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J Biol Chem, Vol. 273, Issue 17, 10709-10718, April 24, 1998

Regions Remote from the Site of Cleavage Determine Macromolecular Substrate Recognition by the Prothrombinase Complex

From the Department of Medicine, Emory University, Atlanta, Georgia 30322

The proteolytic formation of thrombin is catalyzed by the prothrombinase complex of blood coagulation. The kinetics of prethrombin 2 cleavage was studied to delineate macromolecular substrate structures necessary for recognition at the exosite(s) of prothrombinase. The product, -thrombin, was a linear competitive inhibitor of prethrombin 2 activation without significantly inhibiting peptidyl substrate cleavage by prothrombinase. Prethrombin 2 and -thrombin compete for binding to the exosite without restricting access to the active site of factor Xa within prothrombinase. Inhibition by -thrombin was not altered by saturating concentrations of low molecular weight heparin. Furthermore, proteolytic removal of the fibrinogen recognition site in -thrombin only had a modest effect on its inhibitory properties. Both -thrombin and prethrombin 2 were cleaved with chymotrypsin at Trp¹⁴⁸ and separated into component domains. The C-terminal-derived 2 fragment retained the ability to selectively inhibit macromolecular substrate cleavage by prothrombinase, while the 1 fragment was without effect. As the 2 fragment lacks the fibrinogen recognition site, the P1-P3 residues or the intact cleavage site, specific recognition of the macromolecular substrate by the exosite in prothrombinase is achieved through substrate regions, distinct from the fibrinogen recognition or heparin-binding sites, and spatially removed from structures surrounding the scissile bond.

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