Thermal effects on an enzymatically latent conformation of coagulation factor VIIa.
Activation of the zymogen factor VII yields an enzyme form, factor VIIa, with only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Factor VIIa activity measured with a chromogenic peptide substrate is characterized by an unusual temperature dependency which indicates that the activated protease exists in an equilibrium between a latent (enzymatically inactive) and an active conformation. As shown by calorimetry and activity measurements the thermal effects on factor VIIa are fully reversible below the denaturation temperature of 58.1 degrees C. A model for factor VIIa has been proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Biol. Chem. 269, 18891-18898] in which the protease is supposed to exist primarily as a latent enzyme form because of the poor incorporation into the protease 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 the N-terminal Ile153 forms a salt bridge with Asp343. We corroborate the validity of this model by: (a) chemical modification of factor VIIa; this suggests that the thermal effect on the equilibrium between the active and inactive 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 stabilization of the active conformation; and (c) activity measurements of a cross-linked factor VIIa-tissue factor complex; this showed that cross-linking stabilized the active conformation of factor VIIa and essentially prevented its thermally-induced transformation into the inactive state.
Petersen
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Tissue Factor/Factor VII Research, Novo Nordisk A/S Måløv, Denmark. lcp@novo.dk
Persson
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Freskgård
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D000478:Alkylation; D001665:Binding Sites; D002863:Chromogenic Compounds; D003432:Cross-Linking Reagents; D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013925:Thromboplastin / Q000737:chemistry / Q000378:metabolism
D002863:Chromogenic Compounds; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013925:Thromboplastin; D015942:Factor VIIa