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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 to 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-ArgP(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 2nd generation substrate exhibited 6-fold higher catalytic efficiency than the 1st 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.

journal_name

Thromb Res

journal_title

Thrombosis research

authors

Rut W,Nielsen NV,Czarna J,Poreba M,Kanse SM,Drag M

doi

10.1016/j.thromres.2019.08.016

subject

Has Abstract

pub_date

2019-10-01 00:00:00

pages

124-132

eissn

0049-3848

issn

1879-2472

pii

S0049-3848(19)30346-9

journal_volume

182

pub_type

杂志文章

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