Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells

1990 ◽  
Vol 63 (02) ◽  
pp. 303-311
Author(s):  
Tone Børsum
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Rabbit Antiserum ◽  
Platelet Glycoprotein ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoelectrophoretic Analysis ◽  
Glycoprotein Complex ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryHuman endothelial cells isolated from umbilical cordswere solubilized in Triton X-100 and examined by crossedimmunoelec-trophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelialcell proteins with 14Cmannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques.Two monoclonal antibodies against the platelet glycoprotein complex Ilb-IIIa and glycoprotein IlIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex Ilb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.

scholarly journals Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1176-1180 ◽  
Author(s):  
OC Leeksma ◽  
J Zandbergen-Spaargaren ◽  
JC Giltay ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Protein Complex ◽  
Platelet Glycoprotein ◽  
Plasma Membrane Protein ◽  
Human Endothelial Cells ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis

Abstract We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

Electroimmunochemical Characterization of Endothelial Cell Proteins: Antigenic Relationship with Platelet and Erythrocyte Membrane Proteins

1987 ◽  
Vol 58 (02) ◽  
pp. 686-693 ◽  
Author(s):  
Tone Børsum ◽  
Inger Hagen ◽  
Ole J Bjerrum
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Primary Cultures ◽  
Rabbit Antiserum ◽  
Human Platelets ◽  
Erythrocyte Membranes ◽  
Antigenic Relationship ◽  
Platelet Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryHuman endothelial cells isolated from umbilical cords and cultured in primary cultures were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogeneous labelling of the endothelial cell proteins with 35S-methionine or 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 30 or 8 immunoprecipitates, respectively. Antigenic relationship between endothelial cell proteins and proteins in human platelets or erythrocyte membranes was demonstrated by use of the corresponding antisera and by antigen addition experiments. One of the endothelial cell proteins cross-reacted with antiserum against erythrocyte membranes and showed a partial antigenic identity reaction with the band 3 protein complex of erythrocyte membranes. The same protein showed antigenic relationship also with a platelet protein. In addition, endothelial cells contain at least 7 proteins antigenically related to platelet proteins, of which at least 5 were labelled with 14C-mannose and thus were glycoproteins. Three of these glycoproteins were antigenically related to proteins from isolated platelet membranes and three were related to the release products obtained after thrombin treatment of platelets. The present study demonstrated numerous platelet and endothelial cell proteins that were antigenically related, more than previously anticipated.

scholarly journals Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1176-1180
Author(s):  
OC Leeksma ◽  
J Zandbergen-Spaargaren ◽  
JC Giltay ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Protein Complex ◽  
Platelet Glycoprotein ◽  
Plasma Membrane Protein ◽  
Human Endothelial Cells ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

scholarly journals Synthesis and secretion of thrombospondin by cultured human endothelial cells.

1982 ◽  
Vol 93 (2) ◽  
pp. 343-348 ◽  
Author(s):  
D F Mosher ◽  
M J Doyle ◽  
E A Jaffe
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Human Platelet ◽  
Umbilical Vein ◽  
Rabbit Antiserum ◽  
Two Dimensional ◽  
Human Endothelial Cells ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine-labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin.

Antithrombin III, the Major Modulator of Intravascular Coagulation, Is Synthesized by Human Endothelial Cells

1981 ◽  
Vol 46 (02) ◽  
pp. 504-506 ◽  
Author(s):  
T K Chan ◽  
Vivian Chan
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vascular Endothelium ◽  
Antithrombin Iii ◽  
Heparin Binding ◽  
Human Endothelial Cells ◽  
Intravascular Coagulation ◽  
Cells In Culture ◽  
Crossed Immunoelectrophoresis ◽  
At Iii

SummaryHuman endothelial cells in culture is shown to synthesize antithrombin III (At-III). The endothelial cell At-III(EC-At-III) consists of a small fraction similar to plasma At-III and a larger fraction with decreased heparin-binding as tested by crossed immunoelectrophoresis. However, both the anti-Xa and thrombin-neutralizing activities of the EC-At-III were rapid and active even in the absence of added heparin. It is concluded that the major portion was probably bound to endogenous heparin-like substance, thus accounting for its decreased exogenous heparin binding. The presence of At-III and other antithrombotic factors in the vascular endothelium offer protection against thrombosis and possibly atherosclerosis.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

scholarly journals Direct Activation of Human Endothelial Cells by Plasmodium falciparum-Infected Erythrocytes

2005 ◽  
Vol 73 (6) ◽  
pp. 3271-3277 ◽  
Author(s):  
Nicola K. Viebig ◽  
Ulrich Wulbrand ◽  
Reinhold Förster ◽  
Katherine T. Andrews ◽  
Michael Lanzer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasmodium Falciparum ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Activation ◽  
Physical Contact ◽  
Intercellular Adhesion Molecule ◽  
Immune Reactivity ◽  
Human Endothelial Cells ◽  
Tnf Α

ABSTRACT Cytoadherence of Plasmodium falciparum-infected erythrocytes (PRBC) to endothelial cells causes severe clinical disease, presumably as a of result perfusion failure and tissue hypoxia. Cytoadherence to endothelial cells is increased by endothelial cell activation, which is believed to occur in a paracrine fashion by mediators such as tumor necrosis factor alpha (TNF-α) released from macrophages that initially recognize PRBC. Here we provide evidence that PRBC directly stimulate human endothelial cells in the absence of macrophages, leading to increased expression of adhesion-promoting molecules, such as intercellular adhesion molecule 1. Endothelial cell stimulation by PRBC required direct physical contact for a short time (30 to 60 min) and was correlated with parasitemia. Gene expression profiling of endothelial cells stimulated by PRBC revealed increased expression levels of chemokine and adhesion molecule genes. PRBC-stimulated endothelial cells especially showed increased expression of molecules involved in parasite adhesion but failed to express molecules promoting leukocyte adhesion, such as E-selectin and vascular cell adhesion molecule 1, even after challenge with TNF-α. Collectively, our data suggest that stimulation of endothelial cells by PRBC may have two effects: prevention of parasite clearance through increased cytoadherence and attenuation of leukocyte binding to endothelial cells, thereby preventing deleterious immune reactivity.

scholarly journals Mapping the endothelial cell S-Sulfhydrome highlights the crucial role of integrin sulfhydration in vascular function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
D R Bibli ◽  
D R Hu ◽  
D R Looso ◽  
D R Weigert ◽  
D R Wittig ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Vascular Disease ◽  
Disulfide Bond ◽  
Mesenteric Arteries ◽  
Human Endothelial Cells ◽  
Β3 Integrin ◽  
Static Conditions ◽  
Protein Disulfide

Abstract Background In vascular endothelial cells, cysteine metabolism by the cystathionine-γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the “S-sulfhydrome” i.e. the spectrum of proteins targeted by H2Sn in human endothelial cells. Methods LC-MS/MS was used to identify S-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intra-protein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements and flow-induced vasodilatation in endothelial cell-specific CSE knock out mice and a small collective of patients with endothelial dysfunction. Results Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low), (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression, and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell “S-sulfhydrome” consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that S-sulfhydration affected intra-protein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between β3 integrin and Gα13, resulting in the constitutive activation of RhoA and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation and a failure to detect β3 integrin S-sulfhydration, all of which were rescued following the administration of an H2S supplement. Conclusions Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

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