Factor VIII Related Antigen (FVIIIRAG) In Porcine Endothelial Cells

1981 ◽  
Author(s):  
J C Giddings ◽  
A L Jarvis ◽  
A L Bloom
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Aortic Endothelial Cells ◽  
Factor Viii Related Antigen ◽  
Umbilical Vein Endothelial Cells ◽  
Porcine Endothelial Cells ◽  
Willebrand Factor ◽  
Porcine Aortic Endothelial Cells

Porcine aortic and umbilical vein endothelial cells were maintained in culture and examined for their ability to synthesise or release factor VIII related antigen (Willebrand factor, FVIIIRAG). Two different antisera raised against purified porcine FVIIIRAG were used in conventional immunodiffusion, immunoelectrophoresis and immunofluorescence techniques. Both antisera were adsorbed with porcine von Willebrand's disease (vWd) plasma and gave identical monospecific immunoprecipitin reactions when tested against normal porcine plasma or concentrated porcine FVIIIRAG and failed to show any precipitin reaction against vWd pig plasma. Primary and extended cultures of umbilical vein endothelial cells showed positive intracellular immunofluorescence with either antiserum and supernatant culture medium from these cells contained FVIIIRAG when tested in immunoprecipitin tests. However, cultures derived from aorta demonstrated a different pattern of results. Antiserum one showed homogenous intracellular immunofluorescence but antiserum two gave an immunofluorescence pattern which was extracellular and fibrillar, similar to that seen with human endothelial cells stained with anti-fibronectin. Culture supernatant and lysedaortic endothelial cells did not contain FVIIIRAG in immunoprecipitin tests with either antiserum. The results indicate either that the antisera were not specific and that the aortic cells were not endothelial or that porcine endothelial cells derived from aorta may be different from those obtained from umbilical vein in their ability to synthesise FVIIIRAG. Immunohistological demonstration of “FVIIIRAG” cannot therefore be used as the only criterion for identifying porcine aortic endothelial cells.

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.

Biosynthesis of Factor VIII Related Antigen (VIII. RAG) by Porcine Aortic Endothelial Cells in Culture

1983 ◽  
Vol 64 (2) ◽  
pp. 67P-67P
Author(s):  
J.M. Seid ◽  
R.G. Malia ◽  
B. Sampson ◽  
J. Ball ◽  
F.E. Preston ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Aortic Endothelial Cells ◽  
Factor Viii Related Antigen ◽  
Cells In Culture ◽  
Porcine Aortic Endothelial Cells ◽  
Aortic Endothelial

scholarly journals 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

Abstract 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.

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

2017 ◽  
Vol 44 (5) ◽  
pp. 531-537 ◽  
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

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

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 196 ◽  
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.

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

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.

scholarly journals Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

2003 ◽  
Vol 71 (5) ◽  
pp. 2365-2372 ◽  
Author(s):  
Murray W. Stinson ◽  
Susan Alder ◽  
Sarmishtha Kumar
Keyword(s):  
Endothelial Cells ◽  
Infective Endocarditis ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Bovine Liver ◽  
Surface Proteins ◽  
Oral Streptococci ◽  
Bovine Liver Catalase ◽  
Umbilical Vein Endothelial Cells ◽  
Viridans Group Streptococci

ABSTRACT Colonization of the cardiovascular endothelium by viridans group streptococci can result in infective endocarditis and possibly atherosclerosis; however, the mechanisms of pathogenesis are poorly understood. We investigated the ability of selected oral streptococci to infect monolayers of human umbilical vein endothelial cells (HUVEC) in 50% human plasma and to produce cytotoxicity. Planktonic Streptococcus gordonii CH1 killed HUVEC over a 5-h period by peroxidogenesis (alpha-hemolysin) and by acidogenesis but not by production of protein exotoxins. HUVEC were protected fully by addition of supplemental buffers and bovine liver catalase to the culture medium. Streptococci were also found to invade HUVEC by an endocytic mechanism that was dependent on polymerization of actin microfilaments and on a functional cytoskeleton, as indicated by inhibition with cytochalasin D and nocodazole. Electron microscopy revealed streptococci attached to HUVEC surfaces via numerous fibrillar structures and bacteria in membrane-encased cytoplasmic vacuoles. Following invasion by S. gordonii CH1, HUVEC monolayers showed 63% cell lysis over 4 h, releasing 64% of the total intracellular bacteria into the culture medium; however, the bacteria did not multiply during this time. The ability to invade HUVEC was exhibited by selected strains of S. gordonii, S. sanguis, S. mutans, S. mitis, and S. oralis but only weakly by S. salivarius. Comparison of isogenic pairs of S. gordonii revealed a requirement for several surface proteins for maximum host cell invasion: glucosyltransferase, the sialic acid-binding protein Hsa, and the hydrophobicity/coaggregation proteins CshA and CshB. Deletion of genes for the antigen I/II adhesins, SspA and SspB, did not affect invasion. We hypothesize that peroxidogenesis and invasion of the cardiovascular endothelium by viridans group streptococci are integral events in the pathogenesis of infective endocarditis and atherosclerosis.

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