scholarly journals Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 234-237 ◽  
Author(s):  
JD Sprandio ◽  
SS Shapiro ◽  
P Thiagarajan ◽  
S McCord
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Single Class ◽  
Human Umbilical Vein ◽  
Sds Page ◽  
Umbilical Vein Endothelial Cells ◽  
Triton X

Abstract Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.

scholarly journals Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 234-237 ◽  
Author(s):  
JD Sprandio ◽  
SS Shapiro ◽  
P Thiagarajan ◽  
S McCord
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Single Class ◽  
Human Umbilical Vein ◽  
Sds Page ◽  
Umbilical Vein Endothelial Cells ◽  
Triton X

Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.

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

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2660-2669 ◽  
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.

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

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2660-2669 ◽  
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

AbstractThe 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.

Endothelial cells from human umbilical vein secrete functional transcobalamin II

1989 ◽  
Vol 256 (2) ◽  
pp. C296-C303 ◽  
Author(s):  
E. V. Quadros ◽  
S. P. Rothenberg ◽  
E. A. Jaffe
Keyword(s):  
Endothelial Cells ◽  
Mammalian Cells ◽  
Umbilical Vein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Transcobalamin Ii

Transcobalamin II (TCII) is a cobalamin (Cbl) binding protein in the plasma that mediates the cellular uptake of Cbl. Although the synthesis of TCII by a variety of cultured mammalian cells and by some isolated perfused organs has been reported, no single tissue has been identified as the source of TCII in vivo. In this study, we demonstrate that cultured human umbilical vein endothelial cells secrete a protein that binds CN[57Co]Cbl, elutes from a Sephacryl S-200 column in the same position as TCII, and precipitates with an antiserum to purified human TCII. The biosynthesis of TCII by these cells was confirmed by demonstrating the incorporation of [35S]methionine into a nascent protein that immunoprecipitated with anti-TCII and which, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) had an Mr of 43,000, the same as human TCII. This secreted protein also had the functional properties of TCII because it facilitated the uptake of CN[57Co]Cbl by the same endothelial cells that secreted it as well as other cell lines that express the membrane receptor for TCII. We also present evidence that the venous endothelium could be the source of TCII in vivo by showing that an intact umbilical vein in an isolated umbilical cord, when perfused with medium containing [35S]methionine, secretes a radiolabeled nascent protein with the same immunoreactive and electrophoretic properties as human TCII. These studies demonstrate that the endothelial cell, which has been shown to secrete a number of plasma proteins, also synthesizes and secretes TCII both in vitro and as an intact endothelium in situ, and therefore, could be the source of circulating TCII in vivo.

Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

1989 ◽  
Vol 61 (01) ◽  
pp. 101-105 ◽  
Author(s):  
Bonnie J Warn-Cramer ◽  
Fanny E Almus ◽  
Samuel I Rapaport
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Phorbol Ester ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Factor Xa ◽  
Extrinsic Pathway ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

Partial Characterization of a Fibrinolytic Inhibitor Produced by Cultured Endothelial Cells Derived from Human Umbilical Vein

1982 ◽  
Vol 47 (02) ◽  
pp. 128-131 ◽  
Author(s):  
F Esnard ◽  
E Dupuy ◽  
A M Dosne ◽  
E Bodevin
Keyword(s):  
Endothelial Cells ◽  
Primary Culture ◽  
Ammonium Sulphate ◽  
Concanavalin A ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Preliminary Characterization ◽  
Umbilical Vein Endothelial Cells ◽  
Ammonium Sulphate Saturation

SummaryA preliminary characterization of a fibrinolytic inhibitor released by human umbilical vein endothelial cells in primary culture is reported. This molecule of Mr comprised between 2 × 105 and 106 and of μ2 mobility precipitates at 43% ammonium sulphate saturation and is totally adsorbed on Concanavalin A Sepharose 4 B. A possible relationship with a macroglobulins is discussed.

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