Fast large-scale plasma proteomic profiling in post–cardiac arrest care

Abstract

Post–cardiac arrest syndrome represents a systemic response to ischemia–reperfusion injury and is a key determinant of outcome. While post-resuscitation care relies on manipulating fundamental physiological variables such as oxygen tension and arterial pressure, the biological effects of these interventions and the broader molecular determinants of outcome remain poorly defined. We therefore applied large-scale plasma proteomics in a randomized trial of comatose patients resuscitated after out-of-hospital cardiac arrest (OHCA) to examine associations with treatment allocation and 1-year mortality.

Methods

This post-hoc exploratory study of the BOX trial (Kjaergaard et al. NEJM 2022 and Schmidt et al. NEJM, 2022) in comatose adults resuscitated from OHCA, randomized in a 2×2 factorial design to MAP targets of 63 vs 77 mmHg and to restrictive (PaO₂ 9–10 kPa) vs liberal (PaO₂ 13–14 kPa) oxygen therapy. Blood was sampled at 48 hours and plasma stored at −80°C. Mortality was assessed at 1 year. Plasma proteomics was performed using high-throughput data-independent acquisition mass spectrometry (Evosep platform) and DIA-NN for raw data processing. Of 789 patients, 628 had a 48-hour sample and 603 were included after quality control. In total, 505 proteins were quantified, of which 224-227 met completeness criteria for differential analysis. Log-transformed intensities were analysed using linear models. Functional enrichment was performed using Gene Ontology and Reactome pathways.

Results

Baseline characteristics were balanced between intervention groups. Median age was 63 years (IQR 53–72), 19% were women, median time to return of spontaneous circulation was 18 min. (IQR 12–25), median left ventricular ejection fraction 35% (IQR 25–45), and 90% underwent acute coronary angiography.

At 1 year, 199/603 (33%) patients had died. Differential abundance analysis identified 58 proteins significantly associated with 1-year mortality after FDR correction (76 nominal, P<0.05) (Figure 1). Enrichment analysis of mortality-associated proteins demonstrated overrepresentation of innate immune/acute-phase and hemostasis pathways. Directionally, non-survivors showed higher abundance of inflammatory and vascular injury markers (e.g., B2M, S100A9, VCAM1), whereas survivors had higher levels of several protective/homeostatic proteins (e.g., GSN, SELENOP, GPX3).

Among 227 analysed proteins, 20 showed nominal differential abundance by the MAP intervention and 15 by the PaO₂ target intervention. After FDR correction, only B2M remained associated with MAP (~20% higher in low MAP; Figure 2), compatible with reduced glomerular filtration.

Conclusion

Large-scale plasma proteomics revealed limited molecular effects of the randomized MAP and oxygen targets but a pronounced association between mortality and inflammatory–vascular pathways. This highlights the dominant role of post–cardiac arrest syndrome biology in determining long-term outcome.Figure 1Figure 2