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J. Biol. Chem., Vol. 281, Issue 2, 1169-1178, January 13, 2006

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Novel Fluorescent Prothrombin Analogs as Probes of Staphylocoagulase-Prothrombin Interactions^*

Peter Panizzi1, Rainer Friedrich, Pablo Fuentes-Prior¶, Heather K. Kroh, Judy Briggs, Guido Tans||, Wolfram Bode, and Paul E. Bock2

From the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, the Proteinase Research Group, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany, the ^¶Cardiovascular Research Center, Institut Català de Ciències Cardiovasculars-Consejo Superior de Investigaciones Cientificas, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain, and the ^||Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, 6200MD Maastricht, The Netherlands

Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1-325 (SC-(1-325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1-146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147-325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1-325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1-325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1-325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17-72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with 130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1-325) binding. The SC-(1-325)·(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1-325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1-325)·(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.

Received for publication, July 21, 2005 , and in revised form, October 14, 2005.

^* This work was supported by National Institutes of Health Grants HL038779 and HL071544 (to P. E. B.) and by the SFB 469 of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to W. 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.

¹ Supported in part by National Institutes of Health Training Grant HL07751.

² 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: 615-322-1855; E-mail: paul.bock{at}vanderbilt.edu.

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