title: Identification of novel downstream molecules of tissue factor activation by comparative proteomic analysis.
abstract: Tissue factor (TF) is both an initiator of blood coagulation and a signaling receptor. Using a proteomic approach, we investigated the role of TF in cell signaling when stimulated by its ligand, activated factor VII (FVIIa). From a 2-D difference gel electrophoresis (DIGE) study we found forty one spots that were differentially regulated over time in FVIIa stimulated cells or in comparison to nonstimulated cells. Mass spectrometry identifies 23 out of these as 13 different proteins. One of them, elongation factor 2 (EF-2), was investigated in greater detail by Western blot, a protein synthesis assay and cell cycle analysis. When tissue factor was stimulated by FVIIa, the phosphorylation of EF-2 increased which inactivates this protein. Analyzing the effect using site inactivated FVIIa (FVIIai), as well as the protease activated receptor 2 (PAR-2) agonist SLIGKV, indicated that the inactivation was not PAR-2 dependent. A panel of tissue factor mutants was analyzed further to try to pinpoint what part of the cytoplasmic domain that is needed for this effect. Performing a protein synthesis assay in two different cell lines we could confirm that protein synthesis decreased upon stimulation by FVIIa. Cell cycle analysis showed that FVIIa also promotes a higher degree of cell proliferation.
authors.affiliation: Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University , 751 85 Uppsala, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Kask
authors.forename: Anneli
authors.initials: A
authors.lastname: Jorsback
authors.forename: Maria
authors.initials: M
authors.lastname: Winkvist
authors.forename: Jenny
authors.initials: J
authors.lastname: Alfredsson
authors.forename: Bo
authors.initials: B
authors.lastname: Ek
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergquist
authors.forename: Agneta
authors.initials: A
authors.lastname: Siegbahn
mesh_terms: D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D040901:Proteomics; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D013925:Thromboplastin