Structure-function studies on the serine protease domain of Factor VII activating protease (FSAP).

Abstract

Factor VII activating protease (FSAP) is a circulating serine protease involved in stroke pathophysiology. This effect is likely mediated by the cleavage of substrates such as fibrinogen and extracellular histones. Our goal was to modify FSAP properties through mutagenesis to explore its different functions. Therefore, we expressed the serine protease domain (SPD) of FSAP with mutations and measured the cleavage of chromogenic substrate (S-2288), pro-urokinase (uPA), histones, and fibrinogen, as well as their inhibition by aprotinin, tissue factor pathway inhibitor (TFPI), and C1 inhibitor (C1Inh). Mutations are denoted using chymotrypsin numbering. Mutants KRHK63-65SSSS and K63aA/R63cA in the 60-loop were resistant to TFPI but not to aprotinin or C1Inh. Cleavage of pro-uPA and fibrinogen decreased, while histone and S-2288 cleavage remained normal. Mutants E96S, EDE96-99SSS, and D173A/E99S in the 99-loop produced a protease with reduced cleavage of histones and fibrinogen. EDE96-99SSS showed resistance to aprotinin, TFPI, and C1Inh, had lower affinity for S-2288, and exhibited less pro-uPA activation. The Q192D mutation was resistant to inhibition by aprotinin, TFPI, and C1Inh, but substrate cleavage remained unaffected. E218A was resistant to C1Inh inhibition but not to aprotinin or TFPI and showed normal substrate cleavage. R84S was resistant to aprotinin and TFPI but not C1Inh, with normal substrate cleavage. TFPI and C1Inh inhibited full-length FSAP more effectively than SPD-FSAP, though cleavage of histones and fibrinogen was more efficient with SPD-FSAP. These findings will enhance the understanding of the functional landscape of SPD-FSAP and help in developing mutants with altered properties to investigate FSAP functions.