scholarly journals PADGEM protein in human erythroleukemia cells

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 722-728 ◽  
Author(s):  
E Yeo ◽  
BC Furie ◽  
B Furie
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Protein A ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Cell Content ◽  
Granule Membrane ◽  
Hel Cells

Abstract PADGEM protein, a platelet alpha granule membrane glycoprotein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion and platelet activation. PADGEM protein is expressed on the surface of activated platelets but not on the surface of resting platelets. Human erythroleukemia (HEL) cells contain platelet alpha granule-like organelles, alpha granule proteins, and express platelet membrane glycoproteins GPIIb/IIIa and GPIb. We demonstrate that HEL cells express a protein that has a molecular weight identical to that of PADGEM and binds to anti-PADGEM antibodies. The exposure of HEL cells in culture to dimethylsulfoxide (DMSO) increased the number of cells expressing PADGEM. Fluorescence activated flow cytometric analysis demonstrated an increase in mean surface expression of PADGEM in DMSO-exposed cells compared to noninduced cells. Total cell content of PADGEM was increased 5.3-fold after DMSO exposure, as determined by radioimmunoassay. Direct binding experiments with the monoclonal anti-PADGEM antibody KC4 demonstrated specific, saturable, and time-dependent interaction of KC4 with HEL cells. A Kd of 7 nM was estimated. There were 14,000 surface binding sites per cell in noninduced cells and 24,000 surface binding sites per cell in DMSO- induced HEL cells. Surface expression of PADGEM protein on HEL cells was not increased with platelet agonists, including thrombin, epinephrine, ADP, nor cytokines, including IL-1, IL-2, tissue necrosis factor. The presence of PADGEM protein in HEL cells should facilitate the elucidation of the function of PADGEM protein.

scholarly journals PADGEM protein in human erythroleukemia cells

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 722-728
Author(s):  
E Yeo ◽  
BC Furie ◽  
B Furie
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Protein A ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Cell Content ◽  
Granule Membrane ◽  
Hel Cells

PADGEM protein, a platelet alpha granule membrane glycoprotein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion and platelet activation. PADGEM protein is expressed on the surface of activated platelets but not on the surface of resting platelets. Human erythroleukemia (HEL) cells contain platelet alpha granule-like organelles, alpha granule proteins, and express platelet membrane glycoproteins GPIIb/IIIa and GPIb. We demonstrate that HEL cells express a protein that has a molecular weight identical to that of PADGEM and binds to anti-PADGEM antibodies. The exposure of HEL cells in culture to dimethylsulfoxide (DMSO) increased the number of cells expressing PADGEM. Fluorescence activated flow cytometric analysis demonstrated an increase in mean surface expression of PADGEM in DMSO-exposed cells compared to noninduced cells. Total cell content of PADGEM was increased 5.3-fold after DMSO exposure, as determined by radioimmunoassay. Direct binding experiments with the monoclonal anti-PADGEM antibody KC4 demonstrated specific, saturable, and time-dependent interaction of KC4 with HEL cells. A Kd of 7 nM was estimated. There were 14,000 surface binding sites per cell in noninduced cells and 24,000 surface binding sites per cell in DMSO- induced HEL cells. Surface expression of PADGEM protein on HEL cells was not increased with platelet agonists, including thrombin, epinephrine, ADP, nor cytokines, including IL-1, IL-2, tissue necrosis factor. The presence of PADGEM protein in HEL cells should facilitate the elucidation of the function of PADGEM protein.

PADGEM PROTEIN EXPRESSION IN HUMAN ERYTHROLEUKEMIA (HEL) CELLS

1987 ◽  
Author(s):  
E Yeo ◽  
B C Furie ◽  
B Furie
Keyword(s):  
Molecular Weight ◽  
Fold Increase ◽  
Integral Membrane Protein ◽  
Surface Expression ◽  
Surface Binding ◽  
Platelet Factor ◽  
Activated Platelets ◽  
Hel Cells ◽  
Direct Binding ◽  
Granule Secretion

