Tissue factor (TF) is the essential cofactor for FVIIa. Binding to transmembrane tissue factor increases the catalytic efficiency of FVIIa allowing activation of FX and FIX which initiates coagulation and propagates stable clot. Transmembrane TF resides in a cryptic configuration on the cell surface and in the circulation with low procoagulant activity. However TF can be rapidly switched to an active configuration in order to contribute to thrombus propagation. The precise nature of this switch is unknown, however it is known to be an extracellular event. The extracellular part of TF consists of 2 fibronectin type III domains. The disulfidebond in the membrane proximal domain (Cys186-Cys209) is a cross-strand bond which links adjacent strands in the same ?? sheet. It has the configuration, characteristic dihedral strain energy and bond length of an allosteric disulfide bond. This indicates that it has the potential to undergo thiol/disulfide exchange to change the function of the TF protein. We confirm that the integrity of the Cys186-Cys209 disulfide is required for coagulant function and that tissue factor contains free thiols in the cryptic state which are lost when TF becomes de-encrypted. Membrane based tissue factor procoagulant activity is blocked by the mono-thiol alkylators N-ethylmaleimide and methyl methanethiosulfonate; but increased by ECl/formation of the disulfide via the thiol oxidiser, HgCb or thiol cross-linkers, eimidohexane and bismalemidoethane. The increase in activity correlates with a conformation change in the TF protein adjacent to the disulfide. We show that redox active protein disulfide isomerase is associated with cryptic tissue factor and propose that the cryptic conformation of tissue factor is maintained through formation of an Snitrosylated complex with protein disulfide isomerase. Our results indicate that the activation of TF involves a change of conformation of the domain 2 of TF caused by formation of the cross-strand Cys186-Cys209 disulfide bond. We suggest that this is likely to be the physiological change that facilitates productive binding of FIX and FX in coagulation.
| Identifer | oai:union.ndltd.org:ADTP/258320 |
| Date | January 2007 |
| Creators | Chen, Vivien Mun Yee, Medical Sciences, Faculty of Medicine, UNSW |
| Publisher | Awarded by:University of New South Wales. Medical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Chen Vivien Mun Yee., http://unsworks.unsw.edu.au/copyright |
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