Sequential coagulation factor VIIa domain binding to tissue factor |
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Authors: | Osterlund Maria Persson Egon Carlsson Uno Freskgård Per-Ola Svensson Magdalena |
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Institution: | a IFM-Department of Chemistry, Linköping University, SE-581 83 Linkoping, Sweden b Vascular Biochemistry, Novo Nordisk A/S, DK-2760 Malov, Denmark c Protein Biotechnology, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark |
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Abstract: | Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the γ-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced. |
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Keywords: | Tissue factor Factor VIIa Protein-protein interaction Probe Kinetics |
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