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Carlsson, Karin
Publications (7 of 7) Show all publications
Carlsson, K., Persson, E., Østergaard, H., Lindgren, M., Carlsson, U. & Svensson, M. (2011). Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching. Biochemical and Biophysical Research Communications - BBRC, 413(4), 545-549
Open this publication in new window or tab >>Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 413, no 4, p. 545-549Article in journal (Refereed) Published
Abstract [en]

The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 1044-1053), can influence the apparent  fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.

Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.

Place, publisher, year, edition, pages
Elsevier, 2011
Keywords
Factor VIIa, Fluorescence quenching, Fluorescence resonance energy transfer, Tissue factor, Fluorescein, Rhodamine, Conformational dynamics
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-63686 (URN)10.1016/j.bbrc.2011.08.135 (DOI)000295912800010 ()
Note
Funding agencies|Swedish Research Council||Knut and Alice Wallenbergs Foundation||Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2017-12-11Bibliographically approved
Carlsson, K. (2010). Tissue Factor in Complex: Studies of interactions between blood coagulation proteins. (Doctoral dissertation). Linköping: Linköping University Electronic Press
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. p. 75
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1329
National Category
Natural Sciences
Identifiers
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)
Opponent
Supervisors
Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2010-12-30Bibliographically approved
Carlsson, K., Persson, E., Carlsson, U. & Svensson, M. (2006). Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor. Biochemical and Biophysical Research Communications - BBRC, 349(3), 1111-1116
Open this publication in new window or tab >>Inhibitors of factor VIIa affect the interface between the protease domain and tissue factor
2006 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 349, no 3, p. 1111-1116Article in journal (Refereed) Published
Abstract [en]

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). The γ-carboxyglutamic acid-rich domain of FVIIa docks with the C-terminal domain of TF, the EGF1 domain of FVIIa contacts both domains of TF, and the EGF2 domain and protease domain (PD) form a continuous surface that sits on the N-terminal domain of TF. Our aim was to investigate the conformational changes that occur in the sTF·PD binding region when different types of inhibitors, i.e., one active-site inhibitor (FFR-chloromethyl ketone (FFR)), two different peptide exosite inhibitors (E-76 and A-183), and the natural inhibitor tissue factor pathway inhibitor (TFPI), were allowed to bind to FVIIa. For this purpose, we constructed two sTF mutants (Q37C and E91C). By the aid of site-directed labeling technique, a fluorescent label was attached to the free cysteine. The sTF·PD interface was affected in position 37 by the binding of FFR, TFPI, and E-76, i.e., a more compact structure was sensed by the probe, while for position 91 located in the same region no change in the surrounding structure was observed. Thus, the active site inhibitors FFR and TFPI, and the exosite inhibitor E-76 have similar effects on the probe in position 37 of sTF, despite their differences in size and inhibition mechanism. The allosteric changes at the active site caused by binding of the exosite inhibitor E-76 in turn induce similar conformational changes in the sTF·PD interface as does the binding of the active site inhibitors. A-183, on the other hand, did not affect position 37 in sTF, indicating that the A-183 inhibition mechanism is different from that of E-76.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-36179 (URN)10.1016/j.bbrc.2006.08.148 (DOI)30411 (Local ID)30411 (Archive number)30411 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2018-03-23
Wirehn, J., Carlsson, K., Herland, A., Persson, E., Carlsson, U., Svensson, M. & Hammarström, P. (2005). Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W. Biochemistry, 44(18), 6755-6763
Open this publication in new window or tab >>Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W
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2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 18, p. 6755-6763Article in journal (Refereed) Published
Abstract [en]

Tissue factor (TF), a small transmembrane receptor, binds factor VIIa (FVIIa), and the formed complex initiates blood coagulation by proteolytic activation of substrate factors IX and X. A naturally occurring mutation in the human TF gene was recently reported, where a single-base substitution results in an R200W mutation in the TF extracellular domain [Zawadzki, C., Preudhomme, C., Gavériaux, V., Amouyel, P., and Jude, B. (2002) Thromb. Haemost. 87, 540-541]. This mutation appears to be associated with low monocyte TF expression and may protect against thrombosis but has not been associated with any pathological condition, and individuals who present the heterozygous trait appear healthy. Here, we report the activity, folding, and aggregation behavior of the R200W mutant of the 219-residue soluble extracellular domain of TF (sTFR200W) compared to that of the wild-type protein (sTF wt). No differences in stability or FVIIa cofactor activity but an impaired ability to promote FX activation at physiological conditions between the sTFR200W mutant and sTFwt were evident. Increased binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to sTFR200W indicated a population of partially folded intermediates during denaturation. sTFR200W showed a dramatically increased propensity for aggregate formation compared to sTFwt at mildly acidic pHs, with an increased rate of aggregation during conditions, promoting the intermediate state. The lowered pH resistance could explain the loss of sTFR200W in vivo because of aggregation of the mutant. The intrinsic structure of the sTF aggregates appears reminiscent of amyloid fibrils, as revealed by thioflavin T fluorescence, atomic force microscopy, and transmission electron microscopy. We conclude that the lowered activity for FX activation and the propensity of the mutant protein to misfold and aggregate will both contribute to decreased coagulation activity in TFR200W carriers, which could protect from thrombotic disease. © 2005 American Chemical Society.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-45448 (URN)10.1021/bi047388l (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2018-04-25
Carlsson, K., Freskgard, P.-O., Persson, E., Carlsson, U. & Svensson, M. (2003). Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor. European Journal of Biochemistry, 270(12), 2576-2582
Open this publication in new window or tab >>Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor
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2003 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 12, p. 2576-2582Article in journal (Refereed) Published
Abstract [en]

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.

Keywords
Fluorescence, Local probing, Protein-protein interactions, Site-directed labeling
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-46604 (URN)10.1046/j.1432-1033.2003.03625.x (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
Carlsson, K., Osterlund, M., Persson, E., Freskgard, P.-O., Carlsson, U. & Svensson, M. (2003). Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains. Biochimica et Biophysica Acta - Proteins and Proteomics, 1648(1-2), 12-16
Open this publication in new window or tab >>Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains
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2003 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1648, no 1-2, p. 12-16Article 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.

Keywords
Ca2+ dependence, Factor VIIa, Fluorescence, Protein-protein interaction, Tissue factor, Titration
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-46620 (URN)10.1016/S1570-9639(03)00025-6 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
Osterlund, M., Owenius, R., Carlsson, K., Carlsson, U., Persson, E., Lindgren, M., . . . Svensson, M. (2001). Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa. Biochemistry, 40(31), 9324-9328
Open this publication in new window or tab >>Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa
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2001 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 31, p. 9324-9328Article in journal (Refereed) Published
Abstract [en]

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF-FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-49196 (URN)10.1021/bi010283n (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically approved
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