liu.seSearch for publications in DiVA
Change search
ReferencesLink to record
Permanent link

Direct link
Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
University Holding, Teknikringen 7, SE-581 83 Linköping, Sweden.
Vascular Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
Freskgård, P.-O., Protein Biotechnology, Novo Nordisk A/S, Novo Allé, DK-2880 Bagsværd, Denmark, Molecular Biology, Maxygen ApS, DK-2970, Hørsholm, Denmark.
Show others and affiliations
2003 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1648, no 1-2, 12-16 p.Article in journal (Refereed) Published
Abstract [en]

We have used the site-directed labeling approach to study the Ca 2+-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca2+ binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the ?-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca2+ titration is initiated upon Ca2+ binding to EGF1, the domain containing the site of highest Ca2+ affinity. Besides we showed that a Ca 2+-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca2+ binding to the PD seems to be of some importance for the docking of this domain to sTF. © 2003 Elsevier Science B.V. All rights reserved.

Place, publisher, year, edition, pages
2003. Vol. 1648, no 1-2, 12-16 p.
Keyword [en]
Ca2+ dependence, Factor VIIa, Fluorescence, Protein-protein interaction, Tissue factor, Titration
National Category
Engineering and Technology
URN: urn:nbn:se:liu:diva-46620DOI: 10.1016/S1570-9639(03)00025-6OAI: diva2:267516
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2012-02-17
In thesis
1. Tissue Factor in Complex: Studies of interactions between blood coagulation proteins
Open this publication in new window or tab >>Tissue Factor in Complex: Studies of interactions between blood coagulation proteins
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many biological processes rely on specific protein-protein interactions, for example immune responses, cell signaling, transcription, and blood coagulation. Blood coagulation is initiated when a vessel wall is damaged, exposing tissue factor (TF) to the circulating factor VII/factor VIIa (FVII/FVIIa) which results in the formation of the TF:FVIIa complex and thereby the initiation of blood coagulation. One of the substrates for the TF:FVIIa complex is factor X (FX), which is activated to factor Xa (FXa), subsequently leading to a series of reactions resulting in clot formation. Tissue factor pathway inhibitor (TFPI) is the major physiological inhibitor of the sTF:FVIIa complex, involved in regulation of coagulation by forming the TF:FVIIa:FXa:TFPI complex. Occasionally, the blood coagulation mechanism malfunctions, resulting in conditions such as the inability to stop bleeding or thrombosis. The fact that TF is the main initiator of the coagulation makes this an interesting protein to study, in the hunt for means to interfere with players involved in the blood clotting process.

Throughout the studies included in this thesis the site-directed labeling technique is utilized to attach spectroscopic probes to cysteines, introduced at specific positions by mutagenesis, in the protein of interest. These fluorescent or spin-probes are sensitive for changes in their immediate environment and can thus, for example be used to monitor protein-protein complex formation and conformational changes.

No complete structure has been obtained as yet for the large complex involving sTF, FVIIa, FXa, and TFPI. Therefore, we introduced a fluorescent probe at specific positions in soluble tissue factor (sTF) and the changes in fluorescence emission were detected upon sTF:FVIIa:FXa:TFPI complex formation. From these measurements it was concluded that not only parts of the C-terminal domain of sTF (TF2), but also residues in the N-terminal domain (TF1) are involved in binding to FXa in the quaternary complex.

In order to investigate conformational changes occurring in the extended interface between sTF and FVIIa upon binding of different inhibitors spectroscopic probes were introduced in sTF, in the vicinity of the interaction region. From the obtained data it was concluded that the exosite-binding inhibitor E-76 induces equivalent structural changes at the interface of sTF and the protease domain (PD) of FVIIa, as do the active-site inhibitors FFR and TFPI, i.e. makes the region around the active-site more compact. Binding of these inhibitors shows similar effects despite their differences in size, binding site, and inhibitory mechanism.

In addition, the Ca2+ dependence of the formation of the sTF:FVIIa complex was studied. Association between sTF and FVIIa during Ca2+ titration begins by Ca2+ binding to the first EGF-like domain of FVIIa. However, Ca2+ saturation of the γ-carboxyglutamic acid-rich (Gla) domain of FVIIa is required for complete sTF:FVIIa complex formation, and we were also able to detect that a Gla domain with vacant Ca2+ sites hinders the docking to sTF.

Finally, we investigated the structural changes of free inhibited FVIIa upon sTF and Ca2+ binding by FRET and quenching measurements. From this it was concluded that inhibited FVIIa does not seem to undergo large global structural changes upon binding to sTF, when taking the dynamics of free FVIIa into account. However, Ca2+ binding induces minor local conformational changes in the active-site region of the PD of inhibited FVIIa and subsequent binding of sTF causesfurther structural rearrangements in this area.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2010. 75 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1329
National Category
Natural Sciences
urn:nbn:se:liu:diva-63688 (URN)978-91-7393-355-1 (ISBN)
Public defence
2010-10-22, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2010-12-30Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Carlsson, KarinOsterlund, MariaCarlsson, UnoSvensson, Magdalena
By organisation
BiochemistryThe Institute of Technology
In the same journal
Biochimica et Biophysica Acta - Proteins and Proteomics
Engineering and Technology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 75 hits
ReferencesLink to record
Permanent link

Direct link