Targeted quantitative proteomics of anticoagulated patients

Oral anticoagulants (OACs) are commonly prescribed medications to treat and prevent thromboembolic complications. The two most common indications are the prevention of ischemic stroke in patients with atrial fibrillation (AF) and the treatment or prevention of venous thromboembolism (VTE). Despite their beneficial antithrombotic effect, OACs treatment also leads to severe adverse events, such as occurrence of major bleeding in around 3% of patients each year. Moreover, OACs are not always effective, as breakthrough strokes or VTE still occur despite treatment. The frequency of these adverse events calls for more research towards the individualization of anticoagulant therapy, through better characterization of the population and understanding of differences in risks of developing adverse events.

While a few proteomics studies in patients on OAC treatment were published recently, such as patients with AF (PMID: 34353540, 34390530) or VTE (PMID: 29296750), the potential of proteomics characterization for these patients is currently underutilized. We used targeted proteomics with internal standards to measure 270 proteins, including 64 in the complement and coagulation cascade, in a prospective case-cohort from BLEEDS (PMID: 27935941), which enrolled 16,570 patients initiating VKAs between 2012-2014. The case-cohort included 370 random subcohort members, 250 major bleeding, and 59 stroke cases occurring during follow-up. Plasma concentrations of 270 proteins were measured at baseline (i.e., before the bleeding) using quantitative targeted proteomics with internal standards. To investigate subgroups differences, protein concentrations in the subcohort were compared by age, sex, treatment indication, comorbidities and comedications with Wilcoxon rank-sum test (p-value≤0.05 adjusted for false discovery rate). 139 proteins passed all our quantification criteria. The measurements were not influenced by patients" characteristics or occurrence of clinical outcomes. Proteomic profiles were age- and sex-specific in the 370 subcohort patients. Older patients had higher levels of alpha-2-macroglobulin, fibulin and complement factors. VTE patients had elevated levels of apolipoprotein C-I, B-100, L-1 and A-II compared with AF patients. Patients with pulmonary embolism had a similar profile compared to deep vein thrombosis patients. Neuropilin-2, insulin-like growth factor-binding protein 2, protein AMBP, β-2-microglobulin and leucine-rich α-2-glycoprotein were, among others, ≥15% increased in the major bleeding patients compared to the subcohort.

Our results show how precise proteomic characterization of patient plasma samples on anticoagulation treatment can identify novel potential biomarkers for VTE and major bleeding. The attractiveness of using targeted assays with internal standard is due to the ability of faster adaptation of assays in clinical settings after required validation.