The Preparation of Fluorescence-Quenched Probes for Use in the Characterization of Human Factor Xa Substrate Binding Domains

Bromfield, Karen M.; Cianci, Julia; Duggan, Peter J.

doi:10.3390/90600427

Cited by 6 publications

(2 citation statements)

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“…The active site concentration of the enzymes was determined by titration against antithrombin of known concentration. The synthesis, purification and characterization of all peptide substrates was undertaken as previously described (15). The fluorescence‐quenched substrate library used to determine the specificity of fXa had the following sequence: Abz‐Ile‐Glu‐Gly‐Arg‐P1′‐P2′‐P3′‐Ser‐Lys(Dnp)‐AspOH, where alanine was used as the default amino acid at the prime side positions not under investigation.…”

Section: Methodsmentioning

confidence: 99%

“…A stock solution of all fluorescence‐quenched substrates was prepared in dimethylformamide (DMF) and the concentration determined using a spectrophotometer and an extinction coefficient of 10 4 / m /cm at 360 nm (16). The synthesis of the β ‐homoarginine containing fXa ( 1c ) and thrombin ( 2c ) sequences was also carried out as described previously (15), but the fluorophore (2‐aminobenzoyl, Abz) and quencher [Lys(Dnp)] were omitted, and Fmoc‐ l ‐ β ‐Homoarg(Pmc)OH (supplied by Sigma Aldrich, Castle Hill, NSW, Australia) was utilized for coupling at the P1 position. These two sequences were also obtained from Auspep (Parkville, Vic, Australia) as the equivalent α ‐peptides ( 1b and 2b ), and these four peptides were prepared as stock solutions in 10–20% DMF/H 2 O.…”

Section: Methodsmentioning

confidence: 99%

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Approaches to Selective Peptidic Inhibitors of Factor Xa

et al. 2006

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Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro-and anticoagulant roles and thus fXa is thought to represent an ideal target. Discrete k cat and K m values for cleavage of a library of fluorescence-quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa-specific peptidic inhibitors. We hypothesized that K m rather than k cat /K m values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence-quenched substrates, standard a-peptides and peptides containing a b-homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence-quenched substrates to be the best inhibitors, while the inhibitory effect of the b-homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the b-peptides than the corresponding a-peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a b-amino acid at the cleavage site.Key words: b-peptide, factor Xa, fluorescence-quenched substrates, protease inhibitor, protease specificity, serine protease Factor Xa (fXa) and thrombin are mammalian trypsin-type serine proteases that have a central role in the blood coagulation cascade. Factor Xa, together with phospholipids and factor Va, forms the prothrombinase complex, which activates thrombin by cleaving prothrombin at two positions (1). Thrombin has a complex role in blood coagulation, behaving as both a procoagulant and anticoagulant, as well as having an effect on platelet function (2). Factor Xa has a more specific physiological role and therefore, fXa-specific inhibitors are expected to be promising candidates as anticoagulation agents (3). A number of highly specific small-molecule fXa inhibitors have been reported (3-5) which bind in the non-prime subsites 1 of the enzyme. The tick anticoagulant peptide from Ornithodoros moubata is also a highly potent inhibitor of fXa and binds to both an exosite and the active site of the enzyme (6). Our main interest in this area involves an examination of the binding properties of the prime subsites of human fXa and the use of this information in the development of inhibitors that span the enzyme's cleavage site. The initial goal was to produce peptidic inhibitors, which might provide leads for the development of non-peptidic inhibitors.Recent studies on bovine and human fXa prime subsites reveal that the enzyme is non-specific in its preferences for peptide substrates on the prime side of the cleavage site, especially when compared with thrombin, which is highly specific (8,9). As a consequence, it appears that the design of specific, high affinity peptide-based inhibitors of fXa that span the...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Approaches to Selective Peptidic Inhibitors of Factor Xa

et al. 2006

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FRET‐based and other fluorescent proteinase probes

Gehrig

Reither

et al. 2014

Biotechnology Journal

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The continuous detection of enzyme activities and their application in medical diagnostics is one of the challenges in the translational sciences. Proteinases represent one of the largest groups of enzymes in the human genome and many diseases are based on malfunctions of proteolytic activity. Fluorescent sensors may shed light on regular and irregular proteinase activity in vitro and in vivo and provide a deeper insight into the function of these enzymes and their role in pathophysiological processes. The focus of this review is on Förster resonance energy transfer (FRET)-based proteinase sensors and reporters because these probes are most likely to provide quantitative data. The medical relevance of proteinases are discussed using lung diseases as a prominent example. Probe design and probe targeting are described and fluorescent probe development for disease-relevant proteinases, including matrix-metalloproteinases, cathepsins, caspases, and other selected proteinases, is reviewed.

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