RESUMEN
The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C (PC). Interactions between PC and extended surfaces, likely contributed by TM within the IIa.TM complex, have been proposed to play a key role in PC activation. Initial velocities of PC activation at different concentrations of PC and TM could be accounted for by a model that did not require consideration of direct binding interactions between PC and TM. Reversible inhibitors directed toward the active site of IIa within the IIa.TM complex behaved as classic competitive inhibitors of both peptidyl substrate cleavage as well as PC activation. The ability of these small molecule inhibitors to block PC binding to the enzyme points to a principal role for active site-dependent substrate recognition in determining the affinity of IIa.TM for its protein substrate. Selective abrogation of active site docking by mutation of the P1 Arg in PC to Gln yielded an uncleavable derivative (PC(R15Q)). PC(R15Q) was a poor inhibitor (K(i) >or= 30 microm) of PC activation as well as peptidyl substrate cleavage by IIa.TM. Thus, inhibition by PC(R15Q) most likely results from its ability to weakly interfere with active site function rather than by blocking extended interactions with the enzyme complex. The data suggest a primary role for active site-dependent substrate recognition in driving the affinity of the IIa.TM complex for its protein substrate. Interactions between PC and extended surfaces contributed by IIa and/or TM within the IIa.TM complex likely contribute in a secondary or minor way to protein substrate affinity.