title: The Eph tyrosine kinase receptors EphB2 and EphA2 are novel proteolytic substrates of tissue factor/coagulation factor VIIa.
abstract: Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2.
authors.affiliation: From the Department of Medical Sciences, Clinical Chemistry, Uppsala University Hospital, SE-751 85 Uppsala, Science for Life Laboratory, Uppsala University, Sweden.
authors.forename: Oskar
authors.initials: O
authors.lastname: Eriksson
authors.affiliation: the Department of Chemistry, Analytical Chemistry, BMC, SE-751 24 Uppsala, and Science for Life Laboratory, Uppsala University, Sweden.
authors.forename: Margareta
authors.initials: M
authors.lastname: Ramström
authors.affiliation: the Department of Chemistry, Analytical Chemistry, BMC, SE-751 24 Uppsala, and Science for Life Laboratory, Uppsala University, Sweden.
authors.forename: Katarina
authors.initials: K
authors.lastname: Hörnaeus
authors.affiliation: the Department of Chemistry, Analytical Chemistry, BMC, SE-751 24 Uppsala, and Science for Life Laboratory, Uppsala University, Sweden.
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergquist
authors.affiliation: From the Department of Medical Sciences, Clinical Chemistry, Uppsala University Hospital, SE-751 85 Uppsala, Science for Life Laboratory, Uppsala University, Sweden.
authors.forename: Dariush
authors.initials: D
authors.lastname: Mokhtari
authors.affiliation: From the Department of Medical Sciences, Clinical Chemistry, Uppsala University Hospital, SE-751 85 Uppsala, Science for Life Laboratory, Uppsala University, Sweden agneta.siegbahn@medsci.uu.se.
authors.forename: Agneta
authors.initials: A
authors.lastname: Siegbahn
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement; D002478:Cells, Cultured; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005167:Factor VII; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D036104:Receptor, EphA2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D036183:Receptor, EphB2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D004220:Disulfides; D005167:Factor VII; D013925:Thromboplastin; D036104:Receptor, EphA2; D036183:Receptor, EphB2; D015942:Factor VIIa
keywords: Coagulation Factor; Mass Spectrometry (MS); Receptor Tyrosine Kinase; Serine Protease; Tissue Factor