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      Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

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          Abstract

          Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca 2+-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only in vitro, i.e., for serum or plasma stability assays, but of lower importance in vivo.

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          Most cited references30

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          Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors

          The MEROPS database (http://merops.sanger.ac.uk) is an integrated source of information about peptidases, their substrates and inhibitors, which are of great relevance to biology, medicine and biotechnology. The hierarchical classification of the database is as follows: homologous sets of sequences are grouped into a protein species; protein species are grouped into a family; families are grouped into clans. There is a type example for each protein species (known as a ‘holotype’), family and clan, and each protein species, family and clan has its own unique identifier. Pages to show the involvement of peptidases and peptidase inhibitors in biological pathways have been created. Each page shows the peptidases and peptidase inhibitors involved in the pathway, along with the known substrate cleavages and peptidase-inhibitor interactions, and a link to the KEGG database of biological pathways. Links have also been established with the IUPHAR Guide to Pharmacology. A new service has been set up to allow the submission of identified substrate cleavages so that conservation of the cleavage site can be assessed. This should help establish whether or not a cleavage site is physiologically relevant on the basis that such a cleavage site is likely to be conserved.
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            Overview of the coagulation system

            Coagulation is a dynamic process and the understanding of the blood coagulation system has evolved over the recent years in anaesthetic practice. Although the traditional classification of the coagulation system into extrinsic and intrinsic pathway is still valid, the newer insights into coagulation provide more authentic description of the same. Normal coagulation pathway represents a balance between the pro coagulant pathway that is responsible for clot formation and the mechanisms that inhibit the same beyond the injury site. Imbalance of the coagulation system may occur in the perioperative period or during critical illness, which may be secondary to numerous factors leading to a tendency of either thrombosis or bleeding. A systematic search of literature on PubMed with MeSH terms ‘coagulation system, haemostasis and anaesthesia revealed twenty eight related clinical trials and review articles in last 10 years. Since the balance of the coagulation system may tilt towards bleeding and thrombosis in many situations, it is mandatory for the clinicians to understand physiologic basis of haemostasis in order to diagnose and manage the abnormalities of the coagulation process and to interpret the diagnostic tests done for the same.
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              Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.

              Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum. In the majority of cases, fusion proteins are soluble in aqueous solutions and can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilised glutathione. Using batch wash procedures several fusion proteins can be purified in parallel in under 2 h with yields of up to 15 micrograms protein/ml of culture. The vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin or blood coagulation factor Xa, following which, the carrier and any uncleaved fusion protein can be removed by absorption on glutathione-agarose. This system has been used successfully for the expression and purification of more than 30 different eukaryotic polypeptides.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2 June 2017
                2017
                : 12
                : 6
                : e0178943
                Affiliations
                [1 ]Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig, Germany
                [2 ]Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany
                Institut d'Investigacions Biomediques de Barcelona, SPAIN
                Author notes

                Competing Interests: RH is a cofounder of AMP Therapeutics (Leipzig, Germany) and a member of its scientific advisory board. D.K. was a part-time coworker of AMPT. R.B. has no conflict of interest to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                • Conceptualization: DK RB.

                • Data curation: RB DK.

                • Formal analysis: RB DK.

                • Funding acquisition: RH.

                • Investigation: RB DK.

                • Methodology: DK RB RH.

                • Project administration: RH DK.

                • Resources: RH.

                • Supervision: RH.

                • Validation: DK.

                • Visualization: RB DK.

                • Writing – original draft: DK.

                • Writing – review & editing: RH.

                Author information
                http://orcid.org/0000-0002-4661-8979
                Article
                PONE-D-17-05490
                10.1371/journal.pone.0178943
                5456363
                28575099
                ead2f022-8e7d-48dc-afc7-6f183481ad72
                © 2017 Böttger et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 10 February 2017
                : 22 May 2017
                Page count
                Figures: 4, Tables: 3, Pages: 15
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100002347, Bundesministerium für Bildung und Forschung;
                Award ID: 01GU1104A
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: Open Access Publishing
                Award Recipient :
                Funded by: Universität Leipzig
                Award ID: Ph.D. stipend
                Award Recipient :
                This work was supported by the Federal Ministry of Education and Research (BMBF, grant number 01GU1104A to RH) and a PhD-stipend provided by Universität Leipzig to RB are gratefully acknowledged. We acknowledge support from the German Research Foundation (DFG) and Universität Leipzig within the program of Open Access Publishing. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
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                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Biology and Life Sciences
                Physiology
                Body Fluids
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                Medicine and Health Sciences
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                Biology and Life Sciences
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                Peptides
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                Anatomy
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                Body Fluids
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                Heparin
                Biology and Life Sciences
                Biochemistry
                Proteins
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                Medicine and Health Sciences
                Pharmacology
                Pharmacologic Analysis
                Pharmacokinetic Analysis
                Elimination Half-Life Calculation
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