Analysis of the substrate specificity of Factor VII activating protease (FSAP) and design of specific and sensitive peptide substrates

Thromb Haemost. 2017 Aug 30;117(9):1750-1760. doi: 10.1160/TH17-02-0081. Epub 2017 Jul 20.


Factor VII (FVII) activating protease (FSAP) is a circulating serine protease that is likely to be involved in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To date, no systematic information is available about the substrate specificity of FSAP. Applying phage display and positional scanning substrate combinatorial library (PS-SCL) approaches we have characterised the specificity of FSAP towards small peptides. Results were evaluated in the context of known protein substrates as well as molecular modelling of the peptides in the active site of FSAP. The representative FSAP-cleaved sequence obtained from the phage display method was Val-Leu-Lys-Arg-Ser (P4-P1'). The sequence X-Lys/Arg-Nle-Lys/Arg (P4-P1) was derived from the PS-SCL method. These results show a predilection for cleavage at a cluster of basic amino acids on the nonprime side. Quenched fluorescent substrate (Ala-Lys-Nle-Arg-AMC) (amino methyl coumarin) and (Ala-Leu-Lys-Arg-AMC) had a higher selectivity for FSAP compared to other proteases from the hemostasis system. These substrates could be used to measure FSAP activity in a complex biological system such as plasma. In histone-treated plasma there was a specific activation of pro-FSAP as validated by the use of an FSAP inhibitory antibody, corn trypsin inhibitor to inhibit Factor XIIa and hirudin to inhibit thrombin, which may account for some of the haemostasis-related effects of histones. These results will aid the development of further selective FSAP activity probes as well as specific inhibitors that will help to increase the understanding of the functions of FSAP in vivo.

Keywords: Serine protease; library; peptide substrates; phage display.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antithrombins / pharmacology
  • Catalytic Domain
  • Cell Surface Display Techniques
  • Enzyme Activation
  • Hirudins / pharmacology
  • Histones / metabolism
  • Humans
  • Kinetics
  • Molecular Docking Simulation
  • Peptide Library
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism*
  • Plant Proteins / chemistry
  • Plant Proteins / pharmacology
  • Protein Binding
  • Protein Conformation
  • Serine Endopeptidases / blood
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism*
  • Serine Proteinase Inhibitors / chemistry
  • Serine Proteinase Inhibitors / pharmacology
  • Structure-Activity Relationship
  • Substrate Specificity
  • Thrombin / antagonists & inhibitors
  • Thrombin / metabolism


  • Antithrombins
  • Hirudins
  • Histones
  • Peptide Library
  • Peptides
  • Plant Proteins
  • Serine Proteinase Inhibitors
  • trypsin inhibitor, Zea mays
  • HABP2 protein, human
  • Serine Endopeptidases
  • Thrombin