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Activity, folding, misfolding, and aggregation in vitro of the naturally occurring human tissue factor mutant R200W
Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry.
Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics .
Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark.
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2005 (English)In: Biochemistry, ISSN 0006-2960, Vol. 44, no 18, 6755-6763 p.Article 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.

Place, publisher, year, edition, pages
2005. Vol. 44, no 18, 6755-6763 p.
National Category
Engineering and Technology
URN: urn:nbn:se:liu:diva-45448DOI: 10.1021/bi047388lOAI: diva2:266344
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2011-01-12

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Carlsson, KarinHerland, AnnaCarlsson, UnoSvensson, MagdalenaHammarström, Per
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