Residual endothelial prothrombotic imbalance after patent foramen ovale closure

Background Patent Foramen Ovale (PFO) is associated with migraine with aura and a systemic prothrombotic milieu. Beyond previously described platelet activation, endothelial dysfunction may contribute to this phenotype. Endothelial colony-forming cells (ECFCs) provide a patient-specific model to investigate endothelial thromboinflammatory properties and their modulation after PFO closure. Purposes To characterize the procoagulant phenotype, shear-stress response, and transcriptomic profile of ECFCs from PFO patients before and after closure compared with healthy subjects (HS). Methods ECFCs were obtained from 35 PFO patients at baseline (T0) and 6 months after closure (T1), and from 25 HS. Intracellular Tissue Factor (TF) and Tissue Factor Pathway Inhibitor (TFPI) were measured by flow cytometry and ELISA, and thrombin generation by calibrated automated thrombography. ECFCs were exposed to high laminar shear stress to assess expression of genes involved in endothelial homeostasis, inflammation, vascular tone, and coagulation (including KLF2, KLF4, NOS3, EDN1, HMGB1, TF). Whole-transcriptome analysis compared T0 and T1 profiles. Results ECFCs from PFO patients showed a procoagulant phenotype. A higher proportion of patient-derived ECFC lines generated measurable thrombin compared with HS (29% vs 11%), persisting at T1 (33%). The TF/TFPI ratio was higher in patients than HS (0.18 ± 0.17 vs 0.065 ± 0.043) and remained elevated after closure. TF protein levels were also higher in patients (466.7 ± 233.5 vs 388.3 ± 208.0 pg/mg), whereas TFPI tended to be lower (60.8 ± 49.1 vs 77.5 ± 41.7 ng/mg). Under high laminar shear stress, patient ECFCs showed reduced activation of shear-protective pathways, including lower KLF4 expression, along with persistence of pro-inflammatory/procoagulant signals such as TF and HMGB1. Transcriptomic analysis revealed enrichment at T0 of pathways related to platelet activation, leukocyte adhesion, and cell–matrix interaction, whereas T1 cells showed relative upregulation of immunoregulatory (e.g., IL-10–associated) and vascular repair pathways, including wound-healing and tissue remodeling programs. Conclusions ECFCs from PFO patients exhibit a sustained prothrombotic and thromboinflammatory phenotype characterized by enhanced TF-driven activity and altered shear-stress responses. PFO closure is associated with partial transcriptional reprogramming toward immunoregulation and endothelial repair, although the procoagulant imbalance is not fully normalized, identifying the endothelium as an active contributor to the thromboinflammatory milieu associated with PFO.