文摘
The enzyme complex prothrombinase plays a pivotal role in fibrin clot development throughthe production of thrombin, making this enzyme complex an attractive target for therapeutic regulation.This study both functionally and structurally characterizes a potent, highly selective, active site directedinhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residuesin factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with humanfactor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecularassociation between factor Xa and PD0313052. This interaction was governed by association (kon) anddissociation (koff) rate constants of (1.0 ± 0.1) × 107 M-1 s-1 and (1.9 ± 0.5) × 10-3 s-1, respectively.The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character describedby a Ki*= 0.29 ± 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052also identified a slow-onset mechanism with a Ki* = 0.17 ± 0.03 nM and a kon and koff of (0.7 ± 0.1) ×107 M-1 s-1 and (1.7 ± 0.8) × 10-3 s-1, respectively. Crystals of factor Xa and PD0313052 demonstratedhydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216,as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate thatthe inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicatethat active site residues of human factor Xa, including the catalytic Ser195, are effectively unalteredfollowing assembly into prothrombinase.