PADGEM (Platelet Activation-Dependent Granule ⇒ External Membrane) glycoprotein, a platelet alpha granule integral membrane protein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion. PADGEM protein is expressed solely on activated platelets, but is not on the surface of resting platelets. Because HEL cells contain platelet alpha granule-like organelles and proteins (e.g. platelet factor 4, von Villebrands factor, β-thromboglobulin) and express certain platelet membrane proteins (e.g. GP IIb/IIIa, GPIb), we evaluated induced and uninduced HEL cells for the synthesis and expression of PADGEM protein. HEL cells were induced with 1.25% DMSO for 3-4 days, then grown in the absence of DMSO for 1-3 weeks. After eight cycles of DMSO exposure, the induced HEL cells were found to increase the expression of PADGEM, in contrast to the uninduced cells. Intact fixed and unfixed induced HEL cells were observed by immunofluorescence, utilizing KC4, a monoclonal anti-PADGEM antibody, to express PADGEM while non-induced HFT. cells expressed low levels of PADGEM. Both induced and uninduced HEL cells bound A2A9, an anti—GP Ilb/IIIa monoclonal antibody. Quantitative analysis by fluorescence activated cell sorting demonstrated a 2.5—fold increase in mean surface expression of PADGEM and 3.3—fold mean increase in GP IIb/IIIa surface expression compared to uninduced cells. By fluoresence microscopy, 70% of induced HEL cells expressed PADGEM protein versus 20% of the uninduced cells. GP-IIb/IIIa expression inoreased from 40% in noninduced cells to 90% in induced cells. The induced HEL cells contained PADGEM with a molecular weight identical to that of platelets, as demonstrated by Western blotting using the KC4 antibody. Direct binding experiments with 125I-KC4 antibody demonstrated that surface binding was specific, saturable, and time-dependent. Surface expression of PADGEM protein was not increased with platelet agonists (thrombin, epinephrine, ADP) nor cytokines (IL-1, IL-2, tissue necrosis factor). The surface density of PADGEM protein on induced HEL cells and activated platelets appears similar. HEL cells should provide a useful model to assist in the elucidation of the structure, function and biology of PADGEM protein.

EXPRESSION OF PLATELET ALLOANTIGENS ON HUMAN ENDOTHELIAL CELLS AND HEL CELLS

1987 ◽  
Author(s):  
Y Kawai ◽  
R R Montgomery ◽  
K Furihata ◽  
T J Kunicki
Keyword(s):  
Endothelial Cells ◽  
Protein A ◽  
Cell Types ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoblot Assay ◽  
Sds Page ◽  
Hel Cells ◽  
Erythroleukemia Cell ◽  
Different Cell Types

Analogs of platelet membrane glycoproteins IIb and IIIa (GPIIb-IIIa) have been shown to be synthesized and expressed by human endothelial cells (HEC), a human erythroleukemia cell line (HEL) and various other cells. Previous studies from our laboratory demonstrated that the platelet alloantigen P1A1, is expressed on HEC GPIIIa. Other alloantigen systems, namely, Pen and Bak, are known to be localized on platelet GPIIIa and GPIIb, respectively. Utilizing additional antibodies from patients with PTP specific for Pena, Baka, and Bakb allo-antigens, and isoantibodies (iso-ab) from a patient with Glanzmann's Thrombasthenia (GT), we have studied cultured HEC and HEL cells for expression of epitopes recognized by these antibodies. HEC and HEL cells were meta-bolically labeled with 35S-methionine and lysed in 0.5% TX-100 in the presence of 5mM EDTA. Soluble antigens were immunoprecipitated with these antibodies coupled to Protein A-Sepharose and subjected to SDS-PAGE and fluorography. Anti-Pena and the GT iso-ab reacted with the GPIIb-IIIa complex from both HEC and HEL lysates, but anti-Baka and anti-Bakb failed to immunoprecipitate GPIIb-IIIa analogs from either HEC or HEL. In an immunoblot assay, the GT iso-ab bound to GPIIIa of both HEC and HEL. Anti-Pena failed to react with SDS-denatured proteins. HEL GPIIIa migrates slightly faster than HEC GPIIIa and slightly slower than platelet GPIIIa. These results indicate that the epitopes of platelet GPIIIa recognized by alloantibodies and isoantibodies are shared by GPIIIa analogs of HEC and HEL. GPIIb-associated alloantigens are not expressed by HEC and HEL GPIIb analogs, an observation that is consistent with the decreased structural homology between GPIIb analogs derived from different cell types.

scholarly journals Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 372-379
Author(s):  
MJ Metzelaar ◽  
HF Heijnen ◽  
JJ Sixma ◽  
HK Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Major Protein ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Number Of Binding Sites ◽  
Thrombin Activation ◽  
A Minor

To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

Biochemical Characterization of PECAM-1 (CD31 Antigen) on Human Platelets

1991 ◽  
Vol 66 (06) ◽  
pp. 700-707 ◽  
Author(s):  
Marcel J Metzelaar ◽  
Jeanne Korteweg ◽  
Jan J Sixma ◽  
H Karel Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Biochemical Characterization ◽  
Cell Interaction ◽  
Human Platelets ◽  
Cell Contact ◽  
Membrane Glycoproteins ◽  
Functional Studies ◽  
Granule Membrane ◽  
Cell Cell

