PADGEM protein in human erythroleukemia 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.
Yeo
E
E
Division of Hematology-Oncology, New England Medical Center, Boston, MA 02111.
Furie
B C
BC
Furie
B
B
D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002462:Cell Membrane / Q000276:immunology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000503:physiopathology*; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000502:physiology*; D011863:Radioimmunoassay; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D004121:Dimethyl Sulfoxide