Active Site-Dependent Substrate Recognition Plays a Primary Role in Determining the Affinity of the Thrombin-Thrombomodulin Complex for Protein C.
Authors: Lu Genmin , Chhum Sotheavy , Krishnaswamy Sriram 
Published: April 2020

Abstract
The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C. The TM-dependent enhancement of the anticoagulant function of IIa has alternately been considered to primarily arise from an allosteric modulation of the active site of the protease or from the ability of TM to bind protein C and participate in its presentation to the enzyme. We have applied established kinetic strategies to investigate the relative contributions of active site interactions versus interactions with extended surfaces in the action of the IIa-TM complex on human protein C. Initial velocity studies yielded equivalent kinetic constants for the hydrolysis of L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroanilide (S2366) by IIa alone or IIa saturated with TM. The reversible active site-directed ligands Nα-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581) and 4-amino-phenylpyruvic acid (APPA) were found to act as classical competitive inhibitors of S2366 cleavage by either IIa or IIa-TM. This finding is consistent with the limited interactions expected between S2366 and the active site of either IIa or IIa-TM. Equivalent inhibition constants were obtained for IIa or IIa-TM with either inhibitor that were in good agreement with directly determined equilibrium dissociation constants for I-2581 (IIa: Kd =25 ± 1 nM, IIa-TM: Kd=16 ± 1 nM) or APPA (IIa: Kd = 530 ± 90 nM, IIa-TM: Kd = 370 ± 40 nM). Similar kinetic and equilibrium dissociation constants obtained for IIa and IIa-TM indicate that obvious changes in the active site of IIa induced by TM are not detectable by this series of active-site directed probes. In contrast to peptidyl substrate cleavage, protein C activation by IIa (Km=177 ± 31 μM, Vmax/Et=0.004±0.001 s−1) was greatly enhanced by the addition of saturating concentrations of TM to form the IIa-TM complex (Km=12 ± 1 μM, Vmax/Et=0.28±0.01 s−1). However, both active site-directed reversible inhibitors continued to act as classical competitive inhibitors of protein C activation by IIa-TM (I-2581: Ki = 20 ± 1 nM; APPA: Ki = 470 ± 20 nM). Mutually exclusive binding interactions of protein C and active site-directed inhibitors indicate a principal role for active site-dependent interactions in determining the affinity of IIa-TM for protein C. This conclusion was further investigated by using a recombinant derivative of protein C with the P1 Arg mutated to Gln (PCR169Q) that rendered the zymogen uncleavable by either IIa or the IIa-TM complex. PCR169Q was a poor inhibitor (Ki&gt;&gt;50 μM) of protein C activation or S2236 cleavage implying a greatly reduced affinity for IIa-TM. These data suggest a primary role for active site-dependent substrate recognition in driving the affinity of IIa-TM for protein C. In contrast to the established role for exosites in action of IIa on many of its other substrates, possible interactions between protein C and extended surfaces within the IIa-TM complex likely contribute in a secondary way in determining the affinity of this enzyme complex for its biological substrate.