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J. Biol. Chem., Vol. 281, Issue 11, 7183-7188, March 17, 2006

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Murine Thrombin Lacks Na⁺ Activation but Retains High Catalytic Activity^*

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Human thrombin utilizes Na⁺ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na⁺ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na⁺ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na⁺. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na⁺ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar k_cat/K_m values but activates PAR1 and PAR4 with k_cat/K_m values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.

Received for publication, November 9, 2005 , and in revised form, January 18, 2006.

^* This work was supported in part by National Institutes of Health Research Grants HL49413, HL58141, and HL73813. 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 To whom correspondence should be addressed. Tel.: 314-362-4185; Fax: 314-747-5354; E-mail: enrico{at}wustl.edu.

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