title: The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency.
abstract: We report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild-type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (K(d) from 4.4 nmol/l for FVIIaWT to 17.3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (k(cat)/K(m) from 2.3 x 10(7)/mol/l s for FVIIaWT to 8.7 x 10(5)/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen-like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF-catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the K(m) value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.
authors.affiliation: Angelo Bianchi Bonomi Haemophilia and Thrombosis Centre, Fondazione Luigi Villa, IRCCS Maggiore Hospital and University of Milan, 9-20122 Milan, Italy. flora.peyvandi@unimi.it
authors.forename: Flora
authors.initials: F
authors.lastname: Peyvandi
authors.forename: Raimondo
authors.initials: R
authors.lastname: De Cristofaro
authors.forename: Isabella
authors.initials: I
authors.lastname: Garagiola
authors.forename: Roberta
authors.initials: R
authors.lastname: Palla
authors.forename: Sepideh
authors.initials: S
authors.lastname: Akhavan
authors.forename: Raffaele
authors.initials: R
authors.lastname: Landolfi
authors.forename: Pier Mannuccio
authors.initials: PM
authors.lastname: Mannucci
mesh_terms: D000328:Adult; D001777:Blood Coagulation; D002460:Cell Line; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism; D005168:Factor VII Deficiency / Q000235:genetics* / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D008297:Male; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D005167:Factor VII; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa