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Proteolysis in Septic Shock Patients: Plasma Peptidomic Patterns Are Associated with Mortality J British Journal of Anaesthesia, ▪ (▪): 1e10 (2018) doi: 10.1016/j.bja.2018.05.072 Advance Access Publication Date: xxx Clinical Investigation CLINICAL INVESTIGATION Proteolysis in septic shock patients: plasma peptidomic patterns are associated with mortality J. Bauza-Martinez 1,#, F. Aletti2,#, B. B. Pinto3, V. Ribas4, M. A. Odena1, R. Dı´az1, E. R. Medina5, R. Ferrer6, E. B. Kistler7, G. Tedeschi8,9, G. W. Schmid-Schonbein€ 2, A. Herpain10, K. Bendjelid3,* and E. de Oliveira1,* 1Proteomics Platform, Barcelona Science Park, Barcelona, Spain, 2Department of Bioengineering, University of California San Diego, La Jolla, CA, USA, 3Geneva University Hospital, Geneva, Switzerland, 4Eurecat, Technology Centre of Catalonia, Barcelona, Spain, 5University Hospital Mu´ tua Terrassa, Barcelona, Spain, 6Intensive Care Department, Vall d’Hebron University Hospital, Barcelona, Spain, 7Department of Anesthesiology and Critical Care, VA San Diego Healthcare System, San Diego, CA, USA, 8Dipartimento di Medicina Veterinaria, Universita degli Studi di Milano, Milan, Italy, 9Fondazione Filarete Milano, Milan, Italy and 10Universite Libre de Bruxelles, Brussels, Belgium *Corresponding authors. E-mails: [email protected], [email protected] # These authors contributed equally to this study. Abstract Background: Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome. Methods: Intensive care patients with septic shock (n¼29) or sepsis (n¼6) and non-hospitalised subjects (n¼9) were recruited as part of the prospective observational trial ‘ShockOmics’ (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects. Results: Evidence of systemic proteolysis was indicated by a larger number of circulating peptides in septic shock pa- tients, compared with septic patients and non-hospitalised healthy subjects. The peptide count and abundance in the septic shock patients were greater in patients who died (n¼6) than in survivors (n¼23), suggesting an association between magnitude of proteolysis and outcome. In silico analysis of the peptide sequences and of the sites of cleavage on the proteins of origin indicated a predominant role for serine proteases, such as chymotrypsin, and matrix metalloproteases in causing the observed proteolytic degradation. Conclusions: Systemic proteolysis is a novel fundamental pathological mechanism in septic shock. Plasma peptidomics is proposed as a new tool to monitor clinical trajectory in septic shock patients. Clinical trial registration: NCT02141607. Keywords: in-hospital mortality; mass spectrometry; peptidomics; proteolysis; septic shock Editorial decision: 13 June 2018; Accepted: 13 June 2018 © 2018 The Authors. Published by Elsevier Ltd on behalf of British Journal of Anaesthesia. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). For Permissions, please email: [email protected] 1 2 - Bauza-Martinez et al. received more than four units of red blood cells or any fresh 11 Editor’s key points frozen plasma were excluded. Given the stringency of the exclusion criteria, the thresholds on SOFA and lactate were Recent data have suggested that systemic proteolysis chosen to include severe enough septic shock patients, while occurs in sepsis but its clinical relevance is uncertain. ensuring adequate patient enrolment and recruitment rate. In this observational study, circulating concentrations A convenience sampled cohort of patients with sepsis of peptide were increased in patients with septic (infection with inflammatory response and one organ shock compared with patients with sepsis and healthy dysfunction),11,12 but not in shock, was included as control. controls. Healthy donors were enrolled at Mutua Terrassa Hospital, There was an association between increased peptide Terrassa, Spain for an additional non-sepsis control. Informed concentrations and mortality in patients with septic consent was obtained from patients or proxies. Patients in the shock. ICU were managed according to international guidelines.13 Further translational and clinical research into the Patient characteristics, organ function, and haemodynamic possible role of proteolysis in sepsis is needed. data were prospectively collected into a custom-made elec- tronic case report form. In-hospital mortality was assessed by consultation of the local death registry, or by telephone call to the patient or proxies. Sepsis is a life-threatening condition caused by a dysregulated host response to infection.1 Septic shock is the most severe form of sepsis, characterised by circulatory failure and tissue Sample collection