Journal: Journal of Lipid Research

Article Title: Circulating membrane aminophospholipids contribute to thrombotic risk in rheumatoid arthritis

doi: 10.1016/j.jlr.2025.100842

Figure Lengend Snippet: RA patients show higher coagulation markers and EV counts in plasma, and their EV can support increased in vitro thrombin generation. A and B: d -dimer levels are raised in RA patients. d -dimer levels were measured as described in section, using ELISA in plasma from RA patients (n = 21) and HC (n = 16), showing absolute levels (ng/ml) and frequency of raised levels (%) above clinical cutoff of 500 ng/ml shown. C: TAT complexes are increased in plasma from RA patients . TAT complexes were measured using ELISA in plasma from RA patients (n = 25) and HC (n = 19). D: EVs from RA patients generate more thrombin. The ability of EVs in 6 ml plasma to support coagulation reactions was assessed using the prothrombinase assay, as described in section, for HC (n = 24) and RA patients (n = 22). E and F RA patients have increased plasma EV counts but no increase in diameter. EV particles were counted, from HC (n = 24) and RA (n = 24) plasma, as described in section. G: Thrombin generation is increased in RA plasma because of increased EV levels. Prothrombinase assay, using 10 10 EV particles, was applied to plasma from HC (n = 24) and RA (n = 21). Data were analyzed using Mann-Whitney test (∗ P < 0.05, ∗∗ P < 0.001, ∗∗∗ P < 0.001). H: EV particle counts correlate with thrombin generation and TAT levels in RA. Spearman correlation was performed comparing EV particle counts with TAT complexes (n = 22) in RA plasma. I: Heatmap for external-facing PE and PS levels in RA and HC plasma. EV particles were isolated followed by biotinylation of externalized lipids, as described in section. Lipids were extracted from plasma from HC (n = 19) and RA patients (n = 21) as described in section and analyzed by LC-MS/MS. Externalized aPLs in EV are shown in a heatmap (log10, ng/ml). J: Total aPLs are increased in EVs from RA. Total (internal + external) individual aPLs in EVs were analyzed using LC-MS/MS and shown in a heatmap (log10, ng/ml). K and L: Externalized PS was barely detected in EVs from RA plasma and undetectable in HC, whereas external-facing PE was similar in both groups. Externalized PS and PE levels were determined (ng/ml) for HC (n = 19) and RA (n = 21), using LC-MS/MS as described in section. M and N: Total PS and PE levels are increased in EVs in RA. Total (external and internal) aPLs were quantified as described in section or HC (n = 19) and RA (n = 22) (ng/ml of plasma). O–Q : Externalized PE is decreased in EVs from RA patients following normalization of EV count. Total aPL concentrations were adjusted to EV counts (10 10 EV particles), for HC (n = 18) and RA (n = 21), along with externalized PE for HC (n = 19) and RA patients (n = 20) (ng/10 10 EV particles). Data were analyzed using the Mann-Whitney test (∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001).

Article Snippet: Twenty microliter platelets (4 × 10 6 cells), WBC (8 × 10 4 cells), or EVs (equivalent to 6 ml of plasma) were incubated with 20 μl coagulation factors (1 mM factor II, 50 nM factor Xa, 5 mM CaCl 2 [Enzyme Laboratories, UK], 15 nM factor Va [Haematologic; Cambridge Bioscience]).

Techniques: Coagulation, Clinical Proteomics, In Vitro, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Isolation, Liquid Chromatography with Mass Spectroscopy