FVIIa treatment disrupts caveolin-1 interaction with EPCR and PAR1 and promotes the interaction between EPCR and PAR1. (A) HUVECs were treated with a control vehicle (con), APC (80 nM) or FVIIa (20 nM) for 1 h in serum-free medium and then lysed in RIPA buffer. Total cell lysates were immunoprecipitated with anti-caveolin-1, anti-EPCR, or anti-PAR1 antibodies. The immunoprecipitates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with anti-caveolin-1, anti-EPCR, or anti-PAR1 antibodies raised in a different animal species than that were used for immunoprecipitation (see “Materials and methods”). Each experiment was repeated at least 3 times, and the band intensities were quantified by FIJI software ImageJ2. IP, immunoprecipitation; IB, immunoblotting. (B) HUVECs cultured on a glass coverslip were treated with FVIIa as described in panel A. Cells were fixed and permeabilized and processed for double immunostaining to stain EPCR (green), caveolin-1 (red, left), PAR1 (green), caveolin-1 (red, center), or PAR1 (green), and EPCR (red, right). The images shown are merged. A small portion of the merged image (bordered with the yellow box) was digitally enlarged to illustrate colocalization. Scale bar indicates 50 µm. The colocalization coefficient between the 2 signals was analyzed using Zen 2009 software (Carl Zeiss; n = 30 cells). A Mann-Whitney U test was used to determine the significance between control and FVIIa treatment. ***P < .001.