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J. Biol. Chem., Vol. 280, Issue 33, 29393-29396, August 19, 2005

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Thrombin Functions through Its RGD Sequence in a Non-canonical Conformation^*

Matthew E. Papaconstantinou, Christopher J. Carrell, Agustin O. Pineda, Kevin M. Bobofchak, F. Scott Mathews, Christodoulos S. Flordellis, Michael E. Maragoudakis, Nikos E. Tsopanoglou¶, and Enrico Di Cera||

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 and the Department of Pharmacology, Medical School, University of Patras, 26500 Patras, Greece

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with _v3 and ₅₁ integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187–189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Å resolution crystal structure of free thrombin grown in the presence of high salt (400 mM KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Å away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.

Received for publication, June 10, 2005 , and in revised form, June 30, 2005.

^* This work was supported in part by National Institutes of Health Research Grants HL49413, HL58141, and HL73813 (to E. D. C.) and European Social Fund, Operational Program for Educational and Vocational Training II (EPEAEK II), Program Pythagoras II (to N. E. T.). 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 atomic coordinates and structure factors (code 2A0Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^¶ To whom correspondence may be addressed. Tel.: 30-2610-996171; Fax: 30-2610-994720; E-mail: ntsopan{at}med.upatras.gr. || To whom correspondence may be addressed. Tel.: 314-362-4185; Fax: 314-747-5354; E-mail: enrico{at}wustl.edu.

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