Allosteric activation of human α-thrombin through exosite 2 by suramin analogs

Abstract

Thrombin is a serine protease that plays fundamental roles in hemostasis. We have recently elucidated the crystal structure of thrombin in complex with suramin, evidencing the interaction through the anion binding exosite 2. Here, we show that the activity of thrombin toward natural and synthetic substrates is enhanced by suramin as well as analogs of suramin at a low micromolar range prior to an inhibitory component at higher concentrations. Suramin analogs substituted by phenyl and chlorine instead of methyl were the most efficient in promoting allosteric activation, with an enhancement of enzymatic activity of 250% and 630% respectively. We discuss the importance of exosite 2 as a regulatory site for ligands in both the procoagulant and inhibitory scenarios.

Immobilization of Thrombin to Alginate Gel and in vitro Application of Immobilized Thrombin

“…The procedure for determination of thrombin activity was briefly given at Table 1. The standard curve was obtained from different concentrations of p-nitroanilide to calculate thrombin activity [7][8][9][10][11][12]. One unit of enzyme activity was defined as the amount of enzyme catalyzing the hydrolysis of 1 µmol chromogenic substrate per minute at 37°C under standard assay conditions.…”