Design, synthesis, and characterization of chromogenic substrates of coagulation factor XIIIa

Hardes, Kornelia; Becker, Gero L.; Hammamy, M. Zouhir; Steinmetzer, Torsten

doi:10.1016/j.ab.2012.05.023

Cited by 6 publications

(5 citation statements)

References 38 publications

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“…This finding indicates a one‐step mechanism for inactivation of both guinea pig and human TGase 2 by iodoacetamide and suggests that no stable noncovalent enzyme ⋅ inhibitor complex is formed prior to the inactivating alkylation step. This result is in agreement with observations for the inactivation of factor XIIIa by iodoacetate and can be explained by considering that this small‐molecule inhibitor does not provide many contact points for noncovalent interactions.…”

Section: Resultssupporting

confidence: 92%

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

et al. 2016

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Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

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Section: Resultssupporting

confidence: 92%

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

et al. 2016

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“…The activation of FXIII (Fibrogammin, CSL Behring Marburg, Germany) and the chromogenic measurements with the substrate H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-Gly-NH 2 were performed as described previously. 21,28 The fluorogenic assays were done with the newly developed FRET substrates H-Tyr(3-NO 2 )-Glu(NH-(CH 2 ) 4 -NH-Abz)-Val-Lys-Val-Ile-NH 2 (15) and H-Tyr(3-NO 2 )-Glu(NH-(CH 2 ) 4 -NH-Abz)-Val-Lys-Val-Ile-GlyNH 2 (14). 16 Fluorescence readings were performed in black 96 well plates (Nunc, Langenselbold, Germany) at room temperature using a Fluoroskan Ascent plate reader (Thermo Fisher Scientific, Vantaa, Finland) with λ ex = 320 and λ em = 405 nm.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

“…The measurements of enzyme activity were performed with chromogenic and fluorogenic substrates. The activation of FXIII (Fibrogammin, CSL Behring Marburg, Germany) and the chromogenic measurements with the substrate H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-Gly-NH 2 were performed as described previously. , …”

Section: Experimental Sectionmentioning

confidence: 99%

Novel Insights into Structure and Function of Factor XIIIa-Inhibitor Tridegin

et al. 2014

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The inhibition of the final step in blood coagulation, the factor XIIIa (FXIIIa) catalyzed cross-linking of fibrin monomers, is currently still a challenge in medicinal chemistry. We report synthesis, recombinant expression, disulfide connectivity, and biological activity of tridegin, the sole existing peptide representative displaying inhibitory activity on FXIIIa. Inhibition of the enzyme by this 66-mer cysteine-rich peptide is mediated by its C-terminal sequence, while the N-terminal part comprises structural information and contributes to inhibitor binding. Either of the production strategies examined leads to the formation of different disulfide-bridged isomers indicating the requirement of the correct fold for inhibitory activity. Molecular modeling and docking studies confirm disulfide bond isomer preference with respect to binding to FXIIIa, in turn, the knowledge of the enzyme−inhibitor interactions might bring about comprehensive ideas for the design of a suitable lead structure for addressing FXIIIa.

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“…Again the glutamine-containing reaction is not being monitored directly. An additional method is to work with synthetic peptides in which the reactive glutamine is replaced with colorimetric Glu-pNA or fluorimetric Glu-AMC [23; 24]. With such reporter groups, the direct release of p-nitroaniline (pNA) or amidomethylcoumarin (AMC) can finally be monitored and is a real strength of this assay design.…”

Section: Introductory Statementmentioning

confidence: 99%

“…The individual transglutaminase reactions are quenched at distinct time points and MALDI-TOF mass spectra are collected to monitor for loss of Q-substrate and gain of the cross-linked product (Q-substrate)-GEE (gain of 86 m/z). Overall, the MALDI-TOF MS kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on only detecting the final deacylation portions of the transglutaminase reaction [12; 22; 23]. …”

Section: Introductory Statementmentioning

confidence: 99%

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

Doiphode

Malovichko

Mouapi

et al. 2014

Analytical Biochemistry

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Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.

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Design, synthesis, and characterization of chromogenic substrates of coagulation factor XIIIa

Cited by 6 publications

References 38 publications

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2

Novel Insights into Structure and Function of Factor XIIIa-Inhibitor Tridegin

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

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