Identification of novel downstream molecules of tissue factor activation by comparative proteomic analysis.
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Kask L, Jorsback A, Winkvist M, Alfredsson J, Ek B, Bergquist J, Siegbahn A
J. Proteome Res. 13 (2) 477-488 [2014-02-07; online 2013-12-11]
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.
Affinity Proteomics Uppsala [Technology development]
PLA and Single Cell Proteomics [Service]
PubMed 24274763
DOI 10.1021/pr4006298
Crossref 10.1021/pr4006298