Plasma Proteome Profiling: Widespread Oxidative Changes in Endotoxemia Correlate with Intravascular Tissue Factor Response.
Authors: Kasthuri Raj S. , Hysjulien Jessica L. , Norton Laura E. , Rogers Tyson , Jilma Bernd , Key Nigel S. , Nelsestuen Gary L. 
Published: April 2020

Abstract
The initial response to systemic infection in sepsis syndromes is mediated by the innate immune system and results in widespread activation of inflammation and coagulation. Some of these changes are recapitulated in the human Endotoxemia model. Proteomics represents a novel approach to study the pathophysiology of disease processes, and offers the potential for identification of biomarkers that may be used for diagnostic and/or prognostic purposes. Therefore, plasma proteome profiling was performed to study the response to intravenously administered low-dose lipopolysaccharide (LPS; 2ng/kg) in 28 healthy adult male volunteers. Interleukin 6 (IL 6), which mediates the host inflammatory responses in endotoxemia and sepsis, and circulating tissue factor (TF), which initiates coagulation, were also measured as markers of these respective processes. Plasma was obtained at baseline and at 1, 2, 3, 4, 8, and 24 hours following LPS administration. Protein profiling was performed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. All individuals showed qualitative profile changes that peaked at 8 hours after LPS and reverted towards baseline at 24 hours. The spectra obtained consisted of 15 non-redundant peaks that were seen consistently in all samples, and ratios of these peaks were used in data analysis. These peaks consisted of Apolipoproteins CI, CII and CIII, and Transthyretin (TTr) and their various isoforms. Serum Amyloid A appeared at later time points following endotoxin. Six of the 28 subjects demonstrated severe oxidation of plasma proteins detected by the loss of free sulfhydryls of TTr and addition of oxygen to many proteins. Increased proteolysis of plasma proteins was also noted in these subjects, along with the appearance of new protein peaks. These 6 individuals were designated ‘high responders’. Microparticle (MP)-associated TF procoagulant activity (PCA) was measured using a recently described assay [Blood 2004 103: 4545]. The TF PCA peaked between 3-4 hours following endotoxin challenge with a 10 fold increase, on average, compared to baseline (p &lt; 0.001). The mean increase in MP-associated TF PCA at 4 hours after LPS was significantly higher among those subjects classified as high responders by protein profiling (p &lt; 0.05). IL 6 levels, measured in 24 of the 28 subjects, peaked at 2–3 hours following LPS with a mean increase &gt; 175 fold compared to baseline (p &lt; 0.01). This study demonstrates significant increases in the MP-associated TF PCA and IL 6 levels in all subjects. The increase in TF PCA was significantly higher among those with extreme changes in their proteome profile despite the small number of individuals in this category. The magnitude of changes observed among the high responder subset supports the hypothesis that some individuals have a predisposition to increased responsiveness to endotoxin. This response may reflect either a protective effect or an increased susceptibility. Further, based on the findings of this study, this phenomenon can be rapidly determined by a novel technique using a proteomics approach. This approach offers the potential to assess early events that may precede sepsis and warrants further exploration in the clinical setting.