Releasing the brakes in coagulation Factor IXa by co-operative maturation of the substrate-binding site.

Coagulation Factor IX is positioned at the merging point of the intrinsic and extrinsic blood coagulation cascades. Activated Factor IX (FIXa) serves as the trigger for amplification of coagulation through formation of the so-called Xase complex, which is a ternary complex of FIXa, its substrate Factor X, and the cofactor Factor VIIIa on the surface of activated platelets. Within the Xase complex the substrate turnover by FIXa is enhanced 200,000-fold, however, the mechanistic and structural basis for this dramatic enhancement remains only partly understood.  A multi-faceted approach using enzymatic, biophysical and crystallographic methods to evaluate a key set of activity enhanced FIXa variants has demonstrated a delicately balanced, bidirectional network.  Essential molecular interactions across multiple regions of the FIXa molecule cooperate in the maturation of the active site. This maturation is specifically facilitated by long range communication through the Ile212-Ile213 motif unique to FIXa and a flexibility of the 170-loop that is further dependent on the conformation in the Cys168-Cys182 disulphide bond. Ultimately the network consists of compensatory brakes (V16 and I213) and accelerators (Tyr99 and Phe174) that together allow for a subtle fine tuning of enzymatic activity.