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J. Biol. Chem., Vol. 283, Issue 19, 13378-13387, May 9, 2008

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The Origins of Enhanced Activity in Factor VIIa Analogs and the Interplay between Key Allosteric Sites Revealed by Hydrogen Exchange Mass Spectrometry^*

Kasper D. Rand¹, Mette D. Andersen, Ole H. Olsen, Thomas J. D. Jørgensen, Henrik Østergaard, Ole N. Jensen, Henning R. Stennicke, and Egon Persson²

From the Department of Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark and the Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

Factor VIIa (FVIIa) circulates in the blood in a zymogen-like state. Only upon association with membrane-bound tissue factor (TF) at the site of vascular injury does FVIIa become active and able to initiate blood coagulation. Here we used hydrogen exchange monitored by mass spectrometry to investigate the conformational effects of site-directed mutagenesis at key positions in FVIIa and the origins of enhanced intrinsic activity of FVIIa analogs. The differences in hydrogen exchange of two highly active variants, FVIIa_DVQ and FVIIa_VEAY, imply that enhanced catalytic efficiency was attained by two different mechanisms. Regions protected from exchange in FVIIa_DVQ include the N-terminal tail and the activation pocket, which is a subset of the regions of FVIIa protected from exchange upon TF binding. FVIIa_DVQ appeared to adopt an intermediate conformation between the free (zymogen-like) and TF-bound (active) form of FVIIa and to attain enhanced activity by partial mimicry of TF-induced activation. In contrast, exchange-protected regions in FVIIa_VEAY were confined to the vicinity of the active site of FVIIa. Thus, the changes in FVIIa_VEAY appeared to optimize the active site region rather than imitate the TF-induced effect. Hydrogen exchange analysis of the FVIIa_M306D variant, which was unresponsive to stimulation by TF, correlated widespread reductions in exchange to the single mutation in the TF-binding region. These results reveal the delicate interplay between key allosteric sites necessary to achieve the transition of FVIIa into the active form.

Received for publication, November 28, 2007 , and in revised form, February 21, 2008.

^* This work was supported by a fellowship from the Danish Ministry of Science, Technology, and Innovation (to K. D. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence may be addressed. Tel.: 45-6550-2369; Fax: 45-6550-2467; E-mail: kasperd{at}bmb.sdu.dk.

² To whom correspondence may be addressed. Tel.: 45-4443-4351; Fax: 45-4466-3450; E-mail: egpe{at}novonordisk.com.

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