The active conformations of Factor VIIa and Factor IXa: biochemical, biophysical, chemical and crystallographic studies

Abstract number: OC450

Banner^* D. W., Mueller^* F., Brochon† J. C., Huber^* W., Schoenfeld^* H. J., Sichler‡ K., Kopetzki‡ E., Benz^* J., Stihle^* M., Ackermann^* J., Groebke^* K., Alig^* L., Obst^* U., Tschopp^* T.

†Ecole Normale Superieure, France; ‡Roche, Germany ^*Roche, Switzerland;

The proteolytically activated forms of coagulation factors VII and IX, factors VIIa and IXa, share the property that they have limited enzymatic activity in the absence of their protein cofactors, tissue factor and factor VIIIa, respectively, which makes medical sense. In the search for small chemical ligands to act as anti-thrombotic agents it has been possible to determine crystal structures of factor VIIa in complex with small chemical inhibitors either in the presence or absence of tissue factor. The structures are comparable and indicate how tissue factor activates factor VIIa. For factor IXa it has not yet been possible to determine equivalent structures, as factor VIIIa is very large and difficult to work with and factor IXa alone has very poor affinity for potential substrates and inhibitors. Only two factor IXa crystal structures are known, the covalent complex with D-Phe-Pro-Arg-chloromethylketone (PPACK) and the noncovalent complex with para-amino benzamidine (PABA). For factor IXa we have determined inhibition constants and binding constants using enzyme assays, surface plasmon resonance and fluorescence titration for PABA and related compounds. We have determined crystal structures of factor IXa in the presence of PABA and of the best related ligands. A number of novel crystal forms have been identified and a structure of the PABA complex determined. Whilst the PABA complex is the same as that published, we find that for related inhibitors the ‘activation domain’, which consists of loops which together form the primary substrate recognition pocket, is totally disordered and no ligand is bound. It is known that factor VII in its zymogen form has a similarly disordered activation domain and that factor VIIa is in rapid conformational equilibrium between ‘zymogen-like’ and an ‘active enzyme-like’ conformations. We have observed, using fluorescence techniques, the stabilization of the active conformation of factor VIIa on the addition of inhibitors of high affinity. We are currently using fluorescence titration to investigate conformational changes in factor IXa in the presence of ligands such as PABA and PABA analogues and also ethylene glycol, which is known to be an activator. First results will be reported.