Biology subjects

Kanse, S. M.

Publications and source records attributed to Kanse, S. M..

4 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↗

bioRxiv2022

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

Factor VII activating protease (FSAP) is a circulating serine protease, and individuals with the Marburg I (MI) single nucleotide polymorphism (SNP), which results in an inactive enzyme, have an increased risk of stroke. The outcome of ischemic stroke is more marked in FSAP-deficient mice compared to their wild-type (WT) counterparts. Plasma FSAP levels are raised in patients as well as mice after stroke. In vitro, FSAP promotes fibrinolysis by cleavage of fibrinogen, activates protease-activated receptors and decreases the cellular cytotoxicity of histones. Since these are desirable properties in stroke treatment, we tested the effect of recombinant serine protease domain of FSAP (FSAP-SPD) on ischemic stroke in mice. A combination of tissue plasminogen activator (tPA) and FSAP-SPD enhanced clot lysis, improved microvascular perfusion and neurological outcome and reduced infarct volumes in a mouse model of thromboembolic stroke. In the tail bleeding model FSAP-SPD treatment provoked a faster clotting time indicating that it has a pro-coagulant effect that is described before. FSAP-SPD improved stroke outcome and diminished the negative effects of co-treatment with tPA in the transient middle cerebral artery occlusion model. The inactive MI-isoform of FSAP did not have any effects in either model. In mice with FSAP deficiency there were minor differences in the outcomes of stroke but the treatment with FSAP-SPD was equally effective. Thus, FSAP represents a promising novel therapeutic strategy in the treatment of ischemic stroke that requires further evaluation.

biochemistry↗

bioRxiv2022

Identification of a phage display-derived peptide interacting with the N-terminal region of Factor VII activating protease (FSAP) enables characterization of zymogen activation.

Increased Factor VII activating protease (FSAP) activity has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to a single nucleotide polymorphism. The activation of FSAP zymogen in plasma is mediated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear if this activation mechanism is specific and amenable to manipulation. Using a phage display approach we have identified a peptide, NNKC9/41, that activates pro-FSAP in plasma. Other commonly found zymogens in the plasma were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. Blocking the contact pathway of coagulation did not influence pro-FSAP activation by the peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41 and this was reversed by MA-FSAP-38C7 demonstrating the utility of this peptide. Identification of this peptide, and the corresponding interaction site, provides proof of principle that it is possible to activate a single protease zymogen in blood in a specific manner. Peptide NNKC/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP in more detail, elucidate its biological role. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/475526v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@112eea6org.highwire.dtl.DTLVardef@1f1d7eaorg.highwire.dtl.DTLVardef@967b03org.highwire.dtl.DTLVardef@1662c51_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