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. Author manuscript; available in PMC: 2016 Nov 25.
Published in final edited form as: Thromb Haemost. 2015 Aug 13;114(6):1144–1155. doi: 10.1160/TH15-01-0079

Figure 2. Functional analysis of cell surface expressed EPCR-GPI.

Figure 2

To confirm attachment of the GPI anchor to EPCR, HEK-293 cells expressing EPCR-GPI (square), wild type EPCR (circle) or no EPCR (diamond) were treated with PI-PLC. (A) EPCR released in the supernatant upon PI-PLC treatment was determined by ELISA and (B) APC binding on PI-PLC treated cells was analyzed by on-cell Western. To determine whether EPCR-GPI supports PAR cleavage, APC-mediated PAR cleavage on HEK-293 cells expressing SEAP-PAR1 (C), or SEAP-PAR3 (E) with wild type EPCR (circle), EPCR-GPI (square), E86A-EPCR (diamond), or no EPCR (X) was compared. (D, F) The role of EPCR-GPI in PAR1 (D) and PAR3 (F) cleavage was confirmed by incubating the cells with PI-PLC prior to addition of APC (50 nM). SEAP-PAR1 (D) and SEAP-PAR3 (F) cleavage by APC in the absence of PI-PLC is set to 100%. Total PAR1 (D) and total PAR3 (F) is the control for SEAP-PAR1 (D) or SEAP PAR3 (F) expression on the cell surface in the presence of increasing concentrations of PI-PLC, measured as the SEAP activity directly on the cell surface in the absence of APC. Shown are mean ± SEM of three independent experiments.