Thermal effects on an enzymatically latent conformation of coagulation factor VIIa

Activation of the zymogen factor VII yields an enzyme form, factor VIIa, wi th only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Fa ctor VIIa activity measured with a chromogenic peptide substrate is charact erized by an unusual temperature dependency which indicates that the activa ted protease exists in an equilibrium between a latent (enzymatically inact ive) and an active conformation. As shown by calorimetry and activity measu rements the thermal effects, on factor VIIa are fully reversible below the denaturation temperature of 58.1 degrees C. A model for factor VIIa has bee n proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Bi ol. Chem. 269, 18891-18898] in which the protease is supposed to exist prim arily as a latent enzyme form because of the poor incorporation into the pr otease structure of the N-terminal Ile153 released by proteolytic cleavage during activation of factor VII. Binding of tissue factor to factor VIIa is assumed to shift the equilibrium towards an active conformation in which t he N-terminal Ile153 forms a salt bridge with Asp343. We corroborate the va lidity of this model by: (a) chemical modification of factor VIIa; this sug gests that the thermal effect on the equilibrium between the active and ina ctive conformation is reflected in the relative accessibility of the active site and the N-terminal Ile153; (b) measurements of factor VIIa binding to tissue factor indicating that complex formation is favoured by stabilizati on of the active conformation; and (c) activity measurements of a cross-lin ked factor VIIa-tissue factor complex; this showed that cross-linking stabi lized the active conformation of factor VIIa and essentially prevented its thermally-induced transformation into the inactive state.