITIATION SITE

1987 ◽  
Author(s):  
Corolyn J Collins ◽  
Richard B Levene ◽  
Christina P Ravera ◽  
Marker J Dombalagian ◽  
David M Livingston ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Start Site ◽  
Von Willebrand Factor ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Start Site ◽  
Transcription Start ◽  
Von Willebrand's Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Most patients with von Willebrand's disease appear to have a defect affecting the level of expression of the von Willebrand factor (vWf) gene. Thus, an understanding of the pathogenesis of von Willebrand's disease will require an analysis of the structure and function of the vWf gene in normals and in patients. To begin such analyses, we have screened a human genomic cosmid library with probes obtained from vWf cDNA and isolated a colinear segment spanning ≈175 kb in five overlapping clones. This segment extends ≈25 kb upstream and ≈5 kb downstream of the transcription start and stop sites for vWf mRNA, implying the vWf gene has a length of ≈150 kb. Within one of these clones, the vWf transcription initiation sites have been mapped. A portion of the promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the major transcription start site. Primer extension analysis suggests that sequences contained within the downstream repeat of the transcription start site may be used as minor initiation sites in endothelial cells. Transfection studies are underway to evaluate the role of sequences within this promoter region in gene regulatory activity. Comparative restriction analyses of cloned and chromosomal DNA segments strongly suggests that no major alterations ocurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-expressing endothelial cells and non-expressing white blood cells suggests that no major rearrangements are associated with vWf gene expression. Finally, cross hybridization patterns among seven mammalian species suggests a strong conservation of genomic sequences encoding the plasma portion of vWf, but a lower degree of conservation of sequences encoding the N terminal region of provWf.

Unique expression of von Willebrand factor by type IIA von Willebrand's disease endothelial cells

1992 ◽  
Vol 81 (3) ◽  
pp. 401-406 ◽  
Author(s):  
Barry Wilbourn ◽  
Paul Harrison ◽  
Andrew Lawrie ◽  
Frances LeRoy ◽  
Megan Rowley ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

1993 ◽  
Vol 70 (06) ◽  
pp. 1053-1057 ◽  
Author(s):  
Agnès M Journet ◽  
Simin Saffaripour ◽  
Denisa D Wagner
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Deletion Mutants ◽  
Relative Importance ◽  
D2 Domain ◽  
Constitutive Secretion ◽  
Von Willebrand ◽  
And Storage ◽  
Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

Further Studies on Aggregation of Platelet-Type von Willebrand’s Disease Platelets by Human von Willebrand Factor

1986 ◽  
Vol 55 (03) ◽  
pp. 338-341 ◽  
Author(s):  
H Takahashi ◽  
W Tatewaki ◽  
M Hanano ◽  
R Nagayama ◽  
A Shibata
Keyword(s):  
Von Willebrand Factor ◽  
Ricinus Communis ◽  
Divalent Cations ◽  
Peanut Agglutinin ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Ricinus Communis Agglutinin ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

SummaryPlatelet-type von Willebrand’s disease (vWD) is a bleeding disorder characterized by a heightened interaction between platelets and von Willebrand factor (vWF) as the result of an intrinsic platelet abnormality (probably in GPIb). Platelet aggregability was nearly normal in response to thrombin, wheat germ agglutinin and Ricinus communis agglutinin in this disorder. Unmodified platelets showed no aggregation upon the addition of peanut agglutinin. Partially purified human vWF induced little aggregation of washed patient platelets, but the aggregation was greatly enhanced in the presence of plasma devoid of vWF. Monoclonal antibodies directed against GPIb and GPIIb/IIIa as well as EDTA completely inhibited vWF-induced aggregation. These results indicate that human vWF induces aggregation of platelet-type vWD platelets in the presence of divalent cations and some plasma cofactor(s), and that both GPIb and GPIIb/IIIa are involved in this aggregation.

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 released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1531-1534 ◽  
Author(s):  
LA Sporn ◽  
VJ Marder ◽  
DD Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunofluorescent Staining ◽  
Cultured Endothelial Cells ◽  
Human Foreskin Fibroblasts ◽  
Platelet Binding ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

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