Figure 2
Figure 2. Noncanonical cleavage of PAR1 at Arg46 by APC on cells. Cleavage of wt-SEAP-PAR1 and SEAP-PAR1 cleavage site mutants by (A) APC and (B) thrombin in the presence of wt-EPCR and in the absence of EPCR (C). (□, ■) represent wt-SEAP-PAR1; (Δ, ▴), R41Q-SEAP-PAR1; (○, ●), R46Q-SEAP-PAR1; and (♢, ♦), R41Q/R46Q-SEAP-PAR1. (C) Closed symbols indicate thrombin; and open symbols, APC. (D) Mapping of the canonical and noncanonical cleavage site sensitive epitopes of anti-PAR1 antibodies SPAN12, ATAP2, and WEDE15 using synthetic peptides coated onto microtiter plate wells. Peptides represent the N-terminal (TR24-41) or C-terminal fragment (TR42-66) of PAR1 after cleavage at Arg41 or the N-terminal (TR24-47) or C-terminal fragment (TR47-66) of PAR1 after cleavage at Arg46 or the entire cleavage site region of the PAR1 N-terminal tail (TR33-62). (E) OCW of anti-PAR1 antibodies WEDE15 and ATAP2 on EA.hy.926 endothelial cells after incubation with control buffer (NONE), APC (100nM), or thrombin (0.25nM) for 3 hours. Cell-bound anti-PAR1 antibodies were detected with biotinylated goat anti–mouse secondary antibodies and IRDye 800CW streptavidin in the 800-nm channel of the Odyssey Imager (green). Draq5 was used for cell number normalization and detected in the 700-nm channel (red). Overlay of the Draq5 700 channel and anti-PAR1 800-nm channel is indicated in yellow (merge). The presence and loss of PAR1 epitopes on endothelial cell surfaces were determined using OCW quantification of cleavage site sensitive and insensitive PAR1 antibodies on EA.hy926 endothelial cells versus time for incubation of cells with (F) APC (100nM) or (G) thrombin (0.25nM). The cell surface was probed for PAR-1 with (○) WEDE15 (detecting all PAR1 regardless of cleavage at either Arg41 or Arg46), (□) ATAP2 (detecting uncleaved and PAR1 cleaved only at Arg41), or (♢) SPAN12 (detecting only uncleaved PAR1). (D-E) Representative experiment in triplicates. (A-C,F-G) Data points represent the mean ± SEM (N ≥ 3). (F-G) *P < .001, compared with WEDE15. ns indicates not significant.

Noncanonical cleavage of PAR1 at Arg46 by APC on cells. Cleavage of wt-SEAP-PAR1 and SEAP-PAR1 cleavage site mutants by (A) APC and (B) thrombin in the presence of wt-EPCR and in the absence of EPCR (C). (□, ■) represent wt-SEAP-PAR1; (Δ, ▴), R41Q-SEAP-PAR1; (○, ●), R46Q-SEAP-PAR1; and (♢, ♦), R41Q/R46Q-SEAP-PAR1. (C) Closed symbols indicate thrombin; and open symbols, APC. (D) Mapping of the canonical and noncanonical cleavage site sensitive epitopes of anti-PAR1 antibodies SPAN12, ATAP2, and WEDE15 using synthetic peptides coated onto microtiter plate wells. Peptides represent the N-terminal (TR24-41) or C-terminal fragment (TR42-66) of PAR1 after cleavage at Arg41 or the N-terminal (TR24-47) or C-terminal fragment (TR47-66) of PAR1 after cleavage at Arg46 or the entire cleavage site region of the PAR1 N-terminal tail (TR33-62). (E) OCW of anti-PAR1 antibodies WEDE15 and ATAP2 on EA.hy.926 endothelial cells after incubation with control buffer (NONE), APC (100nM), or thrombin (0.25nM) for 3 hours. Cell-bound anti-PAR1 antibodies were detected with biotinylated goat anti–mouse secondary antibodies and IRDye 800CW streptavidin in the 800-nm channel of the Odyssey Imager (green). Draq5 was used for cell number normalization and detected in the 700-nm channel (red). Overlay of the Draq5 700 channel and anti-PAR1 800-nm channel is indicated in yellow (merge). The presence and loss of PAR1 epitopes on endothelial cell surfaces were determined using OCW quantification of cleavage site sensitive and insensitive PAR1 antibodies on EA.hy926 endothelial cells versus time for incubation of cells with (F) APC (100nM) or (G) thrombin (0.25nM). The cell surface was probed for PAR-1 with (○) WEDE15 (detecting all PAR1 regardless of cleavage at either Arg41 or Arg46), (□) ATAP2 (detecting uncleaved and PAR1 cleaved only at Arg41), or (♢) SPAN12 (detecting only uncleaved PAR1). (D-E) Representative experiment in triplicates. (A-C,F-G) Data points represent the mean ± SEM (N ≥ 3). (F-G) *P < .001, compared with WEDE15. ns indicates not significant.

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