Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140.
We have examined the cell activation-dependent redistribution of the intracellular granule membrane protein GMP-140 of human endothelial cells. By dual-label immunofluorescence, the distribution of GMP-140 within cultured human umbilical vein endothelial cells was found to coincide with the distribution of von Willebrand factor (vWF), suggesting that GMP-140 is located in the membranes of vWF-containing storage granules. Stimulation of vWF secretion resulted in an increase in GMP-140 on the cell surface, as detected by increased binding of the monoclonal antibody S12 which recognizes the extracytoplasmic domain of GMP-140. For each agonist tested (histamine, thrombin, phorbol 12-myristate 13-acetate, and the calcium ionophore A23187) a dose-dependent redistribution of GMP-140 to the endothelial surface was observed which closely paralleled the dose-dependent secretion of vWF into the cell supernatant. When cells were maximally stimulated by histamine in the presence of antibody S12, a 4-fold increase in S12 uptake by the cells was observed. This increase occurred rapidly and reached a plateau by 10 min. In contrast, when histamine-stimulated cells were first fixed with paraformaldehyde or chilled to 4 degrees C before addition of antibody S12, only a transient increase in cell surface GMP-140 was detected. Under these conditions of arrested membrane turnover during antibody binding, cell surface GMP-140 was maximal 3 min after histamine stimulation and then declined to control levels by 20 min. These data suggest that stimulated secretion of vWF from endothelial cells entails fusion of vWF-containing storage granules with the plasma membrane. Once inserted into the plasma membrane, GMP-140 is subsequently removed from the endothelial surface, most likely by an endocytic mechanism.
Hattori
R
R
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Hamilton
K K
KK
Fugate
R D
RD
McEver
R P
RP
Sims
P J
PJ
D000911:Antibodies, Monoclonal; D001692:Biological Transport; D000001:Calcimycin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins; D014841:von Willebrand Factor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D000001:Calcimycin; D006632:Histamine; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate