Thrombotic vs obstetric antiphospholipid syndrome: a role for constitutive endothelial dysfunction

Background Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombotic (T-APS) or obstetric (O-APS) manifestations in the presence of antiphospholipid antibodies (aPLs). Endothelial dysfunction (ED), resulting from the interaction between aPLs and endothelial cells, is thought to contribute to APS clinical manifestations. However, it remains unclear whether constitutive ED may further predispose patients to disease onset and contribute to the heterogeneity of clinical phenotypes. In this context, patient-specific endothelial colony-forming cells (ECFCs) represent a valuable model to investigate ED in APS. Aim To determine whether APS-derived ECFCs display constitutive ED and whether distinct ED profiles discriminate T-APS, O-APS, and aPL carriers. Methods ECFCs were isolated from T-APS and O-APS patients, aPL carriers, and healthy donors (HDs). Constitutive ED was investigated by assessing the expression of adhesion and haemostatic markers by flow cytometry. Unbiased FACS-based clustering was performed to identify combinatorial ECFC phenotypic states. Functional ED was evaluated by PBMC adhesion assay, thrombin generation assay (TGA), and thrombogenesis assay under flow conditions. In addition, single-cell RNA sequencing (scRNA-seq) was performed on ECFCs from 8 HDs, 8 T-APS patients, 4 O-APS patients, and 4 patients with unprovoked venous thromboembolism, included as a non-autoimmune thrombotic comparator. Results Integrated supervised and unbiased FACS analyses showed that T-APS ECFCs are shifted toward an adhesion-prone and haemostatic-altered phenotype, characterized by increased ICAM-1/VCAM-1 expression, reduced EPCR expression, increased TF/TM ratio, and enrichment of a VCAM1+ activated adhesive/haemostatic ECFC state. Functionally, T-APS ECFCs promoted faster thrombin generation, as shown by reduced lag time and time to peak. Thrombogenesis assays under flow showed heterogeneous platelet deposition and a trend toward increased fibrin formation on APS-derived ECFC layers. scRNA-seq analysis revealed that ECFCs mainly segregated according to cell-cycle phase, with T-APS ECFCs showing a higher representation of quiescent G1 cells. Within the G1 compartment, T-APS ECFCs displayed enrichment of inflammatory, extracellular matrix-remodelling, EndMT-like, and stress-related transcriptional programs. Gene-score analysis in proliferating S/G2M ECFCs confirmed the enrichment of selected disease-associated G1-derived programs also outside the quiescent compartment. Conclusions APS-derived ECFCs display constitutive ED, with T-APS showing the most evident adhesion-prone, haemostatic-altered, and transcriptionally remodelled phenotype. Integrated phenotypic, functional, and scRNA-seq analyses suggest that APS-associated ED is intrinsically reflected by patient-derived ECFCs and may contribute to impaired endothelial repair and thrombotic susceptibility. These findings support the existence of distinct ED profiles across APS clinical manifestations and aPL carriers.