Biology subjects

Emsley, J.

Publications and source records attributed to Emsley, J..

2 recordsLinked to original sources

bioRxiv2025

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

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.

biochemistry↗

bioRxiv2025

Structure-function analysis of Factor Seven Activating Protease (FSAP) by in-silico methods and its expression in insect cells.

Factor VII Activating Protease (FSAP) is a circulating serine protease in blood and is likely to be involved in regulating hemostasis and inflammatory processes in stroke. The zymogen form of FSAP (Pro-FSAP) is activated by charged molecules, including heparin and histones. Here, we tested the feasibility of FSAP expression in insect cells and analyzed its structure and evolution with in-silico methods. The expression of full-length (FL) FSAP and the serine protease domain (SPD) was very low, and this was slightly improved with the protease-inactivating mutant. The remainder of the protein, comprising the heavy chain (HC), was expressed at high levels and demonstrated binding to pro-FSAP activators, histones, and heparin. Since FL-FSAP was not satisfactorily expressible for structural studies, the AlphaFold structure was used for molecular dynamics simulations and in-silico docking studies with pro-FSAP activators. AlphaFold predicts a globular structure for the EGF, kringle and the trypsin domain as well as a disordered N-terminal region (NTR). Docking studies suggested that the EGF-3 domain interacts with heparin and the NTR with histones. Very likely, this disturbs the globular fold of the enzyme to expose the activation site and promotes autocatalytic activation. FSAP evolved in its current domain configuration in jawed vertebrates about 500 million years ago suggesting an important and conserved function. The insights from these studies will facilitate the expression of recombinant FSAP as well as enable a deeper understanding of its structure and biological functions in the context of stroke.

biochemistry↗