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J. Biol. Chem., Vol. 279, Issue 41, 43237-43244, October 8, 2004

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Molecular Mapping of the Thrombin-Heparin Cofactor II Complex^*

Yolanda M. Fortenberry, Herbert C. Whinna, Holly R. Gentry¶, Timothy Myles||, Lawrence L. K. Leung||, and Frank C. Church¶**

From the Departments of Pathology and Laboratory Medicine, ^¶Pharmacology, and ^**Medicine, Carolina Cardiovascular Biology Center, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, North Carolina 27599-7035 and the ^||Division of Hematology, Department of Medicine, School of Medicine, Stanford University, Stanford, California 94305

We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln²⁴, Lys⁶⁵, His⁶⁶, and Tyr⁷¹ (using the thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 x 10⁸ M^-1 min^-1 for HCII-heparin when compared with 2.36 x 10⁸ M^-1 min^-1 with wild-type thrombin and 0.03-0.53 x 10⁸ M^-1 min^-1 for HCII-dermatan sulfate when compared with 5.23 x 10⁸ M^-1 min^-1 with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp⁵¹, Lys⁵², Lys¹⁴⁵/Thr¹⁴⁷/Trp¹⁴⁸, Asp²³⁴) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp⁵⁰, Glu²⁰², Glu²²⁹, Arg²³³) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-glycosaminoglycan inhibition reactions. Collectively, our results support a "double bridge" mechanism for HCII inhibition of thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the "hirudin-like" domain of HCII with thrombin exosite-1.

Received for publication, June 16, 2004 , and in revised form, August 2, 2004.

^* This research was supported in part by Research Grants HL-32656 and HL-06350 (to F. C. C), HL-04063 (to H. C. W.), HL-57530 (to L. L. K. L.), and F32-HL-076108 (to Y. M. F.) from the National Institutes of Health. 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.

Supported by a stipend provided through the Carolina Minority Postdoctoral Scholars Program from the University of North Carolina Chapel Hill.

To whom correspondence and reprint requests should be addressed: Campus Box 7035, Division of Hematology-Oncology/Medicine, 932 Mary Ellen Jones Bldg., University of North Carolina, Chapel Hill, NC 27599-7035. Fax: 919-966-7639; E-mail: fchurch{at}email.unc.edu.

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