Thromb Haemost 2017; 117(09): 1750-1760
DOI: 10.1160/TH17-02-0081
Coagulation and Fibrinolysis
Schattauer GmbH

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

Emrah Kara
1   Oslo University Hospital and University of Oslo, Norway
,
Dipankar Manna
1   Oslo University Hospital and University of Oslo, Norway
,
Åge Geir Løset
2   Centre for Immune Regulation and Department of Biosciences, University of Oslo, Norway
,
Eric L. Schneider
3   University of California, San Francisco, USA
,
Charles S. Craik
3   University of California, San Francisco, USA
,
Sandip M. Kanse
1   Oslo University Hospital and University of Oslo, Norway
› Author Affiliations

This study was supported by the Helse Sør-Øst and the National Research Council of Norway. CSC and ELS were supported by NIHP41 CA196276.
Further Information

Publication History

Received 02 May 2017

Accepted after major revision: 11 June 2017

Publication Date:
28 November 2017 (online)

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Summary

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 histonetreated 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.

Supplementary Material to this article is available online at www.thrombosis-online.com.