The release of von Willebrand Factor thrombomodulin and p-selectin from cultured endothelial cells induced by cyclosporin

1995 ◽  
Vol 6 (6) ◽  
pp. 595
Author(s):  
S. M. Evans ◽  
J. C. Giddings ◽  
S. L Tanswell ◽  
K. K. Hampton ◽  
J. A. Whittaker
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

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.

Von Willebrand Factor in Cultured Human Vascular Endothelial Cells from Adult and Umbilical Cord Arteries and Veins

1986 ◽  
Vol 56 (02) ◽  
pp. 189-192 ◽  
Author(s):  
Pauline B van Wachem ◽  
Jan Hendrik Reinders ◽  
Marijke F van Buul-Wortelboer ◽  
Philip G de Groot ◽  
Willem G van Aken ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vena Cava ◽  
Ionophore A23187 ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Arteries And Veins

SummaryEndothelial cells were cultured from various human arteries and veins, obtained from adult individuals and from umbilical cords. We compared the storage and secretion of von Willebrand factor by endothelial cells from umbilical veins with that of endothelial cells cultured from a number of adult vessels, including aorta, arteria iliaca, vena saphena magna and vena cava. There were no differences in the way the cultured endothelial cells handled the von Willebrand factor they synthesized. Endothelial cells from the various vessels responded to stimuli in secreting stored von Willebrand factor. The cells also responded to thrombin and ionophore A23187 in producing enhanced amounts of prostacyclin. Thus, cultured umbilical vein endothelial cells have properties that are very similar to those of cultured endothelial cells of various other origins. It is concluded that foetal venous cells provide a representative model for studies of endothelial cell von Willebrand factor biosynthesis and prostacyclin production.

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

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.

scholarly journals Erratum: 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 ◽  
Body Size ◽  
Von Willebrand Factor ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Relative Importance of the Glycoprotein Ib-Binding Domain and the RGD Sequence of von Willebrand Factor for Its Interaction With Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2335-2344 ◽  
Author(s):  
Christelle Perrault ◽  
Hanneke Lankhof ◽  
Dominique Pidard ◽  
Danièle Kerbiriou-Nabias ◽  
Jan J. Sixma ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Binding Domain ◽  
Rgd Sequence ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Endothelial Cell Adhesion

Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.

RISTOCETIN-INDUCED ENDOTHELIAL CELL BINDING OF PLASMA VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
KA Vokac ◽  
J Ferrars ◽  
RR Montgomery
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Mouse Serum ◽  
Type I ◽  
Antibody Technique ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Our laboratory previously used a technique of labeling plasma von Willebrand factor (vWf) with radiolabeled AVW1 - a “ neutral” monoclonal antibody to vWf. This technique has been used to study the binding of plasma vWf to platelets in the plasma milieu. Studies by several laboratories including ours have demonstrated structural glycoproteins on endothelial cells that are analogous to platelet GP Ilb/IIIa and we have shown that the platelet alloantigen Pl-Al is expressed on the surface of cultured endothelial cells. We undertook this study to evaluate the binding of plasma vWf to cultured endothelial cells in confluent monolayer cultures using the “ neutral” monoclonal antibody technique. Plasma vWf was “ labeled” using trace quantities of radiolabeled AVW-1. We then added 80,000 cpm of monoclonal-labeled plasma to 48 well culture plates containing confluent secondary cultures of human umbilical vein endothelial cells. Following the addition of ristocetin, the plates were incubated for 1 hour at room temperature, centrifuged, and the count8 bound and the counts remaining in the supernate were determined. In the presence of ristocetin, 67.5% of the labeled vWf bound to the endothelial cells. When “ labeled“ severe von Willebrand plasma was used or when ristocetin was omitted, less than 5% of the counts bound. Controls using mouse serum or excess mouse IgG to rule out Fc receptor binding and controls to evaluate binding to the subcellular matrix were performed and demonstrated this binding to be vWf and cell surface dependent. Unlike platelet vWf binding, this binding was not inhibited by monoclonal or polyclonal antibodies to platelet GPIb. We studied plasma from patients with type I, a variant of type I, and type Ila vWd and found normal binding with the type I plasma, but reduced binding with the type I variant plasma (14.5%) and the type Ila plasma (7.1%). AVW3, a monoclonal antibody to vWf that blocks vWf binding to platelet GPIb, blocked vWf binding to endothelial cells. Endothelial cells, like platelets, have the ability to bind plasma von Willebrand factor in the presence of ristocetin. This phenomenon occurs on the surface of endothelial cells in culture. Qualitative and quantitative reductions of this vWf binding are found with the plasma of patients with von Willebrand’s disease.

The effect of cultured endothelial cells on factor VIII procoagulant activity

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 560-572
Author(s):  
NW Stead ◽  
PA McKee
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Protease Activity ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Cultured Endothelial Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Cultured human umbilical vein endothelial cells produce a protein that has von Willebrand factor activity and forms immunoprecipitates with rabbit antibody to purified plasma factor VIII/von Willebrand factor (FVIII/vWF) protein, but it has no FVIII procoagulant activity. Of the three characteristics of plasma FVIII/vWF protein, only FVIII procoagulant activity is readily destroyed by trace proteases. A previous report from this laboratory demonstrated protease activity in culture medium under conditions that had been used by others to show that endothelial cells do not synthesize protein with FVIII procoagulant activity. However, even if cultured endothelial cells are placed in protease-free culture medium, no FVIII procoagulant activity can be detected, despite an increase in the level of protein with vWF activity from 0 to 0.57 microgram/ml by 48 hr. This observation and the lack of protease activity in medium left in contact with the cells for 48 hr led to the hypothesis that proteases exist on the surface of cultured umbilical vein endothelial cells. Protease activity was quantitated by the hydrolysis of p-nitroaniline from the substrate, N- benzoyl-phenylalanyl-valyl-arginyl-p-nitroanilide and by degradation of the procoagulant activity of added purified plasma FVIII/vWF protein. In the absence of endothelial cells, no protease activity was present in protease-free culture medium whether or not it had previously overlaid cultured cells. This medium did not cause cleavage of p- nitroaniline from the tripeptide substrate, and 83% of added FVIII procoagulant activity remained after 48 hr. When the synthetic tripeptide was incubated in contact with cultured endothelial cells, 7.3 +/- 0.8 X 10(-10) moles of p-nitroaniline/hr was released; moreover, only 47% of the added FVIII procoagulant activity remained after 48 hr. Given this rate of destruction, it can be calculated that sufficient protease activity exists on the surface of cultured endothelial cells to degrade the procoagulant activity of approximately 1.6 microgram FVIII/vWF protein/hr. This degradation rate is 45 times the rate of release of FVIII/vWF protein from cultured endothelial cells when assessed by the generation of protein with vWF activity. Hence, the detection of FVIII procoagulant activity, if in fact synthesized by cultured endothelial cells, will be most difficult.

scholarly journals Relative Importance of the Glycoprotein Ib-Binding Domain and the RGD Sequence of von Willebrand Factor for Its Interaction With Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2335-2344 ◽  
Author(s):  
Christelle Perrault ◽  
Hanneke Lankhof ◽  
Dominique Pidard ◽  
Danièle Kerbiriou-Nabias ◽  
Jan J. Sixma ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Binding Domain ◽  
Rgd Sequence ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Endothelial Cell Adhesion

AbstractEndothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.

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