Plasma Proteomic Profiling Illustrates Endothelial and Complement Signature in Patients with Alcohol-Associated Cirrhosis

INTRODUCTION

Cirrhosis related coagulation derangements result in altered hemostasis. Endothelial markers such as von Willebrand factor (VWF) antigen and the factor VIII-to-protein C ratio (FVIII/PC) have prognostic significance in patients with cirrhosis. Additional endothelial markers have not been well described in patients with alcohol-associated cirrhosis. We performed plasma proteomic profiling in patients with alcohol-associated cirrhosis to gain insight into the potential roles of endothelial and hemostatic proteins associated with severity of cirrhosis.

METHODS

Two independent cohorts of patients with Child-Pugh class A (CPA) and C (CPC) alcohol-associated cirrhosis were recruited. Controls were patients without underlying liver disease. For plasma proteomic profiling, plasma supernatant was frozen, then sent to Eve Technologies (Calgary, Alberta, Canada). Linear discrimination analysis (LDA) was applied to the first cohort to identify a set of proteins that clustered patients based on the severity of liver disease (CPA vs CPC) and controls. Identified proteins were then validated using a second independent cohort of patients with alcohol-associated cirrhosis to determine if the protein signature was replicable. Boruta feature selection was performed on cohort 2 to identify candidate proteins to explain the differences in FVIII/PC ratio. A p-value of < 0.05 was considered statistically significant.

RESULTS

Cohort 1 included 13 patients: 4 CPA, 9 CPC (median age was 57; 4 women; 9 had dyslipidemia, 2 diabetes mellitus, 2 hypertension, and 2 chronic kidney disease). Cohort 2 included 12 patients: 7 CPA, 5 CPC (median age 50; 5 women; 7 had dyslipidemia, 1 diabetes mellitus, 1 hypertension and 0 chronic kidney disease). For cohort 1 LDA clustered patients based on CPA vs CPC with an endothelial, inflammatory and complement signature (fig.1). The proteins set that achieved robust LDA clustering of CPA, CPC, and control patients in cohort 1 included: sVCAM-1 and PAI-1 (endothelial markers); C3, C4, and CFH (complement cascade factors); IL-8 (inflammatory chemokine), NCAM (neural adhesion maker) and BDNF (neurotrophin); MPO (marker of neutrophilic activation); Eotaxin (eosinophil associated chemokine); MIP-1d (activator of T-cells and monocytes). The same protein profile applied to the validation cohort 2 was also able to cluster CPA, CPC, and control patients (fig. 2). Boruta feature selection for proteins of importance for the FVIII/PC ratio included sVCAM-1; CFI, C4b, C4, C5, CFB (complement factors); IL-6 (inflammatory cytokine), and VWF.

CONCLUSIONS

Using plasma proteomic profiling we describe a signature of endothelial, complement, hemostatic, and inflammatory activation that distinguishes the severity of liver disease in patients with alcohol-associated cirrhosis. Exploratory analyses suggest that endothelial and complement proteins may also contribute to the differences in the FVIII/PC ratio which has prognostic significance in patients with cirrhosis. Future work should continue to highlight the role of endothelium in the progression of liver disease and describe alternative prognostic markers to help guide clinicians.