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A more recent version of this article appeared on March 4, 2005
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Papers In Press, published online ahead of print December 6, 2004
J. Biol. Chem, 10.1074/jbc.M412869200
Submitted on November 15, 2004
Revised on December 6, 2004
Accepted on December 6, 2004

Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C

Hong Xu, Leslie A Bush, Agustin O Pineda, Sonia Caccia, and Enrico Di Cera

Dept. of Biochemistry & Molecular Biophysics, Washington Univ Med School, St Louis, MI 63110

Corresponding Author: enrico{at}wustl.edu

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of seventy-seven Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na+ site (Thr-172, Lys-224, Tyr-225, Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189) and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35, Pro-37) and 60-loop (Phe-60h) produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin: the unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.


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