Label-free comparative proteomic analysis reveals distinct profiles of direct oral anticoagulants in human endothelial cells
- 1Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Dragana Campus
- 2Individualised Medicine and Pharmacological Research Solutions (IMPReS) Center, Dragana Campus
- 3Clinical Pharmacology Unit, Academic General Hospital of Alexandroupolis, Dragana Campus
Abstract
Introduction: Direct Oral Anticoagulants (DOACs) are the first-line treatment for thromboembolism prevention in patients with atrial fibrillation. Clinical and preclinical studies suggest that, beyond their anticoagulant properties, DOACs may have additional vascular effects. However, their effects on endothelial protein-abundance profiles remain incompletely characterized. The objective of this study was to explore the protein-abundance profiles associated with exposure of human umbilical vein endothelial cells (HUVECs) to each of the four DOACs in the absence of external stimuli, using a label-free quantification (LFQ) methodology.
Methods: Cultured HUVECs were exposed for 6 h to concentrations selected to approximate reported therapeutic plasma concentrations of apixaban, rivaroxaban, edoxaban, and dabigatran. All experiments were carried out in serum-free media, after a starvation period of 18 h. After protein extraction using the TRIzol Reagent, label-free quantitative proteomic analysis was performed using nanoLC-MS/MS, followed by the identification of differentially abundant proteins (DAPs) and bioinformatic enrichment analyses.
Results: We identified 1,643 protein entries, of which 753 were differentially abundant. Of these, 32 showed higher abundance and 26 showed lower abundance than the vehicle control in all four DOAC comparisons. Proteins showing shared abundance changes were mapped to gene identifiers that were enriched for annotations related to autophagy and cardiac development, and cellular activation. Several proteins showing abundance changes in all three FXa-inhibitor comparisons, but not in the dabigatran comparison, were mapped to annotations related to hypoxia response, vascular permeability, and cholesterol homeostasis. However, this comparison is potentially confounded by the absence of an HCl-matched vehicle control for dabigatran. The majority of DAPs were drug specific, with distinct signatures for each DOAC. Exploratory enrichment subnetworks linked mapped gene identifiers from drug specific protein sets to ontology terms including endothelial-cell chemotaxis, sodium-ion transport, necrotic cell death, mRNA splicing, and regulation of gene expression. These cellular functions were not directly measured.
Conclusion: Under the experimental conditions used, exposure to the four DOACs was associated with common and drug-specific protein-abundance patterns in HUVECs. These protein-abundance patterns provide hypothesis-generating observations for subsequent functional investigation.
Related
- Dermatophytes in Argentina: species distribution, comparative identification accuracy of conventional and proteomic methods, introduction of a novel molecular identification approach, and antifungal susceptibility profiles
- Multi-scale analysis of histological, ultrastructural and proteomic regional differences in the Descemet’s membrane-endothelium of Fuchs endothelial corneal dystrophy
- Real-world comparative effectiveness and safety of direct oral anticoagulants versus warfarin in patients with atrial fibrillation
- Plasma proteomic profiles associated with low-dose computed tomography findings and incident lung cancer: a prospective pilot study
- Age-related clonal hematopoiesis and mosaic sex chromosome loss define distinct systemic proteomic programs and disease vulnerabilities
BibTeX
@article{pallikarou2026labelfree,
title = {{Label-free comparative proteomic analysis reveals distinct profiles of direct oral anticoagulants in human endothelial cells}},
author = {Myria Pallikarou and Natalia Atzemian and George Leonidis and Konstantina Portokallidou and Georgia Ragia and Vangelis G. Manolopoulos},
journal = {Frontiers in Pharmacology},
year = {2026},
month = oct,
doi = {10.3389/fphar.2026.1913833},
url = {https://www.frontiersin.org/articles/10.3389/fphar.2026.1913833/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Label-free comparative proteomic analysis reveals distinct profiles of direct oral anticoagulants in human endothelial cells
AU - Myria Pallikarou
AU - Natalia Atzemian
AU - George Leonidis
AU - Konstantina Portokallidou
AU - Georgia Ragia
AU - Vangelis G. Manolopoulos
PY - 2026
DA - 2026/10/28
JO - Frontiers in Pharmacology
DO - 10.3389/fphar.2026.1913833
UR - https://www.frontiersin.org/articles/10.3389/fphar.2026.1913833/full
AB - Introduction: Direct Oral Anticoagulants (DOACs) are the first-line treatment for thromboembolism prevention in patients with atrial fibrillation. Clinical and preclinical studies suggest that, beyond their anticoagulant properties, DOACs may have additional vascular effects. However, their effects on endothelial protein-abundance profiles remain incompletely characterized. The objective of this study was to explore the protein-abundance profiles associated with exposure of human umbilical vein endothelial cells (HUVECs) to each of the four DOACs in the absence of external stimuli, using a label-free quantification (LFQ) methodology. Methods: Cultured HUVECs were exposed for 6 h to concentrations selected to approximate reported therapeutic plasma concentrations of apixaban, rivaroxaban, edoxaban, and dabigatran. All experiments were carried out in serum-free media, after a starvation period of 18 h. After protein extraction using the TRIzol Reagent, label-free quantitative proteomic analysis was performed using nanoLC-MS/MS, followed by the identification of differentially abundant proteins (DAPs) and bioinformatic enrichment analyses. Results: We identified 1,643 protein entries, of which 753 were differentially abundant. Of these, 32 showed higher abundance and 26 showed lower abundance than the vehicle control in all four DOAC comparisons. Proteins showing shared abundance changes were mapped to gene identifiers that were enriched for annotations related to autophagy and cardiac development, and cellular activation. Several proteins showing abundance changes in all three FXa-inhibitor comparisons, but not in the dabigatran comparison, were mapped to annotations related to hypoxia response, vascular permeability, and cholesterol homeostasis. However, this comparison is potentially confounded by the absence of an HCl-matched vehicle control for dabigatran. The majority of DAPs were drug specific, with distinct signatures for each DOAC. Exploratory enrichment subnetworks linked mapped gene identifiers from drug specific protein sets to ontology terms including endothelial-cell chemotaxis, sodium-ion transport, necrotic cell death, mRNA splicing, and regulation of gene expression. These cellular functions were not directly measured. Conclusion: Under the experimental conditions used, exposure to the four DOACs was associated with common and drug-specific protein-abundance patterns in HUVECs. These protein-abundance patterns provide hypothesis-generating observations for subsequent functional investigation.
SN - 1663-9812
ER -