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J. Biol. Chem., Vol. 278, Issue 45, 44489-44495, November 7, 2003

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Role of Prothrombin Fragment 1 in the Pathway of Regulatory Exosite I Formation during Conversion of Human Prothrombin to Thrombin^*

Patricia J. Anderson and Paul E. Bock

From the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin^54–65 labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir^54–65()) or 5-(carboxy)fluorescein ([5F]Hir^54–65()). [NBD]Hir^54–65() distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir^54–65(). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir^54–65() by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin·F1.2 complex bound [5F]Hir^54–65() with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.

Received for publication, June 29, 2003 , and in revised form, August 11, 2003.

^* This work was supported by National Institutes of Health Grant HL38779 (to P. E. B.). 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 postdoctoral fellowship from the American Heart Association, Southeastern Consortium (SE-9820133V) and subsequently by an Institutional National Research Service Award (DK07186). Present address: Howard Hughes Medical Institute, Washington University School of Medicine, 660 S. Euclid, Box 8022, St. Louis, MO 63110.

To whom correspondence should be addressed: Dept. of Pathology, Vanderbilt University School of Medicine, C3321A Medical Center North, Nashville, TN 37232-2561. Tel.: 615-343-9863; Fax: 615-343-7023; E-mail: paul.bock{at}vanderbilt.edu.

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