135355 20260219090934.0 doi 10.1021/acsptsci.4c00065 sideral 138627 ART-2024-138627 eng Senan-Salinas, Ana Selective Detection of Active Extracellular Granzyme A by Using a Novel Fluorescent Immunoprobe with Application to Inflammatory Diseases 2024 Access copy available to the general public Unrestricted Granzymes (Gzms), a family of serine proteases, expressed by immune and nonimmune cells, present perforin-dependent and independent intracellular and extracellular functions. When released in the extracellular space, GzmA, with trypsin-like activity, is involved in the pathophysiology of different inflammatory diseases. However, there are no validated specific systems to detect active forms of extracellular GzmA, making it difficult to assess its biological relevance and potential use as a biomarker. Here, we have developed fluorescence-energy resonance-transfer (FRET)-based peptide probes (FAM-peptide-DABCYL) to specifically detect GzmA activity in tissue samples and biological fluids in both mouse and human samples during inflammatory diseases. An initial probe was developed and incubated with GzmA and different proteases like GzmB and others with similar cleavage specificity as GzmA like GzmK, thrombin, trypsin, kallikrein, or plasmin. After measuring fluorescence, the probe showed very good specificity and sensitivity for human and mouse GzmA when compared to GzmB, its closest homologue GzmK, and with thrombin. The specificity of this probe was further refined by incubating the samples in a coated plate with a GzmA-specific antibody before adding the probe. The results show a high specific detection of soluble GzmA even when compared with other soluble proteases with very similar cleavage specificity like thrombin, GzmK, trypsin, kallikrein, or plasmin, which shows nearly no fluorescence signal. The high specific detection of GzmA was validated, showing that using pure proteins and serum and tissue samples from GzmA-deficient mice presented a significant reduction in the signal compared with WT mice. The utility of this system in humans was confirmed, showing that GzmA activity was significantly higher in serum samples from septic patients in comparison with healthy donors. Our results present a new immunoprobe with utility to detect extracellular GzmA activity in different biological fluids, confirming the presence of active forms of the soluble protease in vivo during inflammatory and infectious diseases. info:eu-repo/grantAgreement/ES/AEI/PID2020-113963RB-I00 info:eu-repo/grantAgreement/ES/DGA/B29-23R info:eu-repo/grantAgreement/ES/DGA/LMP139-21 info:eu-repo/grantAgreement/EC/H2020/859908/EU/Novel Applications in 19F Magnetic Resonance Imaging/NOVA-MRI This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 859908-NOVA-MRI info:eu-repo/grantAgreement/ES/MCIU-AEI/SAF2017-83120-C2-1-R info:eu-repo/grantAgreement/ES/MINECO/FJCI-2017-31629 info:eu-repo/grantAgreement/ES/MINECO/IJC-2019-039192-I info:eu-repo/semantics/openAccess by https://creativecommons.org/licenses/by/4.0/deed.es 3.7 2024 1.23 2024 Pharmacology (medical) 2024 Q1 Pharmacology 2024 Q1 7.5 2024 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Comas, Laura Esteban, Patricia Garzón-Tituaña, Marcela (orcid)0000-0001-6778-0636 Cheng, Zhiming Santiago, Llipsy (orcid)0000-0002-1861-5981 Domingo, Maria Pilar Ramírez-Labrada, Ariel (orcid)0000-0002-3888-7036 Paño-Pardo, José Ramón Universidad de Zaragoza (orcid)0000-0002-9600-8116 Vendrell, Marc Pardo, Julián Universidad de Zaragoza (orcid)0000-0003-0154-0730 Arias, Maykel A. (orcid)0000-0002-9730-2210 Galvez, Eva M. Universidad de Zaragoza 1006 255 Universidad de Zaragoza Dpto. Fisiatría y Enfermería Área Enfermería 1007 610 Universidad de Zaragoza Dpto. Medicina, Psiqu. y Derm. Area Medicina 1011 566 Universidad de Zaragoza Dpto. Microb.Ped.Radio.Sal.Pú. Área Inmunología 7, 5 (2024), 1474-1484 ACS pharmacol. transl. sci. ACS pharmacology & translational science 2575-9108 3369282 http://zaguan.unizar.es/record/135355/files/texto_completo.pdf Versión publicada 3171545 http://zaguan.unizar.es/record/135355/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:135355 articulos driver 2026-02-19-09:00:24 ARTICLE