Abstract

Thrombin is a highly plastic molecule whose activity and specificity are regulated by exosites 1 and 2, positively-charged domains that flank the active site. Exosite binding by substrates and cofactors regulates thrombin activity by localizing thrombin, guiding substrates, and by inducing allosteric changes at the active site. Although inter-exosite and exosite-to-active-site allostery have been demonstrated, the impact of active site ligation on exosite function has not been examined. To address this gap, we used surface plasmon resonance to determine the effects of dabigatran and argatroban, active site-directed inhibitors, on thrombin binding to immobilized γA/γA-fibrin or glycoprotein Ibα peptide via exosite 1 and 2, respectively, and thrombin binding to γA/γ′-fibrin or factor Va, which is mediated by both exosites. Whereas dabigatran attenuated binding, argatroban increased thrombin binding to γA/γA- and γA/γ′-fibrin and to factor Va. The results with immobilized fibrin were confirmed by examining the binding of radiolabeled thrombin to fibrin clots. Thus, dabigatran modestly accelerated the dissociation of thrombin from γA/γA-fibrin clots, whereas argatroban attenuated dissociation. Dabigatran had no effect on thrombin binding to glycoprotein Ibα peptide, whereas argatroban promoted binding. These findings not only highlight functional effects of thrombin allostery, but also suggest that individual active site-directed thrombin inhibitors uniquely modulate exosite function, thereby identifying potential novel mechanisms of action.

Highlights

  • The substrate specificity of thrombin is dependent on exosites 1 and 2, positively-charged domains that flank the active site [1,2]

  • We examined the effects of dabigatran, argatroban, and dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA), active site-directed small molecules that inhibit thrombin with Ki values of 4.5 nM [25], 19 nM [26], and 45 nM [27], respectively, on thrombin binding to immobilized γA/γA-fibrin, γA/γ0-fibrin, factor Va, and GpIbα peptide

  • In contrast to the results with dabigatran, argatroban and DAPA increased thrombin binding to γA/γA-fibrin, γA/γ0-fibrin, and factor Va in a concentration-dependent manner with EC50 values for argatroban of 62.4 ± 4.8 nM, 59.4 ± 5.1 nM, and 23.4 ± 8.8 nM, respectively, and for DAPA of 514.1 ± 24.0 nM, 515.9 ± 31.0 nM, and 565.1 ± 200 nM, respectively (Fig 2)

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Summary

Introduction

The substrate specificity of thrombin is dependent on exosites 1 and 2, positively-charged domains that flank the active site [1,2]. These exosites modulate the reactivity of thrombin by providing initial binding sites for substrates, inhibitors or cofactors, sterically hindering other interactions, and by allosterically modifying the active site. Exosite 1 is more versatile because it (a) serves as a docking site for substrates, (b) redirects thrombin activity by binding thrombomodulin, and (c) mediates thrombin inhibition by hirudin and heparin cofactor II. Exosite 2 has a more limited role because it mainly serves to tether or localize thrombin. Exosite 2 contributes to thrombin binding to platelet glycoprotein (Gp) Ibα [3,4] and, PLOS ONE | DOI:10.1371/journal.pone.0157471 June 15, 2016

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