Figure 1.

Plasma extracellular vesicles (EVs) support elevated thrombin generation in atherosclerotic cardiovascular disease (ASCVD) plasma. A, EVs from patients support higher levels of thrombin generation. The ability of EV membranes to support thrombin generation was assessed using prothrombinase assay as described in Materials and Methods. B, EV counts are significantly higher in patients. To quantify EV, platelet-free plasma (0.5 mL) was processed using size exclusion chromatography (iZON qEV columns) and nanoparticle tracking analysis (Nanosight 300). C, Thrombin generation positively correlates with EV counts. Thrombin generation and EV counts were correlated using Pearson correlation. D, EV surface area is increased in vascular disease. EV surface area was calculated as described in Materials and Methods and plotted as box plots. E, Thrombin generation positively correlates with EV surface area. Thrombin generation and EV surface area were correlated using Pearson correlation. F and G, Thrombin generation was not changed between groups if EVs were normalized by counts or surface area. Thrombin generation on the surface of EV was adjusted by EV counts (per 1×10⁹ EV; F) and surface area (G) and plotted as box plots. H and I, Heatmaps showing aminophospholipid (aPL) molecular species in EV. Heatmaps were drawn using the pheatmap R package as described in Materials and Methods to visualize aPL amounts between groups for all the measured species, analyzed using liquid chromatography tandem mass spectrometry. Statistical significance was tested with 1-way ANOVA and Tukey post hoc test (P<0.05 considered significant). Acute coronary syndrome (ACS; n=24: A, B, D, F, and G; n=21: H and I), coronary artery disease (CAD) but no ACS (n=19: A, B, D, F, and G), positive risk factors (RFs) with no significant CAD (n=23: A, B, D, F, and G; n=22: H and I), and healthy control (HC; n=24: A; n=22: B, D, F, and G; n=23: H and I).