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J. Biol. Chem., Vol. 282, Issue 9, 6556-6563, March 2, 2007

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The Local Phospholipid Environment Modulates the Activation of Blood Clotting^*

Andrew W. Shaw¹, Vincent S. Pureza¹², Stephen G. Sligar, This author has a financial interest (but no managerial or operational role) in Nanodisc, Inc., to whom some of the patents on Nanodisc technology are licensed²³, and James H. Morrissey²⁴

From the Departments of Chemistry and Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Examples abound of membrane-bound enzymes for which the local membrane environment plays an important role, including the ectoenzyme that triggers blood clotting, the plasma serine protease, factor VIIa, bound to the integral membrane protein, tissue factor. The activity of this enzyme complex is markedly influenced by lipid bilayer composition and further by tissue factor partitioning into membrane microdomains on some cell surfaces. Unfortunately, little is known about how membrane microdomain composition controls factor VIIa-tissue factor activity, as reactions catalyzed by membrane-tethered enzymes are typically studied under conditions in which the experimenter cannot control the composition of the membrane in the immediate vicinity of the enzyme. To overcome this problem, we used a nanoscale approach that afforded complete control over the membrane environment surrounding tissue factor by assembling the factor VIIa·tissue factor complex on stable bilayers containing 67 ± 1 phospholipid molecules/leaflet (Nanodiscs). We investigated how local changes in phospholipid bilayer composition modulate the activity of the factor VIIa·tissue factor complex. We also addressed whether this enzyme requires a pool of membrane-bound protein substrate (factor X) for efficient catalysis, or alternatively if it could efficiently activate factor X, which binds directly to the membrane nanodomain adjacent to tissue factor. We have shown that full proteolytic activity of the factor VIIa·tissue factor complex requires extremely high local concentrations of anionic phospholipids and further that a large pool of membrane-bound factor X is not required to support sustained catalysis.

Received for publication, August 21, 2006 , and in revised form, December 26, 2006.

^* This work was supported by Grant HL47014 from the National Institutes of Health (NIH) and Grant 06-2328 from the Roy J. Carver Charitable Trust (to J. H. M.), NIH Grant GM33775 and National Science Foundation Grant SBCNW0830 520N602 (to S. G. S.), and Predoctoral Fellowship 0610028Z from the American Heart Association (to V. S. P.). 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.

¹ These authors contributed equally to this work.

² Co-inventors on patents and patent applications owned by the University of Illinois at Urbana-Champaign pertaining to Nanodiscs.

³ To whom correspondence may be addressed: Biochemistry Dept., University of Illinois at Urbana-Champaign, 116 Morrill Hall, MC-119, 505 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-244-7395; Fax: 217-265-4073; E-mail: s-sligar{at}uiuc.edu. ⁴ To whom correspondence may be addressed: Biochemistry Dept., College of Medicine, University of Illinois at Urbana-Champaign, 417 Medical Sciences Bldg. MC-714, 506 S. Mathews, Urbana, IL 61801. Tel.: 217-265-4036; Fax: 217-265-5290; E-mail: jhmorris{at}uiuc.edu.

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