The platelet glycoprotein IIb/IIIa-like protein in human endothelial cells promotes adhesion but not initial attachment to extracellular matrix.

On platelets the membrane glycoprotein IIb/IIIa complex (GPIIb/IIIa) functions in adhesive interactions with fibrinogen, von Willebrand factor, and fibronectin. However, the function of GPIIb/IIIa-like proteins on endothelial cells, as well as the ligand(s) the complex binds, is unknown. Using a highly specific polyclonal antibody we have explored the function of GPIIb/IIIa-like proteins on human umbilical vein endothelial cells (HUVE). Analysis by immunoblotting shows that this antiserum recognizes the endothelial GPIIIa-like protein of the complex. The IgG fraction of the polyclonal antiserum and its Fab' fragments detach confluent and subconfluent HUVE from extracellular substrata. The effect of the anti-GPIIb/IIIa IgG is not toxic as the detached cells maintain their viability after trypsinization and replating. Anti-GPIIb/IIIa IgG does not inhibit HUVE binding to extracellular matrix or purified fibronectin in an attachment assay despite the presence of intact GPIIb/IIIa on HUVE detached from substrate by various methods. Apparently, the GPIIb/IIIa-like protein on HUVE is important in normal HUVE adhesion to the extracellular matrix, but it is not required in the initial attachment of HUVE to extracellular matrix.

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PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells

Blood ◽

10.1182/blood.v73.5.1109.1109 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1109-1112 ◽

Cited By ~ 346

Author(s):

R Bonfanti ◽

BC Furie ◽

B Furie ◽

DD Wagner

Keyword(s):

Monoclonal Antibody ◽

Endothelial Cells ◽

Umbilical Vein ◽

Polyclonal Antiserum ◽

Human Endothelial Cells ◽

Granule Membrane ◽

Double Label ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor

Abstract PADGEM protein (PADGEM), also known as GMP140, is a platelet alpha- granule membrane protein that is translocated to the external membrane after platelet activation. Although the biosynthesis of this protein was originally thought to be confined to megakaryocytes, the synthesis of PADGEM in endothelial cells was recently demonstrated (McEver et al: Blood 70:1974a, 1987). We now describe the subcellular localization of this protein in endothelial cells. Immunofluorescence staining of permeabilized human umbilical vein endothelial cells with KC4, a well characterized monoclonal antibody to PADGEM, showed positively stained elongated structures similar in distribution and shape to Weibel-Palade bodies. Their identity as Weibel-Palade bodies was confirmed by double label immunofluorescence using KC4 and a polyclonal antiserum to von Willebrand factor (vWf), a protein known to be specifically stored in these organelles. All Weibel-Palade bodies were found to contain PADGEM. In contrast to strong perinuclear staining produced with anti- vWf antibodies, no significant perinuclear staining was obtained with KC4, indicating that relatively little PADGEM is present in the endoplasmic reticulum and in the Golgi apparatus. In endothelial cells treated with secretagogues that stimulate vWf release the elongated structures positive for PADGEM disappeared, further identifying these structures as Weibel-Palade bodies. This observation extends the parallels between Weibel-Palade bodies and alpha-granules and suggests a possible functional association between vWf and PADGEM.

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PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells

Blood ◽

10.1182/blood.v73.5.1109.bloodjournal7351109 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1109-1112 ◽

Cited By ~ 3

Author(s):

R Bonfanti ◽

BC Furie ◽

B Furie ◽

DD Wagner

Keyword(s):

