Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)

Abstract

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.