Heparin enhances the catalytic activity of des-ETW-thrombin

Abstract

The thrombin mutant, des-ETW-thrombin, lacking Glu(146), Thr(147), and Trp(148) within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the K(m) for cleavage of four thrombin-specific substrates by des-ETW-thrombin by 3-4-fold. Similarly, pentosan polysulphate (PPS) decreased the K(m) with these substrates by 8-10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final K(i) in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite I (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The K(i) for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM, and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.

Heparin-induced substrate behavior of antithrombin Cambridge II

“…The change in antithrombin conformation increases the rate of thrombin inhibition by approximately 2-fold, and heparin binding to thrombin also has been associated with an increase in catalytic activity through an allosteric mechanism. 37 Since the stoichiometry of inhibition is dependent on the relative rates of RCL insertion and deacylation, it is likely that either one or both are affected through the binding of heparin. In order to distinguish between the increase in rate of deacylation due to thrombin allostery and the decrease in rate of thrombin translocation due to bridging, stoichiometries were determined after preincubating thrombin with small heparin fragments.…”

Generation of Ligand Conformations in Continuum Solvent Consistent with Protein Active Site Topology: Application to Thrombin

“…For example, binding of thrombin to glycoprotein 1b increases k cat / K m for cleavage of PAR-1 by only 6-fold, while binding of FXa to FVa or FIXa to FVIIIa cause an increase of 3 orders of magnitude . This small scale of allosteric change for thrombin is also reflected in its interactions with thrombomodulin, fibrinogen, the hirudin peptide, and sodium binding and in the interactions of its mutants with these ligands and reflects only small movements of the protein ternary structure . For this reason, the structures with diverse ligands should be comparable, and indeed it has even been possible to identify conserved waters .…”

“…7 This small scale of allosteric change for thrombin is also reflected in its interactions with thrombomodulin, fi-brinogen, the hirudin peptide, and sodium binding 8 and in the interactions of its mutants with these ligands and reflects only small movements of the protein ternary structure. 9 For this reason, the structures with diverse ligands should be comparable, and indeed it has even been possible to identify conserved waters. 10 Several approaches 11,12 have been described to rationalize this extensive data and to allow extrapolation to the design of new inhibitors.…”