Effect of time on MP-associated EPCR release. Time-dependence experiment of PC (dark gray bars) and APC (100 nM; light gray bars) effect on (A) HUVECs and (B) monocytes by flow cytometric analysis of cellular EPCR expression. Results are expressed as percentage of EPCR surface expression and are the mean value of 3 experiments ± SD with the asterisk indicating P < .005. Panels C and D show the corresponding quantitation of MPs isolated from the media after HUVEC and monocyte treatment, respectively, with PC or APC. These were then captured for EPCR and quantified by chromogenic S2366 for the APC bound (nM) and separately for EPCR detected (ng/mL). (E) HUVECs were incubated with APC (100 nM) for up to 2 hours and MPs were isolated and analyzed by Western blotting, with sEPCR and HUVEC cell lysate as reference standards. (F) HUVECs were incubated with APC (100 nM) for up to 24 hours and EPCR mRNA expression was assessed using RT-PCR with ethidium bromide visualization of PCR-amplified products of EPCR and β-actin mRNA after agarose gel electrophoresis.