Monoclonal Antibody ◽

Endothelial Cells ◽

Umbilical Vein ◽

Polyclonal Antiserum ◽

Human Endothelial Cells ◽

Granule Membrane ◽

Double Label ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor

PADGEM protein (PADGEM), also known as GMP140, is a platelet alpha- granule membrane protein that is translocated to the external membrane after platelet activation. Although the biosynthesis of this protein was originally thought to be confined to megakaryocytes, the synthesis of PADGEM in endothelial cells was recently demonstrated (McEver et al: Blood 70:1974a, 1987). We now describe the subcellular localization of this protein in endothelial cells. Immunofluorescence staining of permeabilized human umbilical vein endothelial cells with KC4, a well characterized monoclonal antibody to PADGEM, showed positively stained elongated structures similar in distribution and shape to Weibel-Palade bodies. Their identity as Weibel-Palade bodies was confirmed by double label immunofluorescence using KC4 and a polyclonal antiserum to von Willebrand factor (vWf), a protein known to be specifically stored in these organelles. All Weibel-Palade bodies were found to contain PADGEM. In contrast to strong perinuclear staining produced with anti- vWf antibodies, no significant perinuclear staining was obtained with KC4, indicating that relatively little PADGEM is present in the endoplasmic reticulum and in the Golgi apparatus. In endothelial cells treated with secretagogues that stimulate vWf release the elongated structures positive for PADGEM disappeared, further identifying these structures as Weibel-Palade bodies. This observation extends the parallels between Weibel-Palade bodies and alpha-granules and suggests a possible functional association between vWf and PADGEM.

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Hydrogen peroxide stimulates exocytosis of von Willebrand factor in human umbilical vein endothelial cells

Biology Bulletin ◽

10.1134/s106235901705003x ◽

2017 ◽

Vol 44 (5) ◽

pp. 531-537 ◽

Cited By ~ 2

Author(s):

P. V. Avdonin ◽

A. A. Tsitrina ◽

G. Y. Mironova ◽

P. P. Avdonin ◽

I. L. Zharkikh ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Endothelial Cells ◽

Von Willebrand Factor ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor

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Human Endothelial Cells in Culture and In Vivo Express on Their Surface All Four Components of the Glycoprotein Ib/IX/V Complex

Blood ◽

10.1182/blood.v90.7.2660.2660_2660_2669 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2660-2669 ◽

Cited By ~ 5

Author(s):

Guoxin Wu ◽

David W. Essex ◽

Frank J. Meloni ◽

Toshiro Takafuta ◽

Kingo Fujimura ◽

...

Keyword(s):

Molecular Weight ◽

Endothelial Cells ◽

Umbilical Vein ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Molecular Weights ◽

Hel Cells ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand

The platelet glycoprotein Ib (GpIb) complex is composed of four polypeptides: the disulfide-linked GpIbα and GpIbβ and the noncovalently associated GpIX and GpV. GpIbα contains binding sites for von Willebrand factor and for thrombin and mediates platelet adhesion to the subendothelium under conditions of high shear stress. We have previously shown the presence of GpIbα and GpIbβ mRNA and protein in cultured human umbilical vein endothelial cells (HUVECs) as well as the presence of GpIbα mRNA and protein in tonsillar endothelium. We, therefore, probed ECs for the presence of the other components of the GpIb/IX/V complex. We have identified the presence of GpIX and GpV mRNA in cultured HUVEC monolayers. The sequence of HUVEC GpIX cDNA was identical to the previously published human erythroleukemia (HEL) cell GpIX cDNA sequence. Two species of GpV mRNA, one of 3 kb and one of 4.4 kb, were found in HUVECs, whereas HEL cells displayed only the 4.4-kb species and the megakaryocytic cell line CHRF-288 contained only the 3-kb species. We previously showed that EC GpIbα protein is identical in molecular weight to platelet GpIbα. HUVEC GpIbβ, in contrast to its platelet counterpart, has a molecular weight of 50 kD and forms a correspondingly larger disulfide-bonded complex with EC GpIbα. The molecular weights of GpIX and GpV were 22 and 88 kD, respectively, identical to the corresponding platelet polypeptides. Furthermore, we have identified all four components of the complex in tonsillar vessels. Using flow cytometry, we have established that all four polypeptides of the GpIb/IX/V complex are expressed on the surface membranes of cultured HUVECs and adult aortic ECs. Furthermore, using two-color fluorescence, we have shown that all ECs expressing GpIbα also express GpIX and GpV on their surface. The ratio of GpIbα:GpIX:GpV is 1:1:0.5, which is identical to the ratio present in platelets. None of the polypeptides of the GpIb complex could be identified on the surface of human smooth muscle cells or lymphocytes. The presence of all members of the GpIb complex in the EC membrane suggests that this complex may play a role in endothelial function in vivo.

