Mechanism of Activation of Tissue Factor

Abstract number: OR075

Chen¹ VMY, Ruf² W, Hogg¹ PJ

¹¹University of New South Wales, Sydney, Australia ¹¹University of New South Wales, Sydney, Australia ²²The Scripps Research Institute, La Jolla, USA

Tissue Factor (TF) on the plasma membrane of cells is mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of 2 fibronectin type III domains. The disulphide-bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta-sheet, or is what we have called a cross-strand bond. These domains typically contain one disulphide-bond that links across the two sheets. The Cys186-Cys209 TF bond has the same unusual configuration as the disulphide-bond in the second domain of CD4, which we have shown controls CD4 function by switching between oxidized (disulphide) and reduced (dithiol) states (Matthias et al. Nature Immunol. 3, 727, 2002). The Cys186-Cys209 TF disulphide is exposed to solvent in the crystal structure (Harlos et al. Nature 370, 662, 1994) and ablation of the cross-strand bond by mutating both Cys to Ser severely impairs procoagulant activity (Rehemtulla et al. J. Biol. Chem. 266, 10294, 1991). Residues close to the cross-strand bond have been identified as part of the region of TF that interacts with factors IX and X (Kirchhofer et al. Biochemistry 39, 7380, 2000). By labeling cells with a biotin-linked maleimide, we have demonstrated that the cross-strand bond in cryptic, but not active, TF is reduced on the cell surface. Moreover, the thiol-alkylating agent N-ethylmaleimide blocks TF activation by ionomycin and the thiol-oxidising agent HgCl₂ promotes activation. These findings imply that the disulphide-bond in the membrane-proximal domain is reduced in the cryptic form of TF and activation involves formation of the disulphide. It is likely that formation of this disulphide-bond changes the conformation of the domain which facilitates productive binding of factors IX and X.