injury, and associated with high mortality.2 Despite longstanding efforts to develop new treatments for Blood (total volume 18 ml for the multiomics analyses of the ’ 11 septic shock, interventions are still based on haemodynamic ‘ShockOmics project) was collected from either the arterial or support, antibiotics and anti-infective drugs, and source con- venous line in septic shock and sepsis patients; venous blood trol (e.g. surgery). To date, no therapy addresses the root cause was drawn from healthy donors. For peptidomics, plasma (0.5 of septic shock, largely because of an incomplete under- ml) was isolated within 30 min of sample collection in K2- standing of the cascade leading to multiorgan dysfunction. EDTA tubes (BD Biosciences, San Jose, CA, USA) by centrifu- Uncontrolled proteolysis has recently been proposed as a gation (twice at 1200g for 10 min) to pellet cellular elements. fundamental pathological mechanism in shock, possibly COmplete Protease Inhibitor Cocktail (Roche Applied Sciences, e mediated by digestive enzymes.3 9 Enteral blockade of Mannheim, Germany) was added and samples were stored À pancreatic proteases in animals mitigates organ injury and at 80 C until in-batch analyses. Plasma samples collected e improves outcome.3 9 I.V. serine protease blockade has also from the septic shock patients at three time points were shown some efficacy in severe sepsis.10 Further, we recently analysed: within 16 h of ICU admission (T1); at 48 h (T2) after reported the occurrence of systemwide proteolysis and the ICU admission; and before discharge from the ICU or on Day 7 appearance of a large number of circulating peptides in a rat at latest (T3). Only one sample was collected from the sepsis model of shock.8 patients (at T1) and healthy donors. The objectives of this study are: (i) to investigate proteolysis in septic shock patients compared with septic patients Liquid chromatographyemass spectrometry analysis without shock and healthy donors by assessment of plasma peptide concentrations from mass spectrometry-based pepti- Residual protease activity in the plasma samples was tested by domics, and by in silico analysis of proteolytic activity; and (ii) a fluorometric assay (Supplementary material S.1). Peptides to determine the association between proteolysis and in- were extracted by filtration (Supplementary material S.2), hospital mortality in septic shock. separated by reverse phase liquid chromatography, and ana- lysed by mass spectrometry (Supplementary material S.3)in the Orbitrap (Thermo Fisher Scientific, Bremen, Germany). Methods Study design and participants Peptide and protein identification: qualitative This study is part of the prospective observational trial approach ‘ShockOmics’ (ClinicalTrials.gov Identifier NCT02141607)11 and was approved by: the Geneva Regional Research Ethics Com- Proteins and peptides were identified (Supplementary mittee (study number 14-041); the Ethical committee of material S.4) using Mascot search engine (v2.3.01, Matrix Sci- Hopital^ Erasme-Universite Libre De Bruxelles (study number ence, Boston, MA, USA) against the SwissProt Human (SPH) P2014/171), and the Mutua Terrassa Hospital Institutional Re- database (v160127). Venn diagrams of peptide counts were 14 view Board (study number EO/1407). generated using R packages. Adult (>18 yr old) patients admitted between October 2014 and March 2016 to the ICU of Geneva University Hospitals, Peptide and protein quantification: label-free Geneva, Switzerland (38-bed, mixed) and Erasme University approach Hospital, Brussels, Belgium (36-bed, mixed) with septic shock12 diagnosis, Sequential Organ Failure Assessment (SOFA) score Raw data were processed with Progenesis QI for Proteomics À 6, arterial lactate 2 mmol L 1 and a documented source of (Non-Linear Dynamics, Waters, Milford, MA, USA). Pool sam- infection were screened for enrolment. Patients at high risk of ples were used as alignment reference. A total of 219 825 mass death within the first 24 h after admission, systemic immu- spectra (z>1 and Rank<5) were considered for database search. nosuppression, haematological diseases, metastatic cancer, Mascot search engine was used against SPH (Supplementary pre-existing dialysis, decompensated cirrhosis, or who material S.4). Proteolysis in septic shock and mortality - 3 y Table 1 Clinical parameters. *Mean (SD) and n (%). Blood samples were drawn at three different time points. T1: within 16 h of ICU admission; T2: at 48 h after ICU admission; T3: before discharge from the ICU or on Day 7 at latest. SS, septic shock SS non-Survivors SS survivors Sepsis (n¼6) Healthy controls (n¼6) (n¼23) (n¼9)
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