SummaryThe platelet plasma membrane expresses several membrane glycoproteins with a high molecular weight. In this study we have investigated the properties of the CD31 antigen on platelets and endothelial cells using the monoclonal antibody (MoAb) RUU-PL 7E8. Comparative studies revealed that the CD31 antigen, PECAM-1 and endoCAM are the same protein. The CD31 antigen was immunoprecipitated with a molecular mass of 125 kDa nonreduced and 135 kDa reduced from Nonidet-P40 lysates of surface labeled human platelets. The relative position in two-dimensional nonreduced/reduced SDS-PAGE and IEF-PAGE, compared to other glycoproteins of similar molecular weight, was elucidated. The position of the CD31 antigen was clearly distinct from the position of the platelet membrane glycoproteins Ia, Ib, IIa, IIb, IIIa and the granule membrane protein GMP-140. Native resting platelets bound 7,760 ± 1,670 molecules/platelet, whereas thrombin-stimulated platelets bound 14,500 ± 3,790 molecules/platelet. Immunoelectron microscopy revealed the presence of the CD31 antigen on the membrane of both resting and thrombin-activated platelets. Immunofluorescence studies showed the presence of the CD31 antigen in the membrane of endothelial cells on sites of cell-cell contact, suggesting that the CD31 antigen might be involved in cell-cell interaction. In functional studies, MoAb RUU-PL 7E8 did not inhibit platelet aggregation, platelet adherence to the extracellular matrix of endothelial cells and purified collagen fibrils under flow conditions, nor was any influence found on endothelial cell detachment and growth.

scholarly journals Functional modification of the Chlamydomonas flagellar surface.

1982 ◽  
Vol 93 (1) ◽  
pp. 88-96 ◽  
Author(s):  
R A Bloodgood ◽  
G S May
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Antibody Preparation ◽  
Substrate Interaction ◽  
Rabbit Igg ◽  
Flagellar Membrane ◽  
Flagellar Proteins ◽  
Force Transduction

Chlamydomonas flagella exhibit force transduction in association with their surface. This flagellar surface motility is probably used both for whole cell gliding movements (flagella-substrate interaction) and for reorientation of flagella during mating (flagella-flagella interaction). The present study seeks to identify flagellar proteins that may function as exposed adhesive sites coupled to a motor responsible for their translocation in the plane of the plasma membrane. The principal components of the flagellar membrane are a pair of glycoproteins (approximately 350,000 mol wt), with similar mobility on SDS polyacrylamide gels. A rabbit IgG preparation has been obtained which is specific for these two glycoproteins; this antibody preparation binds to and agglutinates cells by their flagellar surfaces only. Treatment of cells with 0.1 mg/ml pronase results in a loss of motility-coupled flagellar membrane adhesiveness. This effect is totally reversible, but only in the presence of new protein synthesis. The major flagellar protein modified by this pronase treatment is the faster migrating of the two high molecular weight glycoproteins; the other glycoprotein does not appear to be accessible to external proteolytic digestion. Loss and recovery of flagella surface binding sites for the specific antibody parallels the loss and recovery of the motility-coupled flagellar surface adhesiveness, as measured by the binding and translocation of polystyrene microspheres. These observations suggest, but do not prove, that the faster migrating of the major high molecular weight flagellar membrane glycoproteins may be the component which provides sites for substrate interaction and couples these sites to the cytoskeletal components responsible for force transduction.

CHARACTERIZATION OF HUMAN PLATELET ClQ BINDING SITES

1987 ◽  
Author(s):  
E IB PEERSCHKE ◽  
B Ghebrehiwet
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Phosphate Buffer ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Inhibitory Effect ◽  
Polyacrylamide Gel ◽  
Triton X

We have recently shown that platelets possess specific binding sites for Clq, a subcomponent of the first component of complement, Cl, and that occupancy of these receptor sites correlates with the previously described inhibitory effect of Clq on collagen-induced platelet aggregation. To further characterize platelet Clq receptors, washed platelets were solubilized in 5 mM sodium phosphate buffer, pH 7.5 containing 10 mM EDTA, 150 mM NaCl, 10 mM EACA, 0.5 mM PMSF, and 1% Triton X-100. After dialysis against 5 mM phosphate buffer pH 7.5 containing 10 mM EDTA, 20 mM NaCl, 10 mM EACA,0.5 mM PMSF and 0.1% Triton X-100, the lysate was passed over Clq-Sepharose-4B affinity columns. A single protein peak eluted with buffer containing 300 mM NaCl. This peak was composed of two predominant molecular weight species (85-95K, 60-66K) as assessed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. When 125-iodine surface labeled platelets were solubilized and applied to Clq-Sepharose affinity resins, the same two molecular weight species eluted and could be visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. Immunoabsorption studies performed under nondenaturing conditions using protein A and the IIl/Dl monoclonal antibody, which binds specifically to platelets and inhibits platelet-Clq interactions, revealed that the 85-95K molecular weight component was preferentially absorbed, but incomplete immunoabsorption of the 60-66K molecular weight constituent was also noted. Affinity purified Clq binding sites sedimented as a single peak during 5-40% sucrose density ultracentrifugation with an S value of approximately 2.4. In addition, both the 85-95K and the 60-66K molecular weight species coeluted in the void volume of Sephadex G-100 columns. The data suggest that the 85-95K and 60-66K molecular weight species represent platelet membrane Clq binding sites, and that these sites may form weak, noncovalently associated complexes.

scholarly journals Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 372-379 ◽  
Author(s):  
MJ Metzelaar ◽  
HF Heijnen ◽  
JJ Sixma ◽  
HK Nieuwenhuis
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Major Protein ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Microscopic Studies ◽  
Number Of Binding Sites ◽  
Thrombin Activation ◽  
A Minor

Abstract To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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