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VAS2870 Inhibits Histamine-Induced Calcium Signaling and vWF Secretion in Human Umbilical Vein Endothelial Cells

Cells ◽

10.3390/cells8020196 ◽

2019 ◽

Vol 8 (2) ◽

pp. 196 ◽

Cited By ~ 5

Author(s):

Pavel Avdonin ◽

Elena Rybakova ◽

Piotr Avdonin ◽

Sergei Trufanov ◽

Galina Mironova ◽

...

Keyword(s):

Endothelial Cells ◽

Calcium Concentration ◽

Umbilical Vein ◽

Rat Aorta ◽

Human Umbilical Vein ◽

Nox Inhibitors ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor

In this study, we investigated the effects of NAD(P)H oxidase (NOX) inhibitor VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine) on the histamine-induced elevation of free cytoplasmic calcium concentration ([Ca2+]i) and the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) and on relaxation of rat aorta in response to histamine. At 10 μM concentration, VAS2870 suppressed the [Ca2+]i rise induced by histamine. Inhibition was not competitive, with IC50 3.64 and 3.22 μM at 1 and 100 μM concentrations of histamine, respectively. There was no inhibition of [Ca2+]i elevation by VAS2870 in HUVECs in response to the agonist of type 1 protease-activated receptor SFLLRN. VAS2870 attenuated histamine-induced secretion of vWF and did not inhibit basal secretion. VAS2870 did not change the degree of histamine-induced relaxation of rat aortic rings constricted by norepinephrine. We suggest that NOX inhibitors might be used as a tool for preventing thrombosis induced by histamine release from mast cells without affecting vasorelaxation.

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THE EFFECT OF PHORBOL DIESTERS AND INTERLEUKINS ON THE SECRETION OF VON WILLEBRAND FACTOR BY HUMAN ENDOTHELIAL CELLS IN CULTURE

10.1055/s-0038-1642910 ◽

1987 ◽

Author(s):

J C Giddings ◽

L Shall

Keyword(s):

Endothelial Cells ◽

Von Willebrand Factor ◽

Umbilical Vein ◽

Interleukin 1 ◽

Interleukin 2 ◽

Morphological Evidence ◽

Adhesive Properties ◽

Umbilical Vein Endothelial Cells ◽

Von Willebrand ◽

Willebrand Factor

Human umbilical vein endothelial cells (EC) were cultured in the presence of 4p-phorbol 12-myristate 13-acetate (PMA, 10ug/l), interleukin 1 (IL-1, 1 unit/ml) and interleukin 2 (IL-2, 1 unit/ml), and secretion of von Willebrand factor activity (vWF, Ristocetin co-factor) and von Willebrand factor antigen (vWFAG, ELISA Technique) measured at intervals. Confluent control EC were treated with PMA, IL-1 and IL-2, and the supernatant medium assayed for release of vWF and vWFAg. Treated cells were also examined for vWFAg by immuno-fluorescence. The levels of both vWF and vWFAg in cultures containing IL-1 were significantly higher than those in control cultures after 5-6 days growth. Moreover, vWF and vWFAg increased significantly in the supernatant of confluent control EC incubated further in the presence of IL-1. Furthermore, the characteristic fluorescence pattern of endothelial vWFAg was markedly reduced in EC treated with IL-1. The levels of vWF and vWFAg in cultures containing PMA were also significantly higher than those of control cultures. In these conditions, however, the growth of cells appeared to be enhanced, and confluence was observed after about 6 days in the presence of PMA compared to 9 - 10 days in control cultures. The mean levels of vWF and vWFAg in the supernatant of EC incubated with PMA were higher than the control values but the differences were not statistically significant. Immunofluorescence of PMA-treated cells suggested that vWFAg might be less granular than in control cells but the differences were not as marked as those seen with IL-1. The results of all assays in the presence of IL-2 were not significantly different from those of control cells. In all instances no morphological evidence of endothelial injury was observed and more than 90% of cells remained viable at the termination of cultures. The results indicated that the synthesis and release of vWF were increased in the presence of PMA, and secretion of vWF was stimulated by IL-1. The data suggest that secreted vWF might contribute to the previously reported enhanced procoagulant and adhesive properties of EC treated with these substances.

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