Effect of sPLA2-V on EPCR function on endothelial cells. (A) APC binding to HAECs. Cells were incubated with increasing amounts of APC-PP* after 48-hour pretreatment with 0.5μM manoalide (○), 2-hour pretreatment with 20 μg/mL sPLA2-V (gray circle), or no pretreatment (●). APC-PP* binding was assessed by flow cytometry. A representative experiment of 3 independent repeats is shown. MEFL indicates molecules of equivalent fluorescein. (B) Protein C activation on HAECs. Increasing amounts of protein C were incubated with thrombin for 30 minutes in the presence of HAECs, 48 hours pretreated with 0.5μM manoalide (○), 2 hours pretreated with 20 μg/mL sPLA2-V (gray circle), or nonpretreated (●). The amount of APC generated was measured with the chromogenic substrate S-2366. A representative experiment of 3 independent repeats is shown. (C) Inhibitory effect of APC on staurosporine-induced apoptosis in HAECs. Cells were pretreated with manoalide or sPLA2-V as in panels A and B, and then supplemented with 50nM APC for 4 hours after which apoptosis was induced with 10μM staurosporine for 60 minutes. Apoptosis was estimated by assessing the number of cells positive for annexin V–Alexa 647 binding by flow cytometry. A total of 6.6% ± 1.1% of the untreated cells were found to be apoptotic, and this percentage increased to 15.1% ± 1.0% after staurosporine incubation. For clarity purposes, results refer to the untreated cells, whose apoptotic rate was considered 100%. Data are mean ± SD of 3 independent experiments. Mann-Whitney U test was used for statistical comparisons. Sta indicates staurosporine. *P < .05 versus the staurosporine + APC group.
