EC release EMP in vitro. The antigenic phenotype of EMP released from resting and IL-1alpha-stimulated EC differs among each other as well as from resting and stimulated EC, respectively. E-selectin exposed on IL-1alpha-stimulated EC is a valid marker for EMP detection ex vivo to establish endothelial cell activation.

; 24 h). Cell fractions were analyzed by flow cytometry for annexin V binding, propidium iodide (PI) and caspase 3 staining (n ¼ 3). Caspase 3 in EMP was studied using Western blot (n ¼ 6) and flow cytometry (n ¼ 6). Plasma from healthy subjects and systemic lupus erythematosus patients (both n ¼ 3) were analyzed for caspase 3-containing (E)MP. Results: Detached but not adherent cells double-stained for annexin V and PI, confirming the apoptotic conditions of the detached cells and the viable nature of the adherent cells. Caspase 3 was solely present in the detached cells and procaspase 3 in the adherent cells. Caspase 3 was present in EMP from both control and IL1a-treated cultures. Counts of EMP and detached cells, but not adherent cells, highly correlated (r ¼ 0.959, P < 0.0001). In vivo circulating MP from nucleated (endothelial cells, monocytes) and anucleated cells (platelets, erythrocytes) contained caspase 3. Conclusions: EMP contain caspase 3 and may be mainly derived from detached (apoptotic) endothelial cells in vitro. The presence of caspase 3 in MP from anucleated cell types, however, suggests that its presence may not necessarily be related to apoptosis in vivo but may be associated with caspase 3 activation unrelated to apoptosis.

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