Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

The characterisation of protein binding from in vivo paediatric ECMO circuits (#108)

Tengyi Cai 1 2 , Samantha Emery 3 , Rebecca Barton 1 2 4 , Suelyn Van Den Helm 1 2 , Natasha Letunica 1 , Chantal Attard 1 2 , Joppe Drop 1 2 , Simon Collett 2 , Stephen Horton 2 5 , Steve Bottrell 5 , Bradley Schultz 5 , Graeme MacLaren 1 6 7 , Roberto Chiletti 6 8 , Derek Best 6 8 , Fiona Newall 1 2 4 , Yves d’Udekem 1 2 5 , Paul Monagle 1 2 4 , Vera Ignjatovic 2 9 10
  1. Haemotology Research, Murdoch Children Research Institute, Parkville, VIC, Australia
  2. Paediatrics, The University of Melbourne, Parkville, Vic, Australia
  3. Advanced Technology and Biology, Parkville, VIC, Australia
  4. Clinical Haematology, The Royal Children’s Hospital, Parkville, VIC, Australia
  5. Cardiac Surgery, The Royal Children’s Hospital, Parkville, VIC, Australia
  6. Intensive Care, The Royal Children’s Hospital, Parkville, VIC, Australia
  7. Cardiothoracic Intensive Care Unit, National University Health System, Singapore
  8. Paediatric Intensive Care Research Group, Murdoch Children Research Institute, Parkville, VIC, Australia
  9. Johns Hopkins All Children’s Institute for Clinical and Translational Research, , St. Petersburg, Florida, United States
  10. Pediatrics , School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States

Background:

Extracorporeal Membrane Oxygenation (ECMO) is an artificial heart-lung machine for critically ill patients with severe respiratory or cardiac failure (1). Despite providing critical support, there is an increased risk of bleeding and clotting due to the continuous contact between blood and circuit (2). Whilst previous studies have extensively investigated blood samples from patients on ECMO (3, 4), protein and cellular adsorption to ECMO circuit, as a factor that could potentially contribute to clinical complications, has largely been overlooked.

Aims:

To characterise the proteins bound to ECMO circuits collected from children on ECMO.

Methods:

This study was approved by the Royal Children's Hospital ethics committee (HREC/15/RCHM/123). ECMO circuits were collected when removed from patients. For each circuit, samples from 5 different sites were collected (Figure 1). Protein quantification and characterisation was performed using bicinchoninic acid protein assay and Data-independent acquisition Mass Spectrometry (DIA-MS), respectively. Identified proteins were processed using the Reactome Over-representation Pathway Analyses tool (5) to determine corresponding functional pathways.

636c7953b13a6-Figure+1.+The+basic+diagram+of+ECMO+circuit..jpg

Results:

Seven ECMO circuits were collected from six paediatric patients, with Patient 1 requiring a second run of ECMO support (Named as Patient 1.1 and 1.2).

The median protein concentration of all samples (n=35) was 16.3ug/mL (Range: 0~76.9ug/mL). In site-matched patient comparisons, the protein concentrations of Patient 4 were significantly higher than that of Patient 5, while in patient-matched site comparisons, site 1 samples were significantly higher than site 2, 3 and 5 samples.

Samples with at least 20ug protein (n=19) were analysed by DIA-MS. The number of proteins identified within each sample varied, with a median of 2138 (Range: 913~3073). Specifically, albumin, haemoglobin, apolipoprotein E, apolipoprotein A-I, fibrinogen, and band 3 anion transport protein were the most abundant proteins identified in most samples. Hierarchical clustering and principal component analysis showed samples from the same ECMO circuit clustered together (Patient 1.1, 1.2, 3 and 6), except in Patient 4 where samples showed significant differences between sites (Figure 2). The pathway analysis demonstrated that identified proteins were enriched in pathways related to cell cycle, protein metabolism, coagulation, and inflammation.

636c7953b13a6-Figure+2.+The+unsupervised+hierarchical+clustering+and+Principal+component+analysis+of+circuit+samples.jpg

Conclusions:

This is the first study to characterise the proteins bound to ECMO circuits collected from paediatric patients utilising proteomic approaches. Composition of bound proteins was heterogeneous between different patients and between different sites. The bound proteins were enriched in pathways of coagulation and inflammation, which may be associated with clinical outcomes and provide a potential area for further interventions.

  1. Brogan TV, Lequier L, Lorusso R, MacLaren G, Peek G. Extracorporeal life support: the ELSO red book (5th Edition): Extracorporeal Life Support Organization (ELSO); 2017.
  2. Dalton HJ, Reeder R, Garcia-Filion P, Holubkov R, Berg RA, Zuppa A, et al. Factors Associated with Bleeding and Thrombosis in Children Receiving Extracorporeal Membrane Oxygenation. Am J Respir Crit Care Med. 2017;196(6):762-71.
  3. Caprarola SD, Ng DK, Carroll MK, Tekes A, Felling RJ, Salorio CF, et al. Pediatric ECMO: unfavorable outcomes are associated with inflammation and endothelial activation. Pediatr Res. 2022;92(2):549-56.
  4. Van Den Helm S, Yaw HP, Letunica N, Barton R, Weaver A, Newall F, et al. Platelet Phenotype and Function Changes With Increasing Duration of Extracorporeal Membrane Oxygenation. Crit Care Med. 2022;50(8):1236-45.
  5. Gillespie M, Jassal B, Stephan R, Milacic M, Rothfels K, Senff-Ribeiro A, et al. The reactome pathway knowledgebase 2022. Nucleic Acids Research. 2022;50(D1):D687-D92.