Fluorescent activity-based probe for the selective detection of Factor VII activating protease (FSAP) in human plasma

Abstract

The zymogen form of circulating Factor VII activating protease (FSAP) is activated by histones that are released as a consequence of tissue damage or excessive inflammation. This is likely to have consequences in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To investigate the existence, as well as the concentration, of active FSAP (FSAPa) in complex biological systems an active site probe is needed. We used Hybrid Combinatorial Substrate Library (HyCoSuL) to screen for natural and unnatural amino acids that specifically bind to P4-P2 pockets of FSAPa. This information was used for designing a fluorogenic substrate (Ac-Pro-DTyr-Lys-Arg-ACC) as well as an irreversible, fluorogenic activity-based probe Cy5-6-Ahx-Pro-DTyr-Lys-Arg P (OPh)2. In normal human plasma the probe showed very low non-specific reactivity with some plasma proteins but upon activation of pro-FSAP with histones, strong labelling of FSAPa was observed. This labelling could be inhibited by aprotinin and was not found in the plasma of a subject that was homozygous for a polymorphism, which leads to loss of activity, or in plasma that was depleted of FSAP by antibodies. This 2 nd generation substrate exhibited 6-fold higher catalytic efficiency than the 1 st generation substrate and a much higher selectivity for FSAPa over other plasma proteases. This substrate and probe can be useful to detect and localize FSAPa in normal and pathological tissue and plasma to gain more insight into its functions.

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

“…The results obtained with the SPD-isoforms in this study largely match the earlier observations made with plasma-derived proteins 9,27 as well as proteins obtained from mammalian expression system 7,18 . During the course of the preparation of this manuscript we have been able to develop a novel substrate, an active-site probe as well as a specific and selective inhibitor for FSAP 29 . Similarly, we also found that this preparation of FSAP can recapitulate the effects of plasma FSAP on gene expression in cells 30 .…”

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

“…Effect of NNKC9/41 on mouse plasma: Mouse FSAP differs in fours amino acids (29)(30)(31)(32)(33) from human FSAP in the MA-FSAP-38C7 binding sequence in the NTR (Fig. 3C).…”

Coumarin as a structural component of substrates and probes for serine and cysteine proteases

“…Rut et al obtained a synthetic FSAP substrate by means of a HyCoSuL with arginine fixed in P1 position and 19 natural and 108 unnatural amino acids in P2-P4 position. ACC was used as fluorophore [66]. Excitation was conducted at 320 nm and the emission was measured at 460 nm.…”

“…The probe was assembled through the introduction of the linker-connected biotin or Cy5 substructure at the N-terminus of the peptide. A nucleophilic attack of the active-site serine at the warhead's phosphorous leads to irreversible inhibition and labeling of the target protease [66]. This prototypical stepwise approach towards an ABP starting from the identification of a preferred amino acid sequence has frequently been demonstrated.…”

“…Exemplary illustration of a protease-catalyzed cleavage of an ACC-based peptidic substrate. Ac-Pro-D-Tyr-Lys-Arg-ACC was identified by employing a PS-SCL [